Evaluation of the impact of the proteasome inhibitor on calcium channel expression in cardiomyocytes treated with doxorubicin

AbstractOne of the less known mechanisms of doxorubicin action is the effect on the functioning of the ubiquitin-proteasome degradation system (UPS). So far, the role of impaired proteasome activity in the development of anthracycline cardiomyopathy has not been clarified. It has been shown, however, that doxorubicin decreases the expression of proteins, including the expression of the calcium channel. However, it has not been established whether the observed disturbances are due to the activation of the UPS system by doxorubicin, or due to inhibition of translation or transcription. Therefore, the aim of the study was to evaluate the mRNA and protein expression of plasmalemmal (NaCaX, L-type) and sarcoplasmic reticulum (SERCA2, RyR2) channels in rat embryonic cardiomyocytes treated with doxorubicin and the proteasome inhibitor – bortezomib. The study was conducted utilizing the rat cardiomyocyte H9C2 line that was treated with doxorubicin and bortezomib in different concentrations. After 24 hours incubation, mRNA and protein expression analysis followed. The study did not show any universal mechanism of doxorubicin influence on calcium channel expression. With regard to the Na/Ca exchanger, we saw that DOX decreased the protein level in a proteasome activitydependent manner. Moreover, we noted that the SERCA2 protein expression level was regulated by degradation intensity, however at the same time, no significant effect of doxorubicin on the level of this protein was demonstrated.

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The Ubiquitin-Proteasome System Plays an Important Role during Various Stages of the Coronavirus Infection Cycle

Journal of Virology ◽

10.1128/jvi.00485-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7869-7879 ◽

Cited By ~ 55

Author(s):

Matthijs Raaben ◽

Clara C. Posthuma ◽

Monique H. Verheije ◽

Eddie G. te Lintelo ◽

Marjolein Kikkert ◽

...

Keyword(s):

Protein Expression ◽

Ubiquitin Proteasome System ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Infection Cycle ◽

Protein Ubiquitination ◽

Ubiquitin Proteasome ◽

The Impact ◽

In Cells

ABSTRACT The ubiquitin-proteasome system (UPS) is a key player in regulating the intracellular sorting and degradation of proteins. In this study we investigated the role of the UPS in different steps of the coronavirus (CoV) infection cycle. Inhibition of the proteasome by different chemical compounds (i.e., MG132, epoxomicin, and Velcade) appeared to not only impair entry but also RNA synthesis and subsequent protein expression of different CoVs (i.e., mouse hepatitis virus [MHV], feline infectious peritonitis virus, and severe acute respiratory syndrome CoV). MHV assembly and release were, however, not appreciably affected by these compounds. The inhibitory effect on CoV protein expression did not appear to result from a general inhibition of translation due to induction of a cellular stress response by the inhibitors. Stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) generally results in impaired initiation of protein synthesis, but the sensitivity of MHV infection to proteasome inhibitors was unchanged in cells lacking a phosphorylatable eIF2α. MHV infection was affected not only by inhibition of the proteasome but also by interfering with protein ubiquitination. Viral protein expression was reduced in cells expressing a temperature-sensitive ubiquitin-activating enzyme E1 at the restrictive temperature, as well as in cells in which ubiquitin was depleted by using small interfering RNAs. Under these conditions, the susceptibility of the cells to virus infection was, however, not affected, excluding an important role of ubiquitination in virus entry. Our observations reveal an important role of the UPS in multiple steps of the CoV infection cycle and identify the UPS as a potential drug target to modulate the impact of CoV infection.

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Ageratina adenophora Inhibits Spleen Immune Function in Rats via the Loss of the FRC Network and Th1–Th2 Cell Ratio Elevation

Toxins ◽

10.3390/toxins13050309 ◽

2021 ◽

Vol 13 (5) ◽

pp. 309

Author(s):

Zhihua Ren ◽

Pei Gao ◽

Samuel Kumi Okyere ◽

Yujing Cui ◽

Juan Wen ◽

...

Keyword(s):

Protein Expression ◽

Immune Function ◽

Tissue Sample ◽

Th2 Cells ◽

Sample Collection ◽

Functional Protein ◽

Th2 Cell ◽

Ageratina Adenophora ◽

Mrna And Protein Expression ◽

The Impact

The objective of this study was to determine the impact of Ageratina adenophora (A. adenophora) on splenic immune function in a rat model. Rats were fed with 10 g/100 g normal feed and an experimental feed, which was composed of 3:7 A. adenophora powder and normal feed for 60 days. On days 14, 28, and 60, subsets of rats (n = 8 rats/group/time point) were selected for blood and spleen tissue sample collection. The results showed that the proportion of CD3+ T cells in the spleen was decreased at day 60 (vs. control). Also, mRNA and protein expression of chemokines CCL21 and CCL19 and functional protein gp38 in spleen decreased significantly versus the control at day 60. In addition, ER-TR7 antigen protein expression was also decreased at day 60. Levels of T-helper (Th)1 cells significantly increased, whereas those of Th2 cells decreased significantly versus the control at day 60 in spleen. The finding revealed that A. adenophora could affect splenic immune function in rats by altering the fibroblast reticulocyte (FRC) network, as well as by causing an imbalance in Th1/Th2 cell ratios. This research provides new insights into potential mechanisms of spleen immunotoxicity due to exposures to A. Adenophora.

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GCM2 Silencing in Parathyroid Adenoma is associated with Promoter Hypermethylation and Gain of Methylation on Histone 3

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgab374 ◽

2021 ◽

Author(s):

Priyanka Singh ◽

Sanjay Kumar Bhadada ◽

Divya Dahiya ◽

Uma Nahar Saikia ◽

Ashutosh Kumar Arya ◽

...

Keyword(s):

Protein Expression ◽

Parathyroid Adenoma ◽

Dna Methyltransferase ◽

Promoter Hypermethylation ◽

Epigenetic Alterations ◽

Protein Levels ◽

Histone 3 ◽

Parathyroid Adenomas ◽

Mrna And Protein Expression

Abstract Purpose Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of parathyroid glands. We sought to identify if the epigenetic alterations in the GCM2 transcription are involved in the pathogenesis of sporadic parathyroid adenoma. In addition, we examined the association between promoter methylation and histone modifications with disease indices. Experimental design mRNA and protein expression of GCM2 were analyzed by RT-qPCR and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of GCM2 promoter were measured by bisulfite sequencing and ChIP-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in PTH-C1 cells by treating with 5-aza 2’deoxycytidine (DAC) and BRD4770 and assessed for GCM2 mRNA and DNMT1 protein levels. Results mRNA and protein expression of GCM2 were lower in sporadic adenomatous than in control parathyroid tissues. This reduction correlated with hypermethylation (P<0.001) and higher H3K9me3 levels in GCM2 promoter (P<0.04) in adenomas. In PTH-C1 cells, DAC treatment resulted in increased GCM2 transcription and decreased DNMT1 protein expression, while cells treated with the BRD4770 showed reduced H3K9me3 levels but a non-significant change in GCM2 transcription. Conclusion These findings suggest the concurrent association of promoter hypermethylation and higher H3K9me3 with the repression of GCM2 expression in parathyroid adenomas. Treatment with DAC restored GCM2 expression in PTH-C1 cells. Our results showed a possible epigenetic landscape in the tumorigenesis of parathyroid adenoma and also that DAC may be promising avenues of research for parathyroid adenoma therapeutics.

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Parathyroid hormone stimulates endothelial expression of atherosclerotic parameters through protein kinase pathways

AJP Renal Physiology ◽

10.1152/ajprenal.00406.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. F1215-F1218 ◽

Cited By ~ 77

Author(s):

Gloria Rashid ◽

Jacques Bernheim ◽

Janice Green ◽

Sydney Benchetrit

Keyword(s):

Parathyroid Hormone ◽

Protein Kinase ◽

Protein Expression ◽

Mrna Expression ◽

Umbilical Vein ◽

Protein Levels ◽

Glycation End Products ◽

Mrna And Protein Expression ◽

Vascular Structures ◽

The Impact

Parathyroid hormone (PTH), the major systemic calcium-regulating hormone, has been linked to uremic vascular changes. Considering the possible deleterious action of PTH on vascular structures, it seemed logical to evaluate the impact of PTH on the receptor of advanced glycation end products (RAGE) and interleukin 6 (IL-6) mRNA and protein expression, taking into account that such parameters might be involved in the pathogenesis of vascular calcification, atherosclerosis, and/or arteriolosclerosis. Human umbilical vein cord endothelial cells (HUVEC) were stimulated for 24 h with 10−12–10−10 mol/l PTH. The mRNA expression of RAGE and IL-6 was established by reverse transcriptase/PCR techniques. RAGE protein levels were determined by Western blot and IL-6 secretion was measured by ELISA. The pathways by which PTH may have an effect on HUVEC functions were evaluated. PTH (10−11–10−10mol/l) significantly increased RAGE mRNA and protein expression. PTH also significantly increased IL-6 mRNA expression without changes at protein levels. The addition of protein kinase (PKC or PKA) inhibitors or nitric oxide (NO) synthase inhibitors significantly reduced the RAGE and IL-6 mRNA expression and the RAGE protein expression. PTH stimulates the mRNA expressions of RAGE and IL-6 and the protein expression of RAGE. These stimulatory effects are probably through PKC and PKA pathways and are also NO dependent. Such data may explain the possible impact of PTH on the atherosclerotic and arteriosclerotic progression.

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Increased expression of the lipocalin 24p3 as an apoptotic mechanism for MK886

Biochemical Journal ◽

10.1042/bj20021696 ◽

2003 ◽

Vol 372 (1) ◽

pp. 203-210 ◽

Cited By ~ 33

Author(s):

Zhimin TONG ◽

Xuli WU ◽

James P. KEHRER

Keyword(s):

Protein Expression ◽

Protein S ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Mrna And Protein Expression ◽

Induced Apoptosis ◽

Inhibitor Of Protein Synthesis ◽

New Protein ◽

Apoptotic Mechanism

MK886, a strong proapoptotic agent, is an inhibitor of 5-lipoxygenase (LOX) through binding to the 5-LOX-activating protein (FLAP). Although MK886-induced apoptosis is through a FLAP-independent pathway, the precise mechanisms are not understood. In the present study, a possible role of 24p3, a lipocalin, in MK886-induced apoptosis was investigated. Exposure of murine prolymphoid progenitor cells (FL5.12) to 20 μM MK886 for 16 h dramatically increased 24p3 mRNA and protein expression. Induction could also be achieved with another FLAP inhibitor, MK591. The induction of 24p3 by MK886 was dose- and time-dependent. The up-regulated 24p3 mRNA expression by MK886 was enhanced a further 3.1-fold by WY14643, an activator of peroxisome-proliferator-activated receptor α, whereas ciglitazone, an activator of peroxisome-proliferator-activated receptor γ attenuated the MK886-induced 24p3 expression by more than 50%. Neither WY14643 nor ciglitazone alone had any effect on the expression of 24p3. The induction of 24p3 by MK886 was dependent on the synthesis of new protein(s), since cycloheximide, an inhibitor of protein synthesis, prevented this effect. In all cases, including the inhibition of MK886-induced 24p3 protein expression by stable transfection with antisense cDNA of 24p3, the extent of apoptosis closely paralleled 24p3 levels. Apoptosis induced by MK886, or enhanced by WY14643, was accompanied by the cleavage and activation of caspase-3. The overexpression of bcl-2 or bcl-xL in FL5.12 cells inhibited apoptosis induced by MK886 as well as the enhancement of apoptosis by WY14643. Thus 24p3 is an MK886-inducible gene and may play an important role in MK886-induced apoptosis.

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Stretch reduces nephrin expression via an angiotensin II-AT1-dependent mechanism in human podocytes: effect of rosiglitazone

AJP Renal Physiology ◽

10.1152/ajprenal.90423.2008 ◽

2010 ◽

Vol 298 (2) ◽

pp. F381-F390 ◽

Cited By ~ 40

Author(s):

Ilaria Miceli ◽

Davina Burt ◽

Elena Tarabra ◽

Giovanni Camussi ◽

Paolo Cavallo Perin ◽

...

Keyword(s):

Angiotensin Ii ◽

Protein Expression ◽

Slit Diaphragm ◽

Peroxisome Proliferator ◽

Glomerular Permeability ◽

Receptor System ◽

Peroxisome Proliferator Activated Receptor ◽

Ppar Γ ◽

Mrna And Protein Expression

Increased glomerular permeability to proteins is a characteristic feature of diabetic nephropathy (DN). The slit diaphragm is the major restriction site to protein filtration, and the loss of nephrin, a key component of the slit diaphragm, has been demonstrated in both human and experimental DN. Both systemic and glomerular hypertension are believed to be important in the pathogenesis of DN. Human immortalized podocytes were subjected to repeated stretch-relaxation cycles by mechanical deformation with the use of a stress unit (10% elongation, 60 cycles/min) in the presence or absence of candesartan (1 μM), PD-123319 (1 μM), and rosiglitazone (0.1 μM). Nephrin mRNA and protein expression were assessed using quantitative real-time PCR, immunoblotting, and immunofluorescence, and the protein expression of AT1 receptor and angiotensin II secretion were evaluated. Exposure to stretch induced a significant ∼50% decrease in both nephrin mRNA and protein expression. This effect was mediated by an angiotensin II-AT1 mechanism. Indeed, podocyte stretching induced both angiotensin II secretion and AT1 receptor overexpression, podocyte exposure to angiotensin II reduced nephrin protein expression, and both the AT-1 receptor antagonist candesartan and a specific anti-angiotensin II antibody completely abolished stretch-induced nephrin downregulation. Similar to candesartan, the peroxisome proliferator-activated receptor (PPAR)-γ agonist, rosiglitazone, also inhibited stretch-induced nephrin downregulation, suggesting interference with stretch-induced activation of the angiotensin II-AT1 receptor system. Accordingly, rosiglitazone did not alter stretch-induced angiotensin II secretion, but it prevented AT1 upregulation in response to stretch. These results suggest a role for hemodynamic stress in loss of nephrin expression and allude to a role of PPAR-γ agonists in the prevention of this loss.

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Increased Expression of Endothelial and Neuronal Nitric Oxide Synthase In Dura and Pia Mater After Air Stress

Cephalalgia ◽

10.1111/j.1468-2982.2005.00978.x ◽

2006 ◽

Vol 26 (1) ◽

pp. 14-25 ◽

Cited By ~ 7

Author(s):

T Zinck ◽

R Illum ◽

I Jansen-Olesen

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Protein Expression ◽

Dura Mater ◽

Underlying Mechanism ◽

Pia Mater ◽

Mrna And Protein Expression ◽

Migraine Pathogenesis ◽

Oxide Synthase

Stress is the leading precipitating factor for migraine attacks but the underlying mechanism is currently unknown. Nitric oxide (NO) has been implicated in migraine pathogenesis based on the ability of NO donors to induce migraine attacks. In the present study, we investigated in Wistar rats the effect of air stress on nitric oxide synthase (NOS) mRNA and protein expression in dura and pia mater using real-time polymerase chain reaction and Western blotting, respectively. Endothelial (e)NOS protein expression was significantly increased in dura and pia mater after air stress. Significantly augmented neuronal (n)NOS protein expression was detected in pia mater after air stress but not in dura mater. Inducible NOS mRNA and protein expression levels in dura and pia mater were unaffected by stress. The increased expression of eNOS in dura mater and eNOS and nNOS in pia mater seen after stress could not be antagonized by treatment with the migraine drug sumatriptan. These findings point towards the involvement of increased NO concentrations in dura and pia mater in response to air stress. However, the role of these findings in relation to migraine pathophysiology remains unclear.

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Complementary expression and phosphorylation of Cx46 and Cx50 during development and following gene deletion in mouse and in normal and orchitic mink testes

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00152.2015 ◽

2015 ◽

Vol 309 (3) ◽

pp. R255-R276 ◽

Cited By ~ 8

Author(s):

R.-Marc Pelletier ◽

Casimir D. Akpovi ◽

Li Chen ◽

Nalin M. Kumar ◽

María L. Vitale

Keyword(s):

Protein Expression ◽

Germ Cell ◽

Gap Junction ◽

Sertoli Cell ◽

Cell Communication ◽

Seminiferous Tubules ◽

Junction Protein ◽

Mrna And Protein Expression ◽

Autoimmune Orchitis ◽

The Impact

Gap junction-mediated communication helps synchronize interconnected Sertoli cell activities. Besides, coordination of germ cell and Sertoli cell activities depends on gap junction-mediated Sertoli cell–germ cell communication. This report assesses mechanisms underlying the regulation of connexin 46 (Cx46) and Cx50 in mouse testis and those accompanying a “natural” seasonal and a pathological arrest of spermatogenesis, resulting from autoimmune orchitis (AIO) in mink. Furthermore, the impact of deleting Cx46 or Cx50 on the expression, phosphorylation of junction proteins, and spermatogenesis is evaluated. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. During the mink active spermatogenic phase, Cx50 became phosphorylated and localized to the site of the blood-testis barrier. By contrast, Cx46 was dephosphorylated and associated with annular junctions, suggesting phosphorylation/dephosphorylation of Cx46 and Cx50 involvement in the barrier dynamics. Cx46-positive annular junctions in contact with lipid droplets were found. Cx46 and Cx50 expression and localization were altered in mink with AIO. The deletion of Cx46 or Cx50 impacted on other connexin expression and phosphorylation and differently affected tight and adhering junction protein expression. The level of apoptosis, determined by ELISA, and a number of Apostain-labeled spermatocytes and spermatids/tubules were higher in mice lacking Cx46 ( Cx46−/−) than wild-type and Cx50−/− mice, arguing for life-sustaining Cx46 gap junction-mediated exchanges in late-stage germ cells secluded from the blood by the barrier. The data show that expression and phosphorylation of Cx46 and Cx50 are complementary in seminiferous tubules.

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Response of the honey bee (Apis mellifera) proteome to Israeli acute paralysis virus (IAPV) infection

Canadian Journal of Zoology ◽

10.1139/cjz-2014-0181 ◽

2015 ◽

Vol 93 (9) ◽

pp. 711-720 ◽

Cited By ~ 9

Author(s):

Sarah Michaud ◽

Humberto F. Boncristiani ◽

Joost W. Gouw ◽

Micheline K. Strand ◽

Jeffrey Pettis ◽

...

Keyword(s):

Apis Mellifera ◽

Protein Expression ◽

Honey Bee ◽

Host Protein ◽

Colony Losses ◽

Israeli Acute Paralysis Virus ◽

Significant Research ◽

Ubiquitin Proteasome ◽

The Impact ◽

Paralysis Virus

Recent declines in honey bee (Apis mellifera L., 1758) populations worldwide have spurred significant research into the impact of pathogens on colony health. The role of the Israeli acute paralysis virus (IAPV) on hive mortality has become of particular concern since being correlated with colony losses. However, the molecular interactions between IAPV and its host remain largely unknown. To investigate changes in host protein expression during IAPV infection, mass-spectrometry-based quantitative proteomics was used to compare IAPV-infected and healthy pupae. Proteins whose expression levels changed significantly during infection were identified and functional analysis was performed to determine host systems and pathways perturbed by IAPV infection. Among the A. mellifera proteins most affected by IAPV, those involving translation and the ubiquitin–proteasome pathway were most highly enriched and future investigation of these pathways will be useful in identifying host proteins required for infection. This analysis represents an important first step towards understanding the honey bee host response to IAPV infection through the systems-level analysis of protein expression.

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PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

Nature Communications ◽

10.1038/s41467-021-25252-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yilun Sun ◽

Jiji Chen ◽

Shar-yin N. Huang ◽

Yijun P. Su ◽

Wenjie Wang ◽

...

Keyword(s):

Single Molecule ◽

Topoisomerase I ◽

Ubiquitin Proteasome System ◽

Live Cells ◽

Ubiquitin Proteasome ◽

Repair Factor ◽

The Impact ◽

Protein Crosslinks ◽

Potential Regulatory Role

AbstractPoly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs.

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