Exploring the mechanism of PPARγ phosphorylation mediated by CDK5

Filho, Helder Veras Ribeiro; Guerra, João Victor da Silva; Cagliari, R.; Batista, Fernanda Aparecida Heleno; Maire, Albane le; Oliveira, Paulo Sérgio Lopes de; Figueira, Ana Carolina Migliorini

doi:10.1016/j.jsb.2019.07.007

Cited by 15 publications

(24 citation statements)

References 39 publications

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“…A double mutant, designed to have a disulphide bridge that strongly stabilizes the β−sheet, confirmed that the phosphorylation of S245 is completely inhibited if the transient unfolding of β strand by Cdk5 is prevented. As shown by Ribeiro Filho et al 19 , the stabilization of the helix H2' and the loop H2-H2' at the interface between PPARγ and Cdk5/p25 can also affect the phosphorylation rate. This is in accordance with the observation that ligands bound at the hydrophobic region between H3 and β1-β4 of PPARγ, such as 7j, the partial agonists (R)-1 14 , MRL24 12,17 and nTZDpa 41 , are able to reduce the mobility of this region, through vdW interactions (Supporting Information Figure 15)…”

Section: Discussionmentioning

confidence: 85%

“…Particularly, in the model proposed by Mottin et al, the external strand of the PPARγ β-sheet is quite distorted, upon interaction with CDK5, with the disruption of one H-bond. Recently, Ribeiro Filho et al 19 proposed a quite different model of the complex between CDK5/p25 and PPARγ-LBD. They found that PPARγ K261 occupies the P + 3 position of the consensus motif, forming a non-contiguous recognition site, as observed in other kinase substrates 20 .…”

Section: Conformational Dynamics Within Pparγ Are Essential To Enable Phosphorylation Of S245 By Cdk5/p25mentioning

confidence: 99%

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Insights into PPARγ Phosphorylation and Its Inhibition Mechanism

et al. 2020

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PPARγ represents a key target for the treatment of type II diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of anti-diabetic drugs acting through this nuclear receptor. We have employed a multi-disciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternate inhibition mechanism adopted by a ligand bound far from the phosphorylation site.

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Section: Discussionmentioning

confidence: 85%

Section: Conformational Dynamics Within Pparγ Are Essential To Enable Phosphorylation Of S245 By Cdk5/p25mentioning

confidence: 99%

Insights into PPARγ Phosphorylation and Its Inhibition Mechanism

et al. 2020

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“…In the imatinib-bound structure, the methylbenzene ring and pyridylpyrimidine moiety of imatinib occupy the Arm3 region in PPARγ LBD, which is surrounded by helix H2′, the Ω loop, and the four-stranded β-sheet (Figure 3). Recently, Filho et al suggested an indirect mechanism of conformational change of the Ile341 side chain by ligand binding to form a hydrophobic interaction network and stabilize the helix H2′, blocking the interaction between Cdk5 and PPARγ and causing the impairment of Cdk5-mediated phosphorylation [31]. Additionally, we found that imatinib binding strengthened the hydrophobic interaction network comprised of Ile249, Leu255, Ile262, Ile341, and Met348 in the Arm3 region and stabilized the surrounding helix H2′, the Ω loop, and the four-stranded β-sheet (Figure 4B and Figure 6).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Regulation of PPARγ Activity by Imatinib

2019

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Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2′ and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects.

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“…CDK5 mediated phosphorylation of PPARg and of the PPARg complexes with PGC1-a, TIF2, SMRT and NCoR from pull down assays were measured by luminescent detection of ADP produced in the in vitro phosphorylation reaction, as it was described in (27,28). We used ADP-Glo ™ kinase assay (Promega) following manufacturer's instructions, in which 15 mM of purified PPARg LBD and the pull down purified complexes PPARg + PGC1-a, PPARg + TIF2, PPARg + SMRT and PPARg + NCoR were incubated with 25 ng of purified CDK5/p35, at room temperature for 15 min, in the kinase assay reaction buffer (200mM Tris-HCl, pH 7.4, 100mM MgCl2 and 0.5 mg/ml BSA, SignalChem kinase assay buffer III) added 10 mM of ATP, in 12.5 ml of reaction volume.…”

Section: In Vitro Phosphorylation Assaymentioning

confidence: 99%

“…Structural data analysis showed that PPARg ligands that inhibit S273 phosphorylation do not make direct contact with this residue, but induces structural modifications in PPARg:CDK5 interaction interface. Such ligands fit into binding pocket promoting an interaction network that protects S273, blocking its phosphorylation (28). Therefore, the most recent strategy of PPARg modulation have been target the partial agonism of receptor, aiming S273 phosphorylation inhibition.…”

Section: Introductionmentioning

confidence: 99%

PPARγ S273 Phosphorylation Modifies the Dynamics of Coregulator Proteins Recruitment

et al. 2020

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The nuclear receptor PPARγ is essential to maintain whole-body glucose homeostasis and insulin sensitivity, acting as a master regulator of adipogenesis, lipid, and glucose metabolism. Its activation through natural or synthetic ligands induces the recruitment of coactivators, leading to transcription of target genes such as cytokines and hormones. More recently, post translational modifications, such as PPARγ phosphorylation at Ser273 by CDK5 in adipose tissue, have been linked to insulin resistance trough the dysregulation of expression of a specific subset of genes. Here, we investigate how this phosphorylation may disturb the interaction between PPARγ and some coregulator proteins as a new mechanism that may leads to insulin resistance. Through cellular and in vitro assays, we show that PPARγ phosphorylation inhibition increased the activation of the receptor, therefore the increased recruitment of PGC1-α and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, our results show a shift in the coregulators interaction domains preferences, suggesting additional interaction interfaces formed between the phosphorylated PPARγ and some coregulator proteins. Also, we observed that the CDK5 presence disturb the PPARγ-coregulator’s synergy, decreasing interaction with PGC1-α, TIF2, and NCoR, but increasing coupling of SMRT. Finally, we conclude that the insulin resistance provoked by PPARγ phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression.

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Exploring the mechanism of PPARγ phosphorylation mediated by CDK5

Cited by 15 publications

References 39 publications

Insights into PPARγ Phosphorylation and Its Inhibition Mechanism

Insights into PPARγ Phosphorylation and Its Inhibition Mechanism

Structural Basis for the Regulation of PPARγ Activity by Imatinib

PPARγ S273 Phosphorylation Modifies the Dynamics of Coregulator Proteins Recruitment

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