scholarly journals ULK1 Promotes Mitophagy via Phosphorylation and Stabilization of BNIP3.

2021 ◽  
Author(s):  
Logan P. Poole ◽  
Althea Bock-Hughes ◽  
Damian E. Berardi ◽  
Kay F. Macleod
Keyword(s):  
Protein Stability ◽  
Serine Residue ◽  
Initiation Complex ◽  
Protein Levels ◽  
Amino Terminal ◽  
Bh3 Domain ◽  
Cargo Receptor ◽  
Mechanistic Basis ◽  
Lir Motif

Abstract UNC51-like kinase-1 (ULK1) is the catalytic component of the autophagy pre-initiation complex that stimulates autophagy via phosphorylation of ATG14, BECLN1 and other autophagy proteins. ULK1 has also been shown to specifically promote mitophagy but the mechanistic basis of how has remained unclear. Here we show that ULK1 phosphorylates the BNIP3 mitochondrial cargo receptor on a critical serine residue (S17) adjacent to its amino terminal LIR motif. ULK1 similarly phosphorylates BNIP3L on S35. Phosphorylation of BNIP3 on S17 by ULK1 promotes interaction with LC3 and mitophagy. ULK1 interaction also promotes BNIP3 protein stability by limiting its turnover at the proteasome. The ability of ULK1 to regulate BNIP3 protein stability depends on an intact “BH3” domain and deletion of its “BH3” domain reduces BNIP3 turnover and increases BNIP3 protein levels independent of ULK1. In summary ULK1 promotes mitophagy by both stabilization of BNIP3 protein and via phosphorylation of S17 to stimulate interaction with LC3.

scholarly journals FXR activation by obeticholic acid or nonsteroidal agonists induces a human-like lipoprotein cholesterol change in mice with humanized chimeric liver

2018 ◽  
Vol 59 (6) ◽  
pp. 982-993 ◽  
Author(s):  
Romeo Papazyan ◽  
Xueqing Liu ◽  
Jingwen Liu ◽  
Bin Dong ◽  
Emily M. Plummer ◽  
...  
Keyword(s):  
Target Gene ◽  
Ldl Cholesterol ◽  
Ldl Receptor ◽  
Hdl Cholesterol ◽  
Farnesoid X Receptor ◽  
Receptor Protein ◽  
Chimeric Mice ◽  
Obeticholic Acid ◽  
Protein Levels ◽  
Mechanistic Basis

Obeticholic acid (OCA) is a selective farnesoid X receptor (FXR) agonist that regulates bile acid and lipid metabolism. FXR activation induces distinct changes in circulating cholesterol among animal models and humans. The mechanistic basis of these effects has been elusive because of difficulties in studying lipoprotein homeostasis in mice, which predominantly package circulating cholesterol in HDLs. Here, we tested the effects of OCA in chimeric mice whose livers are mostly composed (≥80%) of human hepatocytes. Chimeric mice exhibited a human-like ratio of serum LDL cholesterol (LDL-C) to HDL cholesterol (HDL-C) at baseline. OCA treatment in chimeric mice increased circulating LDL-C and decreased circulating HDL-C levels, demonstrating that these mice closely model the cholesterol effects of FXR activation in humans. Mechanistically, OCA treatment increased hepatic cholesterol in chimeric mice but not in control mice. This increase correlated with decreased SREBP-2 activity and target gene expression, including a significant reduction in LDL receptor protein. Cotreatment with atorvastatin reduced total cholesterol, rescued LDL receptor protein levels, and normalized serum LDL-C. Treatment with two clinically relevant nonsteroidal FXR agonists elicited similar lipoprotein and hepatic changes in chimeric mice, suggesting that the increase in circulating LDL-C is a class effect of FXR activation.

Pro-inflammatory cytokines reduce human TAFI expression via tristetraprolin-mediated mRNA destabilisation and decreased binding of HuR

2015 ◽  
Vol 114 (08) ◽  
pp. 337-349 ◽  
Author(s):  
Dragana Komnenov ◽  
Corey Scipione ◽  
Zainab Bazzi ◽  
Justin Garabon ◽  
Marlys Koschinsky ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Inflammatory Mediators ◽  
Hepg2 Cells ◽  
Inflammatory Cells ◽  
Gene Encoding ◽  
Protein Levels ◽  
Anti Inflammatory ◽  
Mechanistic Basis ◽  
Pro Inflammatory Cytokines

SummaryThrombin activatable fibrinolysis inhibitor (TAFI) is the zymogen form of a basic carboxypeptidase (TAFIa) with both anti-fibrinolytic and anti-inflammatory properties. The role of TAFI in inflammatory disease is multifaceted and involves modulation both of specific inflammatory mediators as well as of the behaviour of inflammatory cells. Moreover, as suggested by in vitro studies, inflammatory mediators are capable of regulating the expression of CPB2, the gene encoding TAFI. In this study we addressed the hypothesis that decreased TAFI levels observed in inflammation are due to post-transcriptional mechanisms. Treatment of human HepG2 cells with pro-inflammatory cytokines TNFα, IL-6 in combination with IL-1β, or with bacterial lipopolysaccharide (LPS) decreased TAFI protein levels by approximately two-fold over 24 to 48 hours of treatment. Conversely, treatment of HepG2 cells with the anti-inflammatory cytokine IL-10 increased TAFI protein levels by two-fold at both time points. We found that the mechanistic basis for this modulation of TAFI levels involves binding of tristetraprolin (TTP) to the CPB2 3′-UTR, which mediates CPB2 mRNA destabilisation. In this report we also identified that HuR, another ARE-binding protein but one that stabilises transcripts, is capable of binding the CBP2 3’UTR. We found that pro-inflammatory mediators reduce the occupancy of HuR on the CPB2 3’-UTR and that the mutation of the TTP binding site in this context abolishes this effect, although TTP and HuR appear to contact discrete binding sites. Interestingly, all of the mediators tested appear to increase TAFI protein expression in THP-1 macrophages, likewise through effects on CPB2 mRNA stability.

scholarly journals VBP1 modulates Wnt/β-catenin signaling by mediating the stability of the transcription factors TCF/LEFs

2020 ◽  
Vol 295 (49) ◽  
pp. 16826-16839
Author(s):  
Haifeng Zhang ◽  
Xiaozhi Rong ◽  
Caixia Wang ◽  
Yunzhang Liu ◽  
Ling Lu ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Stability ◽  
Signaling Pathway ◽  
Cultured Cells ◽  
Critical Role ◽  
Family Members ◽  
Von Hippel Lindau ◽  
T Cell Factor ◽  
Protein Levels ◽  
The Stability

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Glutamine synthetase expression in rat lung is regulated by protein stability

1998 ◽  
Vol 275 (5) ◽  
pp. L877-L886 ◽  
Author(s):  
Brian I. Labow ◽  
Steve F. Abcouwer ◽  
Cheng-Mao Lin ◽  
Wiley W. Souba
Keyword(s):  
Protein Stability ◽  
Physiological Stress ◽  
Control Level ◽  
Methionine Sulfoximine ◽  
Activity Levels ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Protein Levels ◽  
Gs Protein

During physiological stress, the lung increases production of the amino acid glutamine (Gln) using the enzyme Gln synthetase (GS) to maintain Gln homeostasis. Glucocorticoid hormones are considered the principal mediators of GS expression during stress. However, whereas animal studies have shown that glucocorticoids increase lung GS mRNA levels 500–700%, GS activity levels rise only 20–45%. This discrepancy suggests that a posttranscriptional control mechanism(s) ultimately determines GS expression. We hypothesized that the level of GS protein in the lung is governed by the intracellular Gln concentration through a mechanism of protein destabilization, a feedback regulatory mechanism that has been observed in vitro. To test this hypothesis, Sprague-Dawley rats were treated with a Gln-free diet and the GS inhibitor methionine sulfoximine (MSO) to deplete tissue Gln levels and prevent this feedback regulation. Exposure to Gln-free chow and MSO (100 mg/kg body wt) for 6 days decreased plasma Gln levels 50% ( P < 0.01) and decreased lung tissue Gln levels by 70% ( P < 0.01). Although lung GS mRNA levels were not influenced by Gln depletion, there was a sevenfold ( P < 0.01) increase in GS protein. A parenteral Gln infusion (200 mM, 1.5 ml/h) for the last 2 days of MSO treatment replenished lung Gln levels to 65% of control level and blunted the increase in GS protein levels by 33% ( P < 0.05) compared with rats receiving an isomolar glycine solution. The acute effects of glucocorticoid and MSO administration on lung GS expression were also measured. Whereas dexamethasone (0.5 mg/kg) and MSO injections individually augmented lung GS protein levels twofold and fourfold ( P < 0.05), respectively, the combination of dexamethasone and MSO produced a synergistic, 12-fold induction ( P < 0.01) in lung GS protein over 8 h. The data suggest that, whereas glucocorticoids are potent mediators of GS transcriptional activity, protein stability greatly influences the ultimate expression of GS in the lung.

scholarly journals Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

2012 ◽  
Vol 23 (23) ◽  
pp. 4515-4525 ◽  
Author(s):  
Sara Ovejero ◽  
Patricia Ayala ◽  
Avelino Bueno ◽  
María P. Sacristán
Keyword(s):  
Regulatory Network ◽  
Cyclin B1 ◽  
Protein Levels ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Data Support ◽  
Amino Terminal Domain

The activity of Cdk1–cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1–cyclin B1 activity through Wee1 dephosphorylation.

MKP-1 expression and stabilization and cGK Iα prevent diabetes- associated abnormalities in VSMC migration

2004 ◽  
Vol 287 (4) ◽  
pp. C1077-C1086 ◽  
Author(s):  
Asha Jacob ◽  
Albert Smolenski ◽  
Suzanne M. Lohmann ◽  
Najma Begum
Keyword(s):  
Diabetic Rats ◽  
Mapk Phosphorylation ◽  
Protein Levels ◽  
Intimal Injury ◽  
Dependent Protein ◽  
Insulin Dependent ◽  
Mechanistic Basis ◽  
Wistar Kyoto ◽  
Interfering Rna ◽  
Insulin Inhibition

Diabetes mellitus is a major risk factor in the development of atherosclerosis and cardiovascular disease conditions, involving intimal injury and enhanced vascular smooth muscle cell (VSMC) migration. We report a mechanistic basis for divergences between insulin’s inhibitory effects on migration of aortic VSMC from control Wistar Kyoto (WKY) rats versus Goto-Kakizaki (GK) diabetic rats. In normal WKY VSMC, insulin increased MAPK phosphatase-1 (MKP-1) expression as well as MKP-1 phosphorylation, which stabilizes it, and inhibited PDGF-mediated MAPK phosphorylation and cell migration. In contrast, basal migration was elevated in GK diabetic VSMCs, and all of insulin’s effects on MKP-1 expression and phosphorylation, MAPK phosphorylation, and PDGF-stimulated migration were markedly inhibited. The critical importance of MKP-1 in insulin inhibition of VSMC migration was evident from several observations. MKP-1 small interfering RNA inhibited MKP-1 expression and abolished insulin inhibition of PDGF-induced VSMC migration. Conversely, adenoviral expression of MKP-1 decreased MAPK phosphorylation and basal migration rate and restored insulin's ability to inhibit PDGF-directed migration in GK diabetic VSMCs. Also, the proteasomal inhibitors lactacystin and MG132 partially restored MKP-1 protein levels in GK diabetic VSMCs and inhibited their migration. Furthermore, GK diabetic aortic VSMCs had reduced cGMP-dependent protein kinase Iα (cGK Iα) levels as well as insulin-dependent, but not sodium nitroprusside-dependent, stimulation of cGMP. Adenoviral expression of cGK Iα enhanced MKP-1 inhibition of MAPK phosphorylation and VSMC migration. We conclude that enhanced VSMC migration in GK diabetic rats is due at least in part to a failure of insulin-stimulated cGMP/cGK Iα signaling, MKP-1 expression, and stabilization and thus MAPK inactivation.

scholarly journals Evaluating Effects of HypomorphicThoc1Alleles on Embryonic Development inRb1Null Mice

2016 ◽  
Vol 36 (11) ◽  
pp. 1621-1627 ◽  
Author(s):  
Meenalakshmi Chinnam ◽  
Xiaoling Wang ◽  
Xiaojing Zhang ◽  
David W. Goodrich
Keyword(s):  
Embryonic Development ◽  
Protein Interaction ◽  
Tissue Expression ◽  
Regulatory Genes ◽  
Terminal Protein ◽  
Interaction Domain ◽  
Ribonucleoprotein Complex ◽  
Protein Levels ◽  
Amino Terminal ◽  
Expression Of Genes

The Rb1 tumor suppressor protein is a molecular adaptor that physically links transcription factors like E2f with various proteins acting on DNA or RNA to repress gene expression. Loss of Rb1 liberates E2f to activate the expression of genes mediating resulting phenotypes. Most Rb1 binding proteins, including E2f, interact through carboxyl-terminal protein interaction domains, but genetic evidence suggests that an amino-terminal protein interaction domain is also important. One protein that binds Rb1 through the amino-terminal domain is encoded byThoc1, a required component of the THO ribonucleoprotein complex important for RNA processing and transport. The physiological relevance of this interaction is unknown. Here we tested whetherThoc1mediates effects ofRb1loss on mouse embryonic development. We found thatThoc1deficiency delays embryo death, and this delay correlates with reduced apoptosis in the brain. E2f protein levels are reduced inRb1:Thoc1-deficient brain tissue. Expression of apoptotic regulatory genes regulated by E2f, like Apaf1 and Bak1, is also reduced. These observations suggest thatThoc1is required to support increased expression of E2f and apoptotic regulatory genes that trigger apoptosis uponRb1loss. These findings implicate Rb1 in the regulation of the THO ribonucleoprotein complex.

scholarly journals Copper-induced structural conversion templates prion protein oligomerization and neurotoxicity

2016 ◽  
Vol 2 (7) ◽  
pp. e1600014 ◽  
Author(s):  
Chi-Fu Yen ◽  
Dilshan S. Harischandra ◽  
Anumantha Kanthasamy ◽  
Sanjeevi Sivasankar
Keyword(s):  
Prion Protein ◽  
Single Molecule ◽  
Amyloid Formation ◽  
Protein Oligomerization ◽  
Copper Exposure ◽  
Structural Conversion ◽  
Neuronal Tissue ◽  
Amino Terminal ◽  
Mechanistic Basis ◽  
Mediate Inflammation

Prion protein (PrP) misfolding and oligomerization are key pathogenic events in prion disease. Copper exposure has been linked to prion pathogenesis; however, its mechanistic basis is unknown. We resolve, with single-molecule precision, the molecular mechanism of Cu2+-induced misfolding of PrP under physiological conditions. We also demonstrate that misfolded PrPs serve as seeds for templated formation of aggregates, which mediate inflammation and degeneration of neuronal tissue. Using a single-molecule fluorescence assay, we demonstrate that Cu2+ induces PrP monomers to misfold before oligomer assembly; the disordered amino-terminal region mediates this structural change. Single-molecule force spectroscopy measurements show that the misfolded monomers have a 900-fold higher binding affinity compared to the native isoform, which promotes their oligomerization. Real-time quaking-induced conversion demonstrates that misfolded PrPs serve as seeds that template amyloid formation. Finally, organotypic slice cultures show that misfolded PrPs mediate inflammation and degeneration of neuronal tissue. Our study establishes a direct link, at the molecular level, between copper exposure and PrP neurotoxicity.

scholarly journals Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling

PLoS Biology ◽  
2021 ◽  
Vol 19 (9) ◽  
pp. e3001392
Author(s):  
Taylor P. Light ◽  
Delphine Brun ◽  
Pablo Guardado-Calvo ◽  
Riccardo Pederzoli ◽  
Ahmed Haouz ◽  
...  
Keyword(s):  
Viral Entry ◽  
Molecular Mimicry ◽  
Human Herpesvirus ◽  
Targeted Mutagenesis ◽  
Human Herpesvirus 8 ◽  
Tyrosine Kinase Receptor ◽  
Binding Studies ◽  
Herpesvirus 8 ◽  
Amino Terminal ◽  
Mechanistic Basis

Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis, and binding studies, we here show that the HHV-8 envelope glycoprotein complex H and L (gH/gL) binds with subnanomolar affinity to EphA2 via molecular mimicry of the receptor’s cellular ligands, ephrins (Eph family receptor interacting proteins), revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8–related diseases.

scholarly journals Intracellular association of the protein product of the c-myc oncogene with the TATA-binding protein

1994 ◽  
Vol 14 (2) ◽  
pp. 1147-1152
Author(s):  
S Maheswaran ◽  
H Lee ◽  
G E Sonenshein
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Protein Product ◽  
Tata Binding Protein ◽  
Nuclear Proteins ◽  
Initiation Complex ◽  
Amino Terminal ◽  
Wehi 231 ◽  
Transcription Initiation Complex

The c-myc proto-oncogene encodes nuclear phosphoproteins that bind DNA in a sequence-specific fashion and appear to function as transcriptional activators. Here we demonstrate that a 40-kDa nuclear protein coimmunoprecipitated with c-Myc specifically when nuclear proteins, extracted from nuclei of exponentially growing murine B-lymphoma WEHI 231 cells by using procedures for preparation of trans-acting factors, were reacted with anti-c-Myc antibodies made against different regions of the c-Myc protein. In contrast, preparation of nuclear lysates under denaturing conditions significantly reduced this coprecipitation. Upon incubation of WEHI 231 cells with the reversible chemical cross-linking agent dithiobis(succinimidyl propionate), the 40-kDa protein could be cross-linked to c-Myc protein intracellularly. Identification of the 40-kDa protein as the TATA-binding protein (TBP) of the TFIID transcription initiation complex was made by comigration and V-8 protease mapping, which yielded identical peptide fragments upon digestion of the 40-kDa protein and material immunoprecipitated with an anti-TBP specific antibody. Furthermore, in vitro-translated TBP bound to the amino-terminal portion of c-Myc. Column chromatography of cross-linked nuclear proteins showed TBP to be in a large-molecular-weight complex with c-Myc, consistent with a transcription initiation complex. These results indicate that intracellularly, c-Myc interacts with TBP, suggesting a mechanism of interaction of this oncoprotein with the basal transcription machinery.

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