Vitamin E isoforms directly bind PKCα and differentially regulate activation of PKCα

Vitamin E isoforms have opposing regulatory effects on leucocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leucocyte migration across endothelial cells by regulating VCAM (vascular cell-adhesion molecule)-1 activation of endothelial cell PKCα (protein kinase Cα). However, it is not known whether tocopherols directly regulate cofactor-dependent or oxidative activation of PKCα. We report in the present paper that cofactor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10-fold lower concentration than γ-tocopherol. In binding studies, NBD (7-nitrobenz-2-oxa-1,3-diazole)-tagged α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain, but not PKCζ. NBD-tagged α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol and retinol, but not by cholesterol or PS (phosphatidylserine). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid vesicles containing tocopherol without PS. The PKCα-C1b domain did not bind to vesicles containing tocopherol and PS. In summary, α-tocopherol and γ-tocopherol bind the diacylglycerol-binding site on PKCα-C1a and can enhance PKCα-C2 binding to PS-containing vesicles. Thus the tocopherols can function as agonists or antagonists for differential regulation of PKCα.

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Determination of the calcium-binding sites of the C2 domain of protein kinase Cα that are critical for its translocation to the plasma membrane

Biochemical Journal ◽

10.1042/bj3370513 ◽

1999 ◽

Vol 337 (3) ◽

pp. 513-521 ◽

Cited By ~ 28

Author(s):

Senena CORBALÁN-GARCÍA ◽

José A. RODRÍGUEZ-ALFARO ◽

Juan C. GÓMEZ-FERNÁNDEZ

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Hela Cells ◽

Calcium Binding ◽

Lipid Vesicles ◽

Site Directed Mutagenesis ◽

C2 Domain ◽

Protein Kinase Cα

The C2 domain is a conserved protein module present in various signal-transducing proteins. To investigate the function of the C2 domain of protein kinase Cα (PKCα), we have generated a recombinant glutathione S-transferase-fused C2 domain from rat PKCα, PKC-C2. We found that PKC-C2 binds with high affinity (half-maximal binding at 0.6 µM) to lipid vesicles containing the negatively charged phospholipid phosphatidylserine. When expressed into COS and HeLa cells, most of the PKC-C2 was found at the plasma membrane, whereas when the cells were depleted of Ca2+ by incubation with EGTA and ionophore, the C2 domain was localized preferentially in the cytosol. Ca2+ titration was performed in vivo and the critical Ca2+ concentration ranged from 0.1 to 0.32 µM. We also identified, by site-directed mutagenesis, three aspartic residues critical for that Ca2+ interaction, namely Asp-187, Asp-246 and Asp-248. Mutation of these residues to asparagine, to abolish their negative charge, resulted in a domain expressed as the same extension as wild-type protein that could interact in vitro with neither Ca2+ nor phosphatidylserine. Overexpression of these mutants into COS and HeLa cells also showed that they cannot localize at the plasma membrane, as demonstrated by immunofluorescence staining and subcellular fractionation. These results suggest that the Ca2+-binding site might be involved in promoting the interaction of the C2 domain of PKCα with the plasma membrane in vivo.

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Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 Domain

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0499 ◽

2006 ◽

Vol 17 (1) ◽

pp. 56-66 ◽

Cited By ~ 77

Author(s):

John H. Evans ◽

Diana Murray ◽

Christina C. Leslie ◽

Joseph J. Falke

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Membrane Binding ◽

Membrane Lipid ◽

C2 Domain ◽

Plasma Membrane Lipid ◽

Protein Kinase Cα ◽

Golgi Network

The C2 domain of protein kinase Cα (PKCα) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCα and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCα Ca2+-binding loops (CBLs) and PIP2 binding site (β-strands 3–4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCα C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on β-strands 3–4, which bind to plasma membrane PIP2.

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Alternate promoters and variable splicing lead to hNedd4–2 isoforms with a C2 domain and varying number of WW domains

AJP Renal Physiology ◽

10.1152/ajprenal.00203.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. F916-F929 ◽

Cited By ~ 39

Author(s):

Omar A. Itani ◽

Jason R. Campbell ◽

Juan Herrero ◽

Peter M. Snyder ◽

Christie P. Thomas

Keyword(s):

Collecting Duct ◽

Rnase Protection Assay ◽

C2 Domain ◽

Binding Studies ◽

Specific Expression ◽

Rnase Protection ◽

Ww Domains ◽

Rat Thyroid

Mutations that disrupt a PY motif in epithelial Na+ channel (ENaC) subunits increase surface expression of Na+ channels in the collecting duct, resulting in greater Na+ reabsorption. Recently, Nedd4 and Nedd4–2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4–2 (hNedd4–2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb, and contains >40 exons. Multiple 5′-exons were identified by 5′-rapid amplification of cDNA ends, and tissue-specific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3′-untranslated region. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4–2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Coexpression, in Fischer rat thyroid epithelia, of ENaC and Nedd4–2 cDNAs leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na+ transport regulation. In vitro binding studies demonstrated that individual PY motifs of α-, β-, and γ-ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4–2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4–2 in the regulation of ENaC and define protein domains important for Nedd4–2 function.

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1120: Citrate and Vitamin E Blunt the SWL-Induced Free Radical Surge in an In-Vitro MDCK Cell Culture Model

The Journal of Urology ◽

10.1016/s0022-5347(18)38357-5 ◽

2004 ◽

Vol 171 (4S) ◽

pp. 295-295

Author(s):

Fernando C. Delvecchio ◽

Ricardo M. Brizuela ◽

Karen J. Byer ◽

W. Patrick Springhart ◽

Saeed R. Khan ◽

...

Keyword(s):

Cell Culture ◽

Free Radical ◽

Vitamin E ◽

Mdck Cell ◽

Cell Culture Model ◽

Culture Model

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Protective Effect of Vitamin E on Immune Triggered, Granulocyte Mediated Endothelial Injury

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661027 ◽

1984 ◽

Vol 51 (01) ◽

pp. 089-092 ◽

Cited By ~ 24

Author(s):

M A Boogaerts ◽

J Van de Broeck ◽

H Deckmyn ◽

C Roelant ◽

J Vermylen ◽

...

Keyword(s):

Vitamin E ◽

Protective Effect ◽

Umbilical Vein ◽

Endothelial Damage ◽

Endothelial Injury ◽

Mediated Effects ◽

Anti Oxidant ◽

Endothelial Cell Cultures ◽

Vitro Model

SummaryThe effect of alfa-tocopherol on the cell-cell interactions at the vessel wall were studied, using an in vitro model of human umbilical vein endothelial cell cultures (HUEC). Immune triggered granulocytes (PMN) will adhere to and damage HUEC and platelets enhance this PMN mediated endothelial injury. When HUEC are cultured in the presence of vitamin E, 51Cr-leakage induced by complement stimulated PMN is significantly decreased and the enhanced cytotoxicity by platelets is completely abolished (p <0.001).The inhibition of PMN induced endothelial injury is directly correlated to a diminished adherence of PMN to vitamin E- cultured HUEC (p <0.001), which may be mediated by an increase of both basal and stimulated endogenous prostacyclin (PGI2) from alfa-tocopherol-treated HUEC (p <0.025). The vitamin E-effect is abolished by incubation of HUEC with the irreversible cyclo-oxygenase inhibitor, acetylsalicylic acid, but the addition of exogenous PGI2 could not reproduce the vitamin E-mediated effects.We conclude that vitamin E exerts a protective effect on immune triggered endothelial damage, partly by increasing the endogenous anti-oxidant potential, partly by modulating intrinsic endothelial prostaglandin production. The failure to reproduce vitamin E-protection by exogenously added PGI2 may suggest additional, not yet elucidated vitamin E-effects on endothelial metabolism.

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The Identification and Significance of a Thr→Pro Polymorphism in Kringle IV Type 8 of Apolipoprotein(a)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656083 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0949-0954 ◽

Cited By ~ 8

Author(s):

J Prins ◽

F R Lues ◽

Y Y van der Hoek ◽

J J.P Kastelein ◽

B N Bouma ◽

...

Keyword(s):

Case Control Study ◽

Amino Acid Position ◽

Case Control ◽

Binding Studies ◽

Binding Characteristics ◽

Apolipoprotein A ◽

Significant Independent Risk Factor ◽

And Gender ◽

Control Study

SummaryElevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr→Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes.The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed an overall allele frequency for the Thr12-→Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro12-subtype and symptomatic atherosclerosis (p <0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr12 or Pro12 in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des- AA-fibrin (DesafibTM-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafibTM-X was observed for the Pro12-subtype Lp(a).

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Structural and Genetic Aspects of Proline-rich Proteins

Journal of Dental Research ◽

10.1177/00220345870660021201 ◽

1987 ◽

Vol 66 (2) ◽

pp. 457-461 ◽

Cited By ~ 77

Author(s):

A. Bennick

Keyword(s):

General Structure ◽

Single Gene ◽

Conclusive Evidence ◽

In Vitro Translation ◽

Binding Studies ◽

Nmr Studies ◽

Post Translational Modifications ◽

Single Precursor ◽

Made In

Considerable advances have been made in the genetics of salivary proline-rich proteins (PRP). The genes for acidic, basic, and glycosylated PRP have been cloned. They code for precursor proteins that all have an acidic N-terminal followed by proline-rich repeat sequences. Structural studies on secreted proteins have demonstrated that not only acidic but also some basic PRPs have this general structure. It is possible that mRNA for different PRP may have originated from a single gene by differential mRNA splicing, but post-translational cleavages of the primary translation product apparently also occur. In vitro translation of salivary gland mRNA results in a single precursor protein for acidic PRP. Such in vitro translated protein can be cleaved by salivary kallikrein, giving rise to two commonly secreted acidic PRPs, and kallikrein or kallikrein-like enzymes may be responsible for other post-translational cleavages of PRPs. Acidic as well as some basic PRPs are phosphorylated. A protein kinase has been demonstrated in salivary glands which phosphorylates the PRPs and other secreted salivary proteins in a cAMP and Ca2+-calmodulinindependent manner. Knowledge of the conformation of PRPs is limited. There is no conclusive evidence of polyproline-like structure in the proline-rich part of PRPs. Ca2+ binding studies on acidic PRPs indicate that there is interaction between the Ca2+ binding N-terminal end and the proline-rich C-terminal part. This interaction is relieved by modification of arginine side-chains. 1H, 32P, and 43Ca NMR studies have further elucidated the conformation of acidic PRPs in solution. Present evidence shows that salivary PRPs constitute a unique superfamily of proteins which pose a number of interesting questions concerning gene structure, pre- and post-translational modifications, and protein conformation.

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Co-factor-free aggregation of tau into seeding-competent RNA-sequestering amyloid fibrils

Nature Communications ◽

10.1038/s41467-021-24362-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Pijush Chakraborty ◽

Gwladys Rivière ◽

Shu Liu ◽

Alain Ibáñez de Opakua ◽

Rıza Dervişoğlu ◽

...

Keyword(s):

Amyloid Fibrils ◽

Corticobasal Degeneration ◽

Binding Studies ◽

Rigid Core ◽

Tau Aggregation ◽

The Brain ◽

Tau Aggregate ◽

Disease Specific ◽

Tau Fibrils

AbstractPathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.

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Vitamin E-Coated Dialyzer Inhibits Oxidative Stress

Blood Purification ◽

10.1159/000478971 ◽

2017 ◽

Vol 44 (4) ◽

pp. 288-293 ◽

Cited By ~ 4

Author(s):

Shiho Yamadera ◽

Yuya Nakamura ◽

Masahiro Inagaki ◽

Isao Ohsawa ◽

Hiromichi Gotoh ◽

...

Keyword(s):

Oxidative Stress ◽

Vitamin E ◽

Synthetic Polymer ◽

Intracellular Reactive Oxygen Species ◽

Ros Production ◽

Oxygen Species ◽

In Vitro Methods ◽

Stress Effects ◽

Intracellular Ros

Aim: To examine the effects of vitamin E-coated dialyzer on oxidative stress in vitro. Methods: A dialyzer with a synthetic polymer membrane (APS-11SA) and vitamin E-coated dialyzer (VPS-11SA) were connected to a blood tubing line, and U937 cells were circulated in the device. The circulating fluid was collected at 1, 2, 5, 10, 25, and 50 cycles, which are estimated numbers of passes through the dialyzer. Intracellular reactive oxygen species (ROS) production, malondialdehyde (MDA), and Cu/Zn-superoxide dismutase (SOD) were quantified. Results: Intracellular ROS production was increased in the first cycle by APS-11SA and was decreased throughout the experiment by VPS-11SA. Intracellular ROS production in the VPS-11SA device was lower, and MDA levels were decreased. MDA levels were lower during VPS-11SA processing than during APS-11SA processing. Cu/Zn-SOD levels remained unchanged. Conclusion: Our results highlight anti-oxidative-stress effects of a vitamin E-coated dialyzer.

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