scholarly journals Heat-shock proteins attenuate SERCA inactivation by the anti-apoptotic protein Bcl-2: possible implications for the ER Ca2+-mediated apoptosis

2012 ◽  
Vol 444 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Elena S. Dremina ◽  
Victor S. Sharov ◽  
Christian Schöneich
Keyword(s):  
Heat Shock ◽  
Mechanistic Study ◽  
Human Embryonic Kidney ◽  
C2c12 Myoblasts ◽  
Hek 293 ◽  
Functional Interactions ◽  
Apoptotic Protein ◽  
293 Cells ◽  
Shock Proteins

We have demonstrated previously that Bcl-2 and Bcl-2Δ21, a C-terminally truncated Bcl-2 sequence, inactivate SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 1 in isolated SR (sarcoplasmic reticulum), accompanied by a translocation from CRDs (caveolae-related domains) of the SR. In the present study, we obtained evidence for the interaction of Bcl-2 with SERCA2b in C2C12 myoblasts and HEK (human embryonic kidney)-293 cells. Bcl-2 and SERCA2b co-immunoprecipitated from lysate and microsomal fractions of Bcl-2-overexpressing cells. However, Bcl-2 overexpression resulted only in a slight translocation from the CRDs and no significant SERCA inactivation. In isolated HEK-293 cell microsomes, incubation with Bcl-2Δ21 afforded SERCA2b inactivation and some translocation. HSP (heat-shock protein) 70, HSP90, HSP27 and α-crystallin attenuated Bcl-2Δ21-dependent SERCA2b inactivation. An in vitro mechanistic study with the SERCA1 isoform shows that HSP70 (i) protects SERCA1 from the inactivation by Bcl-2Δ21, (ii) inhibits SERCA1 translocation from CRD fractions, and (iii) prevents the Bcl-2Δ21-dependent loss of FITC labelling. Our data demonstrate that the mechanism of SERCA inactivation by Bcl-2 established in vitro for the SERCA1 isoform can be extended to the main housekeeping SERCA2b isoform, and that functional interactions of SERCA2b and Bcl-2 in the cell may be modulated by HSP70 and other chaperones and stress-regulated proteins.

In vitro evaluation of copper oxide nanoparticle-induced cytotoxicity and oxidative stress using human embryonic kidney cells

2019 ◽  
Vol 35 (2) ◽  
pp. 159-164 ◽  
Author(s):  
Anreddy Rama Narsimha Reddy ◽  
Srividya Lonkala
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Membrane Damage ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Cell Membrane Damage ◽  
Cuo Nps ◽  
293 Cells ◽  
And Oxidative Stress

This study aimed to evaluate the in vitro cytotoxicity and oxidative stress induced by the copper oxide nanoparticles (CuO NPs) in human embryonic kidney cell line (HEK-293) cells following exposure. CuO NPs size <50 nm were used in this study. HEK-293 cell cultures were exposed to different concentrations of CuO NPs between 3 µg/ml and 300 µg/ml and quartz (known as cytotoxic agent) and assessed for cell viability-mitochondrial function (MTT assay), cell membrane damage (lactate dehydrogenase (LDH) assay), reduced glutathione (GSH), interleukin-8 (IL-8), and lipid peroxidation levels. The IC50 value of NPs was found to be 65.5 µg/ml. Exposure of HEK cells to CuO NPs (10–300 µg/ml) resulted in concentration-dependent cell membrane damage, increased production of IL-8, increased thiobarbituric acid reactive substance (TBARS), and decreased intracellular GSH levels. The significant increases in IL-8, TBARS, and LDH levels along with decreased GSH levels indicated induction of oxidative stress in cells. Our preliminary data suggest that oxidative stress might contribute to CuO NPs-induced cytotoxicity in HEK-293 cells.

scholarly journals Insulin-induced phospholipase D1 and phospholipase D2 activity in human embryonic kidney-293 cells mediated by the phospholipase Cγ and protein kinase Cα signalling cascade

2000 ◽  
Vol 351 (3) ◽  
pp. 613-619 ◽  
Author(s):  
Rita SLAABY ◽  
Guangwei DU ◽  
Yelena M. ALTSHULLER ◽  
Michael A. FROHMAN ◽  
Klaus SEEDORF
Keyword(s):  
Protein Kinase ◽  
Cell Types ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Phospholipase D1

Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKCα results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)γ has the same effect, while co-expression of PLD2 with PLCγ allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLCγ and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKCα is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKCα and PLCγ, are expressed at sufficient levels.

In Vitro Holdase Activity of E. coli Small Heat-Shock Proteins IbpA, IbpB and IbpAB: A Biophysical Study with Some Unconventional Techniques

2014 ◽  
Vol 21 (6) ◽  
pp. 564-571 ◽  
Author(s):  
Sourav Roy ◽  
Monobesh Patra ◽  
Suman Nandy ◽  
Milon Banik ◽  
Rakhi Dasgupta ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Small Heat Shock Proteins ◽  
E Coli ◽  
Biophysical Study ◽  
Shock Proteins

Translation of Some Maize Small Heat Shock Proteins Is Initiated From Internal In-Frame AUGs

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 471-477
Author(s):  
J Roger H Frappier ◽  
David B Walden ◽  
Burr G Atkinson
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Single Gene ◽  
Small Heat Shock Proteins ◽  
Shock Proteins

Abstract Etiolated maize radicles (inbred Oh43) subjected to a brief heat shock synthesize a family of small heat shock proteins (≃18 kD) that is composed of at least 12 members. We previously described the cDNA-derived sequence of three maize shsp mRNAs (cMHSP18-1, cMHSP18-3, and cMHSP18-9). In this report, we demonstrate that the mRNA transcribed in vitro from one of these cDNAs (cMHSP 18-9) is responsible for the synthesis of three members of the shsp family, and we suggest that cMHSP18-3 may be responsible for the synthesis of three additional members and cMHSP18-1 for the synthesis of two other members of this family. The fact that these genes do not contain introns, coupled with the observations reported herein, suggest that maize may have established another method of using a single gene to produce a number of different proteins.

scholarly journals Preliminary Study on Pasta Samples Characterized in Antioxidant Compounds and Their Biological Activity on Kidney Cells

Nutrients ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 1131 ◽  
Author(s):  
Federico Di Marco ◽  
Francesco Trevisani ◽  
Pamela Vignolini ◽  
Silvia Urciuoli ◽  
Andrea Salonia ◽  
...  
Keyword(s):  
Indoleacetic Acid ◽  
Antioxidant Properties ◽  
Antioxidant Compounds ◽  
Kidney Cells ◽  
Hek 293 ◽  
293 Cells ◽  
High Concentrations ◽  
Β Carotene ◽  
Egg Pasta

Pasta is one of the basic foods of the Mediterranean diet and for this reason it was chosen for this study to evaluate its antioxidant properties. Three types of pasta were selected: buckwheat, rye and egg pasta. Qualitative–quantitative characterization analyses were carried out by HPLC-DAD to identify antioxidant compounds. The data showed the presence of carotenoids such as lutein and polyphenols such as indoleacetic acid, (carotenoids from 0.08 to 0.16 mg/100 g, polyphenols from 3.7 to 7.4 mg/100 g). To assess the effect of the detected metabolites, in vitro experimentation was carried out on kidney cells models: HEK-293 and MDCK. Standards of β-carotene, indoleacetic acid and caffeic acid, hydroalcoholic and carotenoid-enriched extracts from samples of pasta were tested in presence of antioxidant agent to determine viability variations. β-carotene and indoleacetic acid standards exerted a protective effect on HEK-293 cells while no effect was detected on MDCK. The concentrations tested are likely in the range of those reached in body after the consumption of a standard pasta meal. Carotenoid-enriched extracts and hydroalcoholic extracts showed different effects, observing rescues for rye pasta hydroalcoholic extract and buckwheat pasta carotenoid-enriched extract, while egg pasta showed milder dose depending effects assuming pro-oxidant behavior at high concentrations. The preliminary results suggest behaviors to be traced back to the whole phytocomplexes respect to single molecules and need further investigations.

scholarly journals The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

2021 ◽  
Vol 22 (7) ◽  
pp. 3700
Author(s):  
Junna Hayashi ◽  
Jennifer Ton ◽  
Sparsh Negi ◽  
Daniel E. K. M. Stephens ◽  
Dean L. Pountney ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein Aggregation ◽  
Molecular Chaperone ◽  
Cross Linking ◽  
Αb Crystallin ◽  
The Cross ◽  
Shock Proteins ◽  
And Function

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

Serine Mutations in Hsp27 abrogate its ability to inhibit p53 dependent apoptosis and Doxorubicin-induced Heart Failure in mice

2021 ◽  
Author(s):  
Ragu Kanagasabai ◽  
Krishnamurthy Karthikeyan ◽  
Jay L. Zweier ◽  
Govindasamy Ilangovan
Keyword(s):  
Heart Failure ◽  
Heat Shock ◽  
Left Ventricular ◽  
Serine Phosphorylation ◽  
Apoptotic Pathway ◽  
Mtor Phosphorylation ◽  
Inner Diameter ◽  
Shock Proteins ◽  
Parp 1

Small heat shock proteins (sHsps) protect the heart from chemotherapeutics-induced heart failure, by inhibiting p53-dependant apoptosis. However, mechanism of such protection has not been elucidated yet. Here we test a hypothesis that serine phosphorylation of sHsps is essential to inhibit the Doxorubicin-induced p53-dependent apoptotic pathway. Three transgenic mice (TG) lines with cardiomyocyte specific overexpression of human heat shock protein 27 (hHsp27), namely, wild type (MHC-hHsp27), S82A single mutant (MHC-mut-hHsp27(S82A) and tri-mutant (MHC-mut-hHsp27(S15A/S78A/S82A)) were generated. TG mice were treated with Dox (6mg/kg body weight; once in a week; 4 weeks) along with age-matched non-transgenic (Non-TG) controls. The Dox-treated MHC-hHsp27 mice showed improved survival and cardiac function (both MRI and echocardiography), in terms of contractility (%EF) and left ventricular inner diameter (LVID), compared to the Dox-treated Non-TG mice. However, both MHC-mut-hHsp27(S82A) and MHC-mut-hHsp27(S82A/S15A/S76A) mutants overexpressing TG mice did not show such a cardioprotection. Furthermore, transactivation of p53 was found to be attenuated only in Dox-treated MHC-hHsp27 mice-derived cardiomyocytes in vitro, as low p53 was detected in the nuclei, not in mutant hHsp27 overexpressing cardiomyocytes. Similarly, only in MHC-hHsp27 overexpressing cardiomyocytes, low Bax, higher mTOR phosphorylation and low apoptotic PARP-1 cleavage (89kDa fragment) were detected. Pharmacological inhibition of p53 was more effective in mutant-TG mice, compared to MHC-hHsp27 mice. We conclude that phosphorylation of overexpressed Hsp27 at S82 and its association with p53 is essential for the overall cardioprotective effect of Hsp27 against Dox-induced dilated cardiomyopathy. Only phosphorylated Hsp27 protect the heart by inhibiting p53 transactivation.

scholarly journals Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60

2005 ◽  
Vol 391 (2) ◽  
pp. 185-190 ◽  
Author(s):  
Renu Wadhwa ◽  
Syuichi Takano ◽  
Kamaljit Kaur ◽  
Satoshi Aida ◽  
Tomoko Yaguchi ◽  
...  
Keyword(s):  
Heat Shock ◽  
Molecular Interactions ◽  
Heat Shock Protein 60 ◽  
Hsp60 Expression ◽  
Mitochondrial Hsp70 ◽  
Shock Proteins ◽  
First Time

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

scholarly journals Bupivacaine inhibits a small conductance calcium‐activated potassium type 2 channel in human embryonic kidney 293 cells

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Hongfei Chen ◽  
Zhousheng Jin ◽  
Fangfang Xia ◽  
Zhijian Fu
Keyword(s):  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background Bupivacaine blocks many ion channels in the heart muscle, causing severe cardiotoxicity. Small-conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unclear. This study investigated the effect of bupivacaine on SK2 channels. Methods The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch-clamp technique was used to examine the effect of bupivacaine on SK2 channels. The concentration–response relationship of bupivacaine for inhibiting SK2 currents (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined. Results Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine, and lidocaine on SK2 currents was 16.5, 46.5, and 77.8µM, respectively. The degree of SK2 current inhibition by bupivacaine depended on the intracellular concentration of free calcium. Conclusions The results of this study suggested the inhibitory effect of bupivacaine on SK2 channels. Future studies should explore the effects of SK2 on bupivacaine cardiotoxicity.

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