scholarly journals Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity

2013 ◽  
Vol 304 (9) ◽  
pp. L582-L592 ◽  
Author(s):  
James D. Londino ◽  
Ahmed Lazrak ◽  
Asta Jurkuvenaite ◽  
James F. Collawn ◽  
James W. Noah ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ion Channel ◽  
Xenopus Oocytes ◽  
Secretory Pathway ◽  
Matrix Protein ◽  
Protein A ◽  
Dependent Manner ◽  
Hek 293 ◽  
Concanamycin A ◽  
And Function

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl−) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H+) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o−) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H+, did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

scholarly journals Interferon Regulatory Factor 6 Promotes Cell Cycle Arrest and Is Regulated by the Proteasome in a Cell Cycle-Dependent Manner

2008 ◽  
Vol 28 (7) ◽  
pp. 2235-2243 ◽  
Author(s):  
Caleb M. Bailey ◽  
Daniel E. Abbott ◽  
Naira V. Margaryan ◽  
Zhila Khalkhali-Ellis ◽  
Mary J. C. Hendrix
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Cell Cycle Arrest ◽  
Interferon Regulatory Factor ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Regulatory Factor ◽  
Cycle Arrest ◽  
Interferon Regulatory Factor 6 ◽  
And Function

ABSTRACT Interferon regulatory factor 6 (IRF6) is a novel and unique member of the IRF family of transcription factors. IRF6 has not been linked to the regulatory pathways or functions associated with other IRF family members, and the regulation and function of IRF6 remain unknown. We recently identified a protein interaction between IRF6 and the tumor suppressor maspin. To gain insight into the biological significance of the maspin-IRF6 interaction, we examined the regulation and function of IRF6 in relation to maspin in normal mammary epithelial cells. Our results demonstrate that in quiescent cells, IRF6 exists primarily in a nonphosphorylated state. However, cellular proliferation leads to rapid IRF6 phosphorylation, resulting in proteasome-dependent IRF6 degradation. These data are supported in situ by the increased expression of IRF6 in quiescent, differentiated lobuloalveolar cells of the lactating mammary gland compared to its expression in proliferating ductal and glandular epithelial cells during pregnancy. Furthermore, the reexpression of IRF6 in breast cancer cells results in cell cycle arrest, and the presence of maspin augments this response. These data support a model in which IRF6, in collaboration with maspin, promotes mammary epithelial cell differentiation by facilitating entry into the G0 phase of the cell cycle.

scholarly journals Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

1996 ◽  
Vol 109 (6) ◽  
pp. 1479-1495 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Rodriguez-Paris ◽  
J.M. Bush ◽  
L. Zhang ◽  
J.A. Cardelli
Keyword(s):  
Morphological Changes ◽  
Specific Inhibitor ◽  
Fluid Phase ◽  
Contractile Vacuole ◽  
Dependent Manner ◽  
Concanamycin A ◽  
Latex Beads ◽  
And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

scholarly journals Identification and Characterization of a Splicing Variant in the 5′ UTR of the Human TLR5 Gene

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Thi Xoan Hoang ◽  
Cao Nguyen Duong ◽  
Jae Young Kim
Keyword(s):  
Cell Lines ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Bacterial Flagella ◽  
Essential Components ◽  
Alternatively Spliced ◽  
And Function

Toll-like receptors (TLRs) are essential components of the innate immune system. TLR5 is the receptor for flagellin, the principal protein component of bacterial flagella. The TLR5 gene has 6 exons. In an RT-PCR analysis, we found long TLR5 transcripts, in addition to those of the expected size (short TLR5 transcripts). A sequence analysis revealed that the long TLR5 transcripts contain a new exon of 94 nucleotides located between previously reported exons IV and V in the 5′ untranslated region (5′ UTR). A real-time PCR analysis of the two alternatively spliced variants in various cell lines showed that the long TLR5 transcripts are abundantly expressed in nonimmune cells. The ratios of long/short transcripts in human nonimmune cell lines, such as A549, T98G, HaCaT, H460, HEK-293, and Caco-2 cells, and primary mesenchymal stem cells were in the range of 1.25 to 4.31. In contrast, those of human monocytic THP-1 and U937 cells and E6.1 T cells and Ramos B cells were around 0.9. These ratios in human monocytic THP-1 cells were decreased by treatment with IFN-γ in a concentration-dependent manner. Based on our findings, we suggest that the newly found long TLR5 transcripts may be involved in the negative regulation of TLR5 expression and function.

scholarly journals Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells

1992 ◽  
Vol 117 (2) ◽  
pp. 269-278 ◽  
Author(s):  
MD Turner ◽  
ME Rennison ◽  
SE Handel ◽  
CJ Wilde ◽  
RD Burgoyne
Keyword(s):  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Isolated Cells ◽  
Pulse Label ◽  
Constitutive Secretion

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.

MUNC13 REGULATES SECRETION AT THE GOLGI IN A LOCALIZATION-DEPENDENT MANNER

2008 ◽  
Vol 31 (4) ◽  
pp. 11
Author(s):  
Neil M Goldenberg ◽  
Mel Silverman
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Phorbol Esters ◽  
Cell Structure ◽  
Eukaryotic Cell ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
C1 Domain ◽  
Surface Labelling ◽  
And Function

Background: Constitutive secretion is critical for the maintenance of eukaryotic cell structure and function. Our lab has shown that Rab34 is required for secretion at the Golgi^1, and that the C1 domain-containing protein, Munc13, is an effector of Rab34^2. Current studies seek to elucidate potential roles for Munc13in secretion at the Golgi. Methods: Using a temperature-sensitive mutant of the Vesicular Stomatitis G-protein fused to GFP (VSVG-GFP) to monitor secretion, we examinedthe role of Munc13 in secretion in HeLa cells. Cells transfected with VSVG-GFP were treated with Munc13, amutant lacking the C1 domain (C1-less), and the phorbol esters TPA andPDBu. The rate of VSVG-GFP secretion was monitored using surface labelling of plasmalemmal VSVG-GFP and spinning disc confocal microscopy. Results: TPA treatment resulted in an increase in the rate of VSVG-GFP appearance at the plasma membrane. Co-transfection of either Munc13 or C1-less alone also resulted in an increased rate of VSVG-GFP transport. Transfection of Munc13 plus TPA treatment resulted in amarked decrease in the rate of VSVG-GFP transport. Since TPA treatment relocalizes Munc13 to the plasma membrane, this result suggests that the availability of Munc13 in the cytosol is required for its effect on VSVG-GFP secretion. Conclusions: Munc13 over-expression increases the rate of VSVG-GFP secretion to the plasma membrane. Sequestration of Munc13 at the plasma membrane with TPA abrogates thiseffect, and reduces the rate of VSVG-GFP secretion. We propose that Munc13 effects VSVG-GFP secretion via its interaction with Rab34 at the Golgi. References: 1. Goldenberg, NM, S. Grinstein, M. Silverman. Golgi-bound Rab34 is a Novel Member ofthe Secretory Pathway. Mol BiolCell. 18(12):4762-4771 (2007). 2. Speight, P, M. Silverman.Diacylglycerol-Activated Hmunc13 Serves as an Effector of the GTPaseRab34. Traffic.6(10):858-865 (2005).

scholarly journals Diversity in Requirement of Genetic and Epigenetic Factors for Centromere Function in Fungi

2011 ◽  
Vol 10 (11) ◽  
pp. 1384-1395 ◽  
Author(s):  
Babhrubahan Roy ◽  
Kaustuv Sanyal
Keyword(s):  
Dna Sequence ◽  
De Novo ◽  
Protein A ◽  
Fungal Species ◽  
Dependent Manner ◽  
Epigenetic Factors ◽  
Daughter Cells ◽  
Centromeric Protein ◽  
Species Specific ◽  
And Function

ABSTRACT A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for de novo centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species.

scholarly journals Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Links Innate and Adaptive Immunity in a TLR2-dependent Manner and Involvement of IL-12 and Nitric Oxide

2012 ◽  
Vol 287 (15) ◽  
pp. 12589-12601 ◽  
Author(s):  
Debasis Pore ◽  
Nibedita Mahata ◽  
Manoj K. Chakrabarti
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Shigella Flexneri ◽  
Critical Role ◽  
Protein A ◽  
Adaptive Immune Response ◽  
Dependent Manner ◽  
Hek 293 ◽  
Ifn Γ

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4+ T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4+ T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4+ T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4+ T cells, representing that OmpA may enhance IFN-γ expression in CD4+ T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.

scholarly journals ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

2020 ◽  
Vol 40 (1) ◽  
pp. 128-144 ◽  
Author(s):  
Yu Yamazaki ◽  
Mitsuru Shinohara ◽  
Akari Yamazaki ◽  
Yingxue Ren ◽  
Yan W. Asmann ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Cell Phenotype ◽  
Dependent Manner ◽  
Barrier Formation ◽  
Brain Pericytes ◽  
Membrane Components ◽  
And Function

Objective: The ε4 allele of the APOE gene ( APOE4 ) is the strongest genetic risk factor for Alzheimer disease when compared with the common ε3 allele. Although there has been significant progress in understanding how apoE4 (apolipoprotein E4) drives amyloid pathology, its effects on amyloid-independent pathways, in particular cerebrovascular integrity and function, are less clear. Approach and Results: Here, we show that brain pericytes, the mural cells of the capillary walls, differentially modulate endothelial cell phenotype in an apoE isoform-dependent manner. Extracellular matrix protein induction, tube-like structure formation, and barrier formation were lower with endothelial cells cocultured with pericytes isolated from apoE4-targeted replacement (TR) mice compared with those from apoE3-TR mice. Importantly, aged apoE4-targeted replacement mice had decreased extracellular matrix protein expression and increased plasma protein leakages compared with apoE3-TR mice. Conclusions: ApoE4 impairs pericyte-mediated basement membrane formation, potentially contributing to the cerebrovascular effects of apoE4.

scholarly journals Distinct Expression and Function of Alternatively Spliced Tbx5 Isoforms in Cell Growth and Differentiation

2008 ◽  
Vol 28 (12) ◽  
pp. 4052-4067 ◽  
Author(s):  
Romain Georges ◽  
Georges Nemer ◽  
Martin Morin ◽  
Chantal Lefebvre ◽  
Mona Nemer
Keyword(s):  
Heart Development ◽  
Wide Spectrum ◽  
Protein A ◽  
Growth Stimulation ◽  
Underlying Mechanism ◽  
Experimental Models ◽  
Dependent Manner ◽  
Dominant Disease ◽  
And Function

ABSTRACT Mutations in the T-box transcription factor Tbx5 cause Holt-Oram syndrome, an autosomal dominant disease characterized by a wide spectrum of cardiac and upper limb defects with variable expressivity. Tbx5 haploinsufficiency has been suggested to be the underlying mechanism, and experimental models are consistent with a dosage-sensitive requirement for Tbx5 in heart development. Here, we report that Tbx5 levels are regulated through alternative splicing that generates, in addition to the known 518-amino-acid protein, a C-terminal truncated isoform. This shorter isoform retains the capacity to bind DNA, but its interaction with Tbx5 collaborators such as GATA-4 is altered. In vivo, the two spliced isoforms are oppositely regulated in a temporal and growth factor-dependent manner and are present in distinct DNA-binding complexes. The expression of the long isoform correlates with growth stimulation, and its reexpression in postnatal transgenic mouse hearts promotes hypertrophy. Conversely, the upregulation of the short but not the long isoform in C2C12 myoblasts leads to growth arrest and cell death. The results provide novel insight into posttranscriptional Tbx5 regulation and point to an important role not only in cell differentiation but also in cell proliferation and organ growth. The data may help analyze genotype-phenotype relations in patients with Holt-Oram syndrome.

scholarly journals Membrane-Bound Meet Membraneless in Health and Disease

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1000 ◽  
Author(s):  
Chujun Zhang ◽  
Catherine Rabouille
Keyword(s):  
Phase Separation ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Dependent Manner ◽  
P Bodies ◽  
Early Secretory Pathway ◽  
Cell Organization ◽  
Health And Disease ◽  
Membrane Bound ◽  
And Function

Membraneless organelles (MLOs) are defined as cellular structures that are not sealed by a lipidic membrane and are shown to form by phase separation. They exist in both the nucleus and the cytoplasm that is also heavily populated by numerous membrane-bound organelles. Even though the name membraneless suggests that MLOs are free of membrane, both membrane and factors regulating membrane trafficking steps are emerging as important components of MLO formation and function. As a result, we name them biocondensates. In this review, we examine the relationships between biocondensates and membrane. First, inhibition of membrane trafficking in the early secretory pathway leads to the formation of biocondensates (P-bodies and Sec bodies). In the same vein, stress granules have a complex relationship with the cyto-nuclear transport machinery. Second, membrane contributes to the regulated formation of phase separation in the cells and we will present examples including clustering at the plasma membrane and at the synapse. Finally, the whole cell appears to transit from an interphase phase-separated state to a mitotic diffuse state in a DYRK3 dependent manner. This firmly establishes a crosstalk between the two types of cell organization that will need to be further explored.

Sign in / Sign up

Export Citation Format

Share Document