scholarly journals The E3 Ubiquitin Ligase TEB4 Mediates Degradation of Type 2 Iodothyronine Deiodinase

2009 ◽  
Vol 29 (19) ◽  
pp. 5339-5347 ◽  
Author(s):  
Ann Marie Zavacki ◽  
Rafael Arrojo e Drigo ◽  
Beatriz C. G. Freitas ◽  
Mirra Chung ◽  
John W. Harney ◽  
...  
Keyword(s):  
Cell Types ◽  
Iodothyronine Deiodinase ◽  
Hek 293 ◽  
Protein Levels ◽  
Hematopoietic Lineage ◽  
293 Cells ◽  
Brown Adipose ◽  
Critical Instability ◽  
Proteasomal Inhibitor

ABSTRACT The endoplasmic reticulum resident thyroid hormone-activating type 2 deiodinase (D2) is inactivated by ubiquitination via the hedgehog-inducible WSB-1. Ubiquitinated D2 can then be subsequently taken up by the proteasomal system or be reactivated by USP-33/20-mediated deubiquitination. Given that heterologously expressed D2 accumulates in Saccharomyces cerevisiae lacking the E3 ligase Doa10, we tested whether the human Doa10 ortholog, TEB4, plays a role in D2 ubiquitination and degradation. In a setting of transient coexpression in HEK-293 cells, TEB4 and D2 could be coimmunoprecipitated, and additional TEB4 expression decreased D2 activity by ∼50% (P < 0.05). A highly efficient TEB4 knockdown (>90% reduction in mRNA and protein levels) decreased D2 ubiquitination and increased D2 activity and protein levels by about fourfold. The other activating deiodinase, D1, or a truncated D2 molecule (Δ18-D2) that lacks a critical instability domain was not affected by TEB4 knockdown. Furthermore, TEB4 knockdown prolonged D2 activity half-life at least fourfold, even under conditions known to promote D2 ubiquitination. Neither exposure to 1 μM of the proteasomal inhibitor MG132 for 24 h nor RNA interference WSB-1 knockdown resulted in additive effects on D2 expression when combined with TEB4 knockdown. Similar results were obtained with MSTO-211 cells, which endogenously express D2, after TEB4 knockdown using a lentivirus-based transduction strategy. While TEB4 expression predominates in the hematopoietic lineage, both WSB-1 and TEB4 are coexpressed with D2 in a number of tissues and cell types, except the thyroid and brown adipose tissue, where TEB4 expression is minimal. We conclude that TEB4 interacts with and mediates loss of D2 activity, indicating that D2 ubiquitination and degradation can be tissue specific, depending on WSB-1 and TEB4 expression levels.

scholarly journals Statins and Downstream Inhibitors of the Isoprenylation Pathway Increase Type 2 Iodothyronine Deiodinase Activity

Endocrinology ◽  
2012 ◽  
Vol 153 (8) ◽  
pp. 4039-4048 ◽  
Author(s):  
B. T. Miller ◽  
C. B. Ueta ◽  
V. Lau ◽  
K. G. Jacomino ◽  
L. M. Wasserman ◽  
...  
Keyword(s):  
Active Form ◽  
Critical Determinant ◽  
Iodothyronine Deiodinase ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Reductase Inhibitors ◽  
293 Cells ◽  
Brown Adipose ◽  
Prenyl Transferase

The type 2 iodothyronine selenodeiodinase (D2) is a critical determinant of local thyroid signaling, converting T4 to the active form T3 at the cytoplasmic face of the endoplasmic reticulum, thus supplying the nucleus with T3 without immediately affecting circulating thyroid hormone levels. Although inhibitors of the cholesterol synthesis/isoprenylation pathway, such as hydroxy-methyl-glutaryl-coenzyme A reductase inhibitors (statins) have been to shown to down-regulate selenoproteins via interruption of normal selenocysteine incorporation, little is known about the effect of statins on D2. Here, we report that statins and prenyl transferase inhibitors actually increase D2 activity in cells with endogenous D2 expression. Although we confirmed that lovastatin (LVS) decreases the activity of transiently expressed D2 in HEK-293 cells, the prenyl transferase inhibitors increase activity in this system as well. LVS treatment increases endogenous Dio2 mRNA in MSTO-211H cells but does not alter transiently expressed Dio2 mRNA in HEK-293 cells. The prenyl transferase inhibitors do not increase Dio2 mRNA in either system, indicating that a posttranscriptional mechanism must exist. Cotreatment with LVS or the prenyl transferase inhibitors with the proteasome inhibitor MG-132 did not lead to additive increases in D2 activity, indirectly implicating the ubiquitin-proteasomal system in the mechanism. Finally, C57BL/6J mice treated with LVS or farnesyl transferase inhibitor-277 for 24 h exhibited increased D2 activity in their brown adipose tissue. These data indicate that statins and downstream inhibitors of the isoprenylation pathway may increase thyroid signaling via stimulation of D2 activity.

Interleukin 9 induces expression of three cytokine signal inhibitors: cytokine-inducible SH2-containing protein, suppressor of cytokine signalling (SOCS)-2 and SOCS-3, but only SOCS-3 overexpression suppresses interleukin 9 signalling

2000 ◽  
Vol 353 (1) ◽  
pp. 109-116 ◽  
Author(s):  
Diane LEJEUNE ◽  
Jean-Baptiste DEMOULIN ◽  
Jean-Christophe RENAULD
Keyword(s):  
Signal Transduction ◽  
Cell Types ◽  
Janus Kinase ◽  
Signal Attenuation ◽  
Hek 293 ◽  
293 Cells ◽  
Apoptotic Activity ◽  
Induced Signal ◽  
Interleukin 9

Interleukin 9 (IL-9) is a cytokine preferentially produced by T helper type 2 lymphocytes and active on various cell types such as T- and B-lymphocytes, mast cells and haemopoietic progenitors. The IL-9 receptor (IL-9R) belongs to the haemopoietic receptor superfamily and its signal transduction involves mainly the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Here we studied the implication of a novel family of suppressors of cytokine signalling (called CIS, for cytokine-inducible SH2-containing protein, and SOCS, for suppressor of cytokine signalling) in IL-9 signal attenuation. In BW5147 T-cell lymphoma, IL-9 induced the rapid expression of CIS, SOCS-2 and SOCS-3 with a peak after 2h of stimulation. Using IL-9R mutants, we showed that STAT activation is required for CIS/SOCS induction: CIS and SOCS-2 expression was induced either via STAT1 and/or STAT3 or via STAT5 but only STAT1 and/or STAT3 were involved in SOCS-3 expression. The effect of these three proteins on IL-9 signal transduction was assessed by transient transfection in HEK-293 cells expressing the components of the IL-9 signalling pathway and a STAT-responsive reporter construct. These experiments showed that only SOCS-3 is able to inhibit IL-9-induced signal transduction; neither CIS nor SOCS-2 exerted any effect. Stable transfection of CIS and SOCS-3 in BW5147 lymphoma cells showed that only overexpression of SOCS-3 had an inhibitory activity on STAT activation, gene induction and the anti-apoptotic activity of IL-9. By contrast, CIS failed to affect the IL-9 response.

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.

Extracellular ATP dissociates nonmuscle myosin from P2X7complex: this dissociation regulates P2X7pore formation

2009 ◽  
Vol 297 (2) ◽  
pp. C430-C439 ◽  
Author(s):  
Ben J. Gu ◽  
Catherine Rathsam ◽  
Leanne Stokes ◽  
Andrew B. McGeachie ◽  
James S. Wiley
Keyword(s):  
Specific Inhibitor ◽  
Life Time ◽  
Cell Types ◽  
Interferon Γ ◽  
Extracellular Atp ◽  
Nonmuscle Myosin ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Myosin Va

The P2X7receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X7channel and massive K+efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X7were isolated by using anti-P2X7monoclonal antibody-coated Dynabeads from both interferon-γ plus LPS-stimulated monocytic THP-1 cells and P2X7-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X7in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X7receptor by ATP caused dissociation of P2X7from nonmuscle myosin in both cell types. The interaction of P2X7and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X7pore function without any increase in surface expression or ion channel function of P2X7receptors. S- l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X7-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X7and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X7channel to a pore.

scholarly journals Inhibition of the Type 2 Iodothyronine Deiodinase Underlies the Elevated Plasma TSH Associated with Amiodarone Treatment

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5961-5970 ◽  
Author(s):  
Matthew L. Rosene ◽  
Gábor Wittmann ◽  
Rafael Arrojo e Drigo ◽  
Praful S. Singru ◽  
Ronald M. Lechan ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Critical Role ◽  
Feedback Mechanism ◽  
Mrna Levels ◽  
Iodothyronine Deiodinase ◽  
Hek 293 ◽  
Tsh Secretion ◽  
Dependent Inhibition ◽  
Noncompetitive Inhibitors

The widely prescribed cardiac antiarrhythmic drug amiodarone (AMIO) and its main metabolite, desethylamiodarone (DEA), have multiple side effects on thyroid economy, including an elevation in serum TSH levels. To study the AMIO effect on TSH, mice with targeted disruption of the type 2 deiodinase gene (D2KO) were treated with 80 mg/kg AMIO for 4 wk. Only wild-type (WT) mice controls developed the expected approximate twofold rise in plasma TSH, illustrating a critical role for D2 in this mechanism. A disruption in the D2 pathway caused by AMIO could interfere with the transduction of the T4 signal, generating less T3 and softening the TSH feedback mechanism. When added directly to sonicates of HEK-293 cells transiently expressing D2, both AMIO and DEA behaved as noncompetitive inhibitors of D2 [IC(50) of &gt;100 μm and ∼5 μm, respectively]. Accordingly, D2 activity was significantly decreased in the median eminence and anterior pituitary sonicates of AMIO-treated mice. However, the underlying effect on TSH is likely to be at the pituitary gland given that in AMIO-treated mice the paraventricular TRH mRNA levels (which are negatively regulated by D2-generated T3) were decreased. In contrast, AMIO and DEA both exhibited dose-dependent inhibition of D2 activity and elevation of TSH secretion in intact TαT1 cells, a pituitary thyrotroph cell line used to model the TSH feedback mechanism. In conclusion, AMIO and DEA are noncompetitive inhibitors of D2, with DEA being much more potent, and this inhibition at the level of the pituitary gland contributes to the rise in TSH seen in patients taking AMIO.

Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase

2013 ◽  
Vol 394 (5) ◽  
pp. 685-693 ◽  
Author(s):  
Petra Cordes ◽  
Wei Sun ◽  
Rainer Wolber ◽  
Ludger Kolbe ◽  
Gerhard Klebe ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Heterologous Expression ◽  
Western Blotting ◽  
Kluyveromyces Lactis ◽  
Cell Types ◽  
Hek 293 ◽  
Activity Staining ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Human Tyrosinase

Abstract Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.

scholarly journals PAPOLA contributes to Cyclin D1 mRNA alternative polyadenylation and promotes breast cancer cells proliferation

2021 ◽  
pp. jcs.252304
Author(s):  
Chrysoula Komini ◽  
Irini Theohari ◽  
Andromachi Lambrianidou ◽  
Lydia Nakopoulou ◽  
Theoni Trangas
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Alternative Polyadenylation ◽  
Hek 293 ◽  
Protein Levels ◽  
293 Cells ◽  
Candidate Target ◽  
Mcf 7

Poly(A) polymerases add the poly(A) tail at the 3’ end of nearly all eukaryotic mRNA, are associated with proliferation and cancer. To elucidate the role of the most studied mammalian poly(A) polymerase α (PAPOLA) in cancer, we assessed its expression in 221 breast cancer samples and found it to correlate strongly with the aggressive triple-negative subtype. Silencing PAPOLA in MCF-7 and MDA-MB-231 breast cancer cells reduced proliferation and anchorage-independent growth by decreasing steady-state CCND1 mRNA and protein levels. Whereas the length of the CCND1 mRNA poly(A) tail was not affected, its 3' untranslated region (3'UTR) lengthened. Overexpressing PAPOLA caused CCND1 mRNA 3'UTR shortening with a concomitant increase in the corresponding transcript and protein, resulting in growth arrest in MCF-7 cells and DNA damage in HEK-293 cells, whereas in the P53 mutant MDA-MB-231 promoted proliferation.Our data suggest PAPOLA as a possible candidate target for the control of tumor growth, mostly relevant to triple-negative tumors, a group characterized by its overexpression and lacking alternative targeted therapies.

scholarly journals Bupivacaine inhibits a small-conductance calcium-activated potassium type 2 channel (SK2) in HEK293 cells

2020 ◽  
Author(s):  
Hongfei Chen ◽  
Fangfang Xia ◽  
Zhousheng Jin ◽  
Zhijian Fu
Keyword(s):  
Hek293 Cells ◽  
Free Calcium ◽  
Heart Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause 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 unknown. 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 study the effect of bupivacaine on SK2 channels. Concentration-response relationship of bupivacaine for inhibiting SK2 current (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 the SK2 current was 16.5, 46.5, and 77.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium.Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity.

scholarly journals The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue

2001 ◽  
Vol 108 (9) ◽  
pp. 1379-1385 ◽  
Author(s):  
Lucia A. de Jesus ◽  
Suzy D. Carvalho ◽  
Mirian O. Ribeiro ◽  
Mark Schneider ◽  
Sung-Woo Kim ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Iodothyronine Deiodinase ◽  
Adaptive Thermogenesis ◽  
Brown Adipose

scholarly journals The Toxic Effects of Aflatoxin B1 and Aflatoxin M1 on Kidney through Regulating L-Proline and Downstream Apoptosis

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Huiying Li ◽  
Lei Xing ◽  
Muchen Zhang ◽  
Jiaqi Wang ◽  
Nan Zheng
Keyword(s):  
Oxidative Stress ◽  
Aflatoxin B1 ◽  
Relative Concentration ◽  
Toxic Effects ◽  
Aflatoxin M1 ◽  
Mice Model ◽  
Hek 293 ◽  
Treatment Groups ◽  
Protein Levels ◽  
293 Cells

The toxic effects and potential mechanisms of aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), and AFB1+AFM1 in the kidney were studied and compared in HEK 293 cells model and CD-1 mice model. The 35-day subacute toxicity mice model was constructed, biochemical indicators and kidney pathological staining were detected, kidney metabonomics detection was performed, and the metabolites were analyzed, and then the related toxicity mechanism was validated. Results showed that AFB1 (0.5 mg/kg), AFM1 (3.5 mg/kg), and AFB1 (0.5 mg/kg)+AFM1 (3.5 mg/kg) activated oxidative stress and caused renal damage. The relative concentration of the metabolite L-proline was found to be lower in aflatoxins treatment groups when compared with the control (P<0.05). Moreover, with the treatment of aflatoxins, proline dehydrogenase (PRODH) and proapoptotic factors (Bax, Caspase-3) were upregulated, while the inhibitor of apoptosis Bcl-2 was downregulated, at both the mRNA and the protein levels, comparing with the control (P<0.05). In addition, the combined effect of AFB1 and AFM1 was validated, for the toxicity of the combination was stronger than the other two groups. In conclusion, AFB1 and AFM1 caused kidney toxicity by activating oxidative stress through altering expression of PRODH and L-proline levels, which then induced downstream apoptosis.

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