The intracellular mechanism of Berberine-induced inhibition of CYP3A4 activity

2021 ◽  
Vol 27 ◽  
Author(s):  
Pan-Feng Feng ◽  
Long-Xun Zhu ◽  
Jing Jie ◽  
Peng-Xiang Yang ◽  
Xia Chen
Keyword(s):  
Constitutive Androstane Receptor ◽  
Ubiquitin Proteasome System ◽  
Isoquinoline Alkaloid ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Cyp3a4 Gene ◽  
Underlying Mechanisms ◽  
Novel Drug

Background: Berberine (BBR) is an isoquinoline alkaloid extracted from Chinese medicine, exerting various pharmacological effects. BBR is partially metabolized by cytochrome 3A4 (CYP3A4) in vivo. Some reports indicated that BBR could inhibit the activity of CYP3A4. However, the underlying mechanisms are not entirely understood. CYP3A4 is transcriptionally regulated by two nuclear receptors: nuclear transcription X receptor (PXR) and constitutive androstane receptor (CAR). It degraded via the ubiquitin-proteasome system. Hence, we tried to explore the mechanisms of CYP3A4 inhibition on both transcriptive and protein levels. Methods: Western Blot, RT-PCR, and Co-immunoprecipitation were used to perform the experiments. Results: Our results showed that BBR inhibited the transcription of CYP3A4 gene by downregulating PXR. In addition, BBR accelerated the degradation of CYP3A4 protein via the polyubiquitination pathway. Conclusion: These findings may lead to the determination of novel drug-drug interactions with BBR and contribute to the future clinical application of BBR.

scholarly journals P01.05 Deciphering the function of the ubiquitin-proteasome-system in regulating the immune checkpoint protein B7-H3 (CD276) in non-small cell lung cancer

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A5.1-A5
Author(s):  
M Kurz ◽  
L Rieger ◽  
P Giansanti ◽  
B Kuster ◽  
F Bassermann
Keyword(s):  
Lung Cancer ◽  
Immune Responses ◽  
Immune Checkpoint ◽  
Ubiquitin Proteasome System ◽  
Mass Spectrometry Analysis ◽  
Checkpoint Protein ◽  
Spectrometry Analysis ◽  
Protein Levels ◽  
Ubiquitin Proteasome

BackgroundCancer cells use the expression of immune checkpoint proteins on their surface to evade immune responses. Targeting these checkpoints with antibodies has substantially advanced cancer therapy in the past years, especially the treatment of lung cancer. However, the prognosis of metastatic lung cancer patients still remains poor and lung cancer remains to be the leading cause of cancer death worldwide. Further therapeutic concepts are therefore urgently needed.It has been shown that protein expression levels of the immune checkpoint protein PD-L1, a member of the B7 protein family, is regulated by the ubiquitin-proteasome system (UPS). Ubiquitin-ligases (E3-ligases) and deubiquitinating enzymes that regulate immune checkpoint levels on the cell surface are therefore considered promising potential drug targets. Inhibiting enzymes that increase immune checkpoint surface levels might increase the anti-cancer immune response.Here, we investigate whether another B7 family member, immune checkpoint protein B7-H3, is regulated by the UPS in non-small cell lung cancer (NSCLC).Materials and MethodsB7-H3 expression in NSCLC cell lines and patient samples was evaluated using mRNASeq data from open databases. Immunoblotting and FACS were used to analyse total endogenous protein levels and surface expression of B7-H3 in different NSCLC lines under normal growth conditions and in response to various inhibitors (MG-132, Chloroquine (CQ) and Cycloheximide (CHX)). Immunoprecipitation of FLAG-tagged B7-H3 followed by a TUBE IP using ubiquitin-binding beads and in-vivo ubiquitylation assays based on co-overexpression of HA-tagged ubiquitin and/or HA-tagged K48/K63-linkages specific ubiquitin together with FLAG-tagged B7-H3 or FLAG-tagged B7-H3 K526R mutant in HEK-93T cells were performed to analyse ubiquitination on B7-H3. Mass spectrometry analysis of FLAG-purified B7-H3 was performed to identify possible interaction partners.ResultsDatabase analysis revealed that B7-H3 expression is higher in lung cancer samples than in healthy lung tissue. We found that B7-H3 is highly expressed in different NSCLC lines on RNA and protein levels. Treatments with either proteasomal (MG-132) or lysosomal (CQ) degradation inhibitors alone showed only minor effects on B7-H3 protein abundance. However, CHX treatment of H1437 cells decreased B7-H3 over time and this decrease was recovered by adding MG-132 or CQ suggesting that both the lysosome as well as proteasome are involved in the degradation of B7-H3. In vivo ubiquitination and TUBE assay showed K48 and K63 B7-H3 ubiquitination. Mass spectrometry analysis of FLAG-tagged purified B7-H3 revealed E3-ligase Trim21, which has recently been identified as a ligase of PD-L1 in lung cancer lines, as a potential interaction partner. Further experiments are planned to validate the result and to identify other UPS-related enzymes involved in post-translational B7-H3 surface level regulation.ConclusionsOur experiments indicate that immune checkpoint B7-H3 levels are regulated by the ubiquitin-proteasome system in NSCLC lines. With further experiments, we aim to identify UPS-related enzymes that stabilize B7-H3 on the cell surface. Pharmacological inhibition of such enzymes might reduce the immune checkpoint’s surface levels and increase anti-tumour immune responses.Disclosure InformationM. Kurz: None. L. Rieger: None. P. Giansanti: None. B. Kuster: None. F. Bassermann: None.

Abstract 131: PDE1 Inhibition Improves Cardiac Protein Quality Control

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Hanming Zhang ◽  
Xuejun "XJ" Wang
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Ubiquitin Proteasome System ◽  
Misfolded Proteins ◽  
Mrna Levels ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Bona Fide

Protein quality control (PQC) functions to minimize the level and toxicity of misfolded proteins in the cell. PQC relies on molecular chaperones and the targeted degradation of misfolded proteins. The latter is currently known to require the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). Virtually all cardiovascular diseases end up heart failure (HF), the leading cause of death of our society. UPS function insufficiency is implicated in the genesis of a large subset of HF, making cardiac PQC enhancement via promoting UPS and ALP function a promising therapeutic strategy to treat HF. Previously, we have demonstrated that stimulating protein kinase G (PKG) genetically or via inhibition of the type 5 phosphodiesterase (PDE5) improves UPS performance, facilitates the removal of misfolded proteins in cardiomyocytes and slows down the progression of cardiac proteinopathy in a transgenic mouse model (CryAB R120G ). PKA has also been shown to enhance proteasomal function. Our preliminary studies reveal that myocardial protein levels of PDE1A, which suppresses both PKG and PKA, are remarkably elevated in the CryAB R120G mice. Hence we hypothesize that PDE1 inhibition (PDE1I) stimulates cardiac proteasomes via PKG and PKA activation and thereby protects against cardiac proteotoxicity. To test our hypothesis, we took advantage of a proven surrogate UPS substrate (GFPu or GFPdgn) as well as a bona fide misfolded protein (CryAB R120G ) that is known to induce cardiac proteinopathy in human and mice. In cultured cardiomyocytes, PDE1 inhibitor LSN2790158 dose- and time-dependently decreased GFPu. Cycloheximide (CHX) chase assays further confirmed that PDE1I shortened the half-life of GFPu, indicative of improved UPS performance. Furthermore, PDE1I promoted the degradation of CryAB R120G . Our in vivo findings revealed that GFPdgn mice treated with LSN2790158 (3mg/kg, i.p.) displayed a significant reduction of myocardial GFPdgn protein but not mRNA levels. Taken together, our data strongly indicate that PDE1I improves cardiac UPS performance and PDE1 represents a potential target to treat cardiac diseases with elevated proteotoxicity.

scholarly journals Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 110
Author(s):  
Dina Aweida ◽  
Shenhav Cohen
Keyword(s):  
Protein Quality ◽  
Protein Structures ◽  
Ubiquitin Proteasome System ◽  
Surface Proteins ◽  
Catalytic Core ◽  
Complex Protein ◽  
Ubiquitin Proteasome ◽  
Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

scholarly journals Identification of the MuRF1 skeletal muscle ubiquitylome through quantitative proteomics

Function ◽  
2021 ◽  
Author(s):  
Leslie M Baehr ◽  
David C Hughes ◽  
Sarah A Lynch ◽  
Delphi Van Haver ◽  
Teresa Mendes Maia ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Potential Role ◽  
Ubiquitin Proteasome System ◽  
In Vivo Electroporation ◽  
Adult Mice ◽  
Expression Of Genes ◽  
Regulation Of Metabolism ◽  
Ubiquitin Proteasome

Abstract MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere, but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

LncRNA Prostate Cancer Gene Expression Marker 1 (PCGEM1) Down-Regulation Inhibits the Development of Osteoarthritis by Modulating miR-152-3p

2022 ◽  
Vol 12 (2) ◽  
pp. 306-315
Author(s):  
Jie Song ◽  
Cheng Chen ◽  
Hui Zhang
Keyword(s):  
Cell Model ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Protein Levels ◽  
Diphenyltetrazolium Bromide ◽  
Proliferation And Apoptosis ◽  
Underlying Mechanisms ◽  
Commercial Kits ◽  
Related Proteins

Osteoarthritis (OA) is a chronic and inflammatory disease, leading to pain or even disability in severe cases. LncRNA PCGEM1 (PCGEM1) is reported to be dysregulated, serving as critical regulators in various human diseases, including OA. However, the biological role of PCGEM1 and its underlying mechanisms during OA remained unclear. In the present study, CHON-001 cells were exposed to interleukin (IL)-1β to construct the OA cell model. Expression of PCGEM1 and miR-152-3p in cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Corresponding commercial kits were used to measure the expressions of lactate dehydrogenase (LDH), inter-leukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Protein levels of apoptosis-related proteins, cleaved-Caspase3 and Caspase3, were detected by Western blotting. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium (MTT) and flow cytometry assays were utilized for the determination of cell proliferation and apoptosis. The association between PCGEN1 and miR-152-3p was confirmed by a dual-luciferase reporter assay. From the results, PCGEM1 expression was significantly increased while miR-152-3p was inhibited in CHON-001 cells after IL-1β treatment. In addition, silencing of PCGEM1 could promote proliferation, inhibit the apoptosis, suppress LDH level and alleviate inflammation response caused by IL-1β in CHON-001 cells by sponging miR-152-3p. In a word, PCGEM1 down-regulation suppressed OA progression by the regulation of miR-152-3p expression, functioning as a potential therapeutic target for OA clinical treatment.

scholarly journals The ubiquitin proteasome system in synaptic and axonal degeneration

2004 ◽  
Vol 165 (1) ◽  
pp. 27-30 ◽  
Author(s):  
Laura Korhonen ◽  
Dan Lindholm
Keyword(s):  
Neurodegenerative Diseases ◽  
Neurodegenerative Disorders ◽  
Drug Targets ◽  
Axonal Degeneration ◽  
Fruit Fly ◽  
Ubiquitin Proteasome System ◽  
Synaptic Protein ◽  
Ubiquitin Proteasome ◽  
Novel Drug ◽  
Novel Drug Targets

The ubiquitin proteasome system (UPS) contributes to the pathophysiology of neurodegenerative diseases, and it is also a major determinant of synaptic protein degradation and activity. Recent studies in rodents and in the fruit fly Drosophila have shown that the activity of the UPS is involved in axonal degeneration. Increased knowledge of the UPS in synaptic and axonal reactions may provide novel drug targets for treatments of neuronal injuries and neurodegenerative disorders.

Gfi1 ubiquitination and proteasomal degradation is inhibited by the ubiquitin ligase Triad1

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3128-3135 ◽  
Author(s):  
Jurgen A. F. Marteijn ◽  
Laurens T. van der Meer ◽  
Liesbeth van Emst ◽  
Simon van Reijmersdal ◽  
Willemijn Wissink ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Binding Domain ◽  
U937 Cells ◽  
Dna Binding Domain ◽  
Protein Levels ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Proteasome

Abstract Growth factor independence 1 (Gfi1) is a transcriptional repressor essential for the function and development of many different hematopoietic lineages. The Gfi1 protein expression is regulated by the ubiquitin-proteasome system. In granulocytes, Gfi1 is rapidly degraded by the proteasome, while it is more stable in monocytes. How the ubiquitination and degradation of Gfi1 is regulated is unclear. Here, we show that the ubiquitin ligase Triad1 interacts with the DNA-binding domain of Gfi1. Unexpectedly, we found that Triad1 inhibited Gfi1 ubiquitination, resulting in a prolonged half-life. Down-regulation of endogenous Triad1 by siRNAs resulted in increased Gfi1 ubiquitination. In U937 cells, Triad1 caused an increase in endogenous Gfi1 protein levels and slowed cell proliferation in a similar manner when Gfi1 itself was expressed. A Triad1 mutant that lacks the Gfi1-binding domain did not affect Gfi1 levels and proliferation. Because neither proteasome-ubiquitin nor Triad1 ubiquitin ligase activity was required for the inhibition of Gfi1 ubiquitination, these data suggest that Triad1 competes for Gfi1 binding with as yet to be identified E3 ubiquitin ligases that do mark Gfi1 for proteasomal degradation. The finetuning of Gfi1 protein levels regulated by Triad1 defines an unexpected role for this protein in hematopoiesis.

Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

2012 ◽  
Vol 443 (3) ◽  
pp. 681-689 ◽  
Author(s):  
Wan Ning Vanessa Chow ◽  
Hon Wing Luk ◽  
Ho Yin Edwin Chan ◽  
Kwok-Fai Lau
Keyword(s):  
Cell Death ◽  
Degradation Mechanism ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Adaptor Protein ◽  
Ubiquitin Proteasome System ◽  
Exon 1 ◽  
Ubiquitin Proteasome

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW–polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.

scholarly journals Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

2015 ◽  
Vol 26 (24) ◽  
pp. 4325-4332 ◽  
Author(s):  
Mingwei Min ◽  
Tycho E. T. Mevissen ◽  
Maria De Luca ◽  
David Komander ◽  
Catherine Lindon
Keyword(s):  
Cell Cycle Progression ◽  
Degradation Kinetics ◽  
Ubiquitin Proteasome System ◽  
Mitotic Exit ◽  
Anaphase Promoting Complex ◽  
Polyubiquitin Chains ◽  
Substrate Degradation ◽  
Ubiquitin Proteasome ◽  
In Cells

The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.

Diminished proteasomal degradation results in accumulation of Gfi1 protein in monocytes

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Jurgen A. F. Marteijn ◽  
Laurens T. van der Meer ◽  
Liesbeth Van Emst ◽  
Theo de Witte ◽  
Joop H. Jansen ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Myeloid Differentiation ◽  
Mrna Levels ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Monocytic Cell ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Monocytic Lineage

Abstract Gfi1 is a transcriptional repressor essential during myeloid differentiation. Gfi1−/− mice exhibit a block in myeloid differentiation resulting in the accumulation of an immature myelo-monocytic cell population and the complete absence of mature neutrophils. Even though mRNA levels of Gfi1 appear to be very low in monocytes, Gfi1 might play a role in the monocytic lineage as Gfi1−/− mice exhibit diminished monocyte-derived dendritic cells and disturbed cytokine production by macrophages in response to LPS. We show here that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Upon forced monocytic differentiation of U937 cells, Gfi1 mRNA levels dropped but protein levels increased due to diminished proteasomal turnover. Similarly, Gfi1 mRNA levels are low in primary monocytes whereas the protein is clearly detectable. Conversely, Gfi1 mRNA levels are high in granulocytes but the protein is swiftly degraded by the proteasome in these cells. Chromatin immunoprecipitation experiments showed that Gfi1 binds to the promoter of several granulocyte-specific genes in primary monocytes, including C/EBPα, neutrophil elastase, and Gfi1 itself. The binding of the repressor Gfi1 to these promoters correlated with low expression of these genes in monocytes compared with granulocytes. Our data fit a model in which Gfi1 protein levels are induced in primary monocytes, due to diminished proteasomal degradation, to repress genes that play a role in granulocytic differentiation.

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