NRBP1-Containing CRL2/CRL4A Regulates Amyloid β Production by Targeting BRI2 and BRI3 for Degradation

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Exploring QSAR for CYP11B2 binding affinity and CYP11B2/CYP11B1 selectivity of diverse functional compounds using GFA and G/PLS techniques

A data set of a series of 132 structurally diverse compounds with cytochrome 11B2 and 11B1 (CYP11B2 and CYP11B1) enzyme inhibitory activities was subjected to molecular shape analysis to explore contributions of shape features as well as electronic, structural, and physicochemical parameters toward enzyme inhibitory activities, in search of appropriate molecular scaffolds with optimum substitutions for highly potent CYP11B2 inhibitors. Genetic function approximation (GFA) and genetic partial least squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical quality considering both internal and external validation parameters (Q²: 0.514–0.659, R²_pred: 0.510–0.734). The G/PLS models with spline option for CYP11B2 and CYP11B1 inhibition and selectivity modeling appeared to be the best models based on r_m²_(overall) criterion. The study indicates the importance of the pyridinylnaphthalene and pyridylmethylene-indane scaffolds with less polar and electrophilic substituents for optimum CYP11B2 inhibitory activity and CYP11B2/CYP11B1 selectivity.     

Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.     

Quantitative analyses for effects of neddylation on CRL2VHL substrate ubiquitination and degradation

Through catalyzing the ubiquitination of key regulatory proteins, cullin‐RING ubiquitin ligases (CRLs) play essential biological roles and their activities are controlled by multiple mechanisms including neddylation, the conjugation of NEDD8 to cullins. Upon neddylation, a CRL, such as the CUL1‐based CRL1, undergoes conformational changes that accelerate substrate ubiquitination. Given the structural diversity across subfamilies of CRLs and their substrates, to what extent neddylation modulates the activity of individual CRLs remains to be evaluated. Here, through reconstituting the CRL2 ubiquitination reaction in vitro, we showed that neddylation promotes CRL2^VHL‐dependent degradation of both full‐length HIF1α and the degron peptide of HIF1α, resulting in more than 10‐fold increase in the rate of substrate ubiquitination. Consistently, pevonedistat (also known as MLN4924), an inhibitor of neddylation, inhibits the degradation of HIF1α in RCC4 cells stably expressing VHL in cycloheximide chase assays. However, such inhibitory effect of pevonedistat on HIF1α degradation was not observed in HEK293 cells, which was further found to be due to CRL2^VHL‐independent degradation that was active in HEK293 but not RCC4 cells. After truncating HIF1α to its Carboxy‐terminal Oxygen‐Dependent Degradation (CODD) domain, we showed that pevonedistat inhibited the degradation of CODD and increased its half‐life by six‐fold in HEK293 cells. Our results demonstrate that neddylation plays a significant role in activating CRL2, and the cellular activity of CRL2^VHL is better reflected by the degradation of CODD than that of HIF1α, especially under conditions where CRL2‐independent degradation of HIF1α is active.     

Molecular basis for the selective recognition and ubiquitination of centromeric histone H3 by yeast E3 ligase Psh1

Centromeres are chromosomal loci marked by histone variant Cen H3(centromeric histone H3) and essential for genomic stability and cell division. The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeast Cen H3(Cse4) for ubiquitination and controls the cellular level of Cse4 for proteolysis,but the underlying mechanism remains largely unknown. Here, we show that Psh1 uses a Cse4-binding domain(CBD, residues 1-211) to interact with Cse4-H4 instead of H3-H4, yielding a dissociation constant(K_d) of 27 nM. Psh1 recognizes Cse4-specific residues in the L1 loop and a2 helix to ensure Cse4 binding and ubiquitination. We map the Psh1-binding region of Cse4-H4 and identify a wide range of Cse4-specific residues required for the Psh1-mediated Cse4 recognition and ubiquitination. Further analyses reveal that histone chaperone Scm3 can impair Cse4 ubiquitination by abrogating Psh1-Cse4 binding. Together, our study reveals a novel Cse4-binding mode distinct from those of known Cen H3 chaperones and elucidates the mechanism by which Scm3 competes with Psh1 for Cse4 binding.     

Hip2 interacts with cyclin B1 and promotes its degradation through the ubiquitin proteasome pathway

Hip2, a ubiquitin conjugating enzyme, is involved in the suppression of cell death. The present study revealed that Hip2 regulates the stability of the apoptotic and cell cycle regulator cyclin B1. Hip2 was found to interact with cyclin B1 to promote its degradation through the ubiquitin proteasome pathway. As a result, Hip2 significantly blocked cell death induced by the cyclin B1 protein, suggesting that Hip2 is involved in the regulation of cyclin B1-mediated cell death.

Structured summary

MINT-8045218, MINT-8045231: Hip2 (uniprotkb:P61086) physically interacts (MI:0915) with cyclin-B1 (uniprotkb:P14635) by pull down (MI:0096)     

Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate cancer. In the last few decades CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound healing and cardiovascular diseases.In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1 and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously reported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold, leading to new potent 4′-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in selectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4′-substituted derivatives also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17.Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential of this compound as a wound-healing promoter.     

Molecular basis for the recognition of CCN1 by monoclonal antibody 093G9

CCN1, also named Cyr61 (cysteine‐rich protein 61), is the first identified member of the CCN family that is composed of 6 secreted extracellular matrix‐associated glycoproteins. CCN1 has been demonstrated to participate in pathogenesis of rheumatoid arthritis through various pathways. A monoclonal antibody, namely, 093G9, is effective to antagonize the effects of CCN1 and hence has potential therapeutic benefits against rheumatoid arthritis. Here, we show that the epitope recognized by 093G9 is mapped to residues 77 to 80 of CCN1, and a cyclic peptide encompassing residues 75 to 81 of CCN1 displays high binding affinity for 093G9. The crystal structure of the 093G9 Fab in complex with the cyclic peptide was determined at 2.7 Å resolution, which reveals the intensive interactions between CCN1 and 093G9. Particularly, residues Asn79 and Phe80 of CCN1 are inserted into cavities mainly formed by residues of complementarity‐determining region loop L3 and framework region L2 and by residues of complementarity‐determining region loops H2 and H3, respectively, which contribute most of the interactions and therefore are critical for the recognition by 093G9. Together, these findings not only identify the epitope of CCN1 for 093G9 but also reveal the molecular mechanism of recognition and binding of CCN1 by 093G9.     

A newly identified Pirh2 substrate SCYL1-BP1 can bind to MDM2 and accelerate MDM2 self-ubiquitination

The SCY1-like 1 binding protein 1 (SCYL1-BP1) protein was identified as an interacting partner of E3 ligase p53-induced RING H2 protein (Pirh2) and mouse double minute gene number 2 (MDM2) by yeast two-hybrid screening. Further investigation suggested there are two interactions involved in different mechanisms. SCYL1-BP1 can be ubiquitinated and degraded by Pirh2 but not by MDM2, which suggests that SCYL1-BP1 can be regulated by Pirh2. On the other hand, while SCYL1-BP1 binds to ubiquitin E3 ligase MDM2, it promotes MDM2 self-ubiquitination and results in a reduction of MDM2 protein level.

Structured summary

MINT-7904819, MINT-7904837, MINT-7904806, MINT-7904715: MDM2 (uniprotkb:Q00987) physically interacts (MI:0915) with SCYL1-BP1 (uniprotkb:Q5T7V8) by anti tag coimmunoprecipitation (MI:0007)MINT-7904857, MINT-7904899: SCYL1-BP1 (uniprotkb:Q5T7V8) physically interacts (MI:0915) with MDM2 (uniprotkb:Q00987) by anti bait coimmunoprecipitation (MI:0006)     

Polymyxin B exerts nephrotoxicity effects via inhibition of the Nrf2/NQO1 pathway-mediated antioxidant response

Polymyxin B (PMB) is a polypeptide antibiotic widely used in treating multidrug-resistant Gram-negative bacteria. However, nephrotoxicity is a serious adverse effect that limits its clinical use. Therefore, clarification of the molecular mechanism of PMB-induced renal injury is essential. Our study aimed to explore possible mechanisms of PMB-induced nephrotoxicity in vivo and in vitro. Mice were treated with PMB to construct the kidney injury model. The antioxidant capacity was assessed by measuring the superoxide dismutase (SOD) and catalase (CAT) activities and the glutathione (GSH) and malondialdehyde (MDA) contents. The pathway of the nuclear factor erythroid 2-related factor 2/NADH quinone oxidoreductase 1 (Nrf2/NQO1) was examined after PMB treatment in NRK-52E cells and mice. Finally, the expressions of genes and proteins (Bax, Bcl-2, Caspase-3, Caspase-9) related to apoptosis were evaluated through quantitative polymerase chain reaction and western blot assay. The study verified PMB-induced nephrotoxicity in mice and NRK-52E cells in a dose- and time-dependent manner. PMB treatment significantly decreased the expression of Nrf2 and its downstream target gene NQO1 and increased the apoptosis-related proteins expression. In summary, our results suggested that PMB-induced oxidative stress damage by inhibiting the Nrf2/NQO1 pathway and promoting apoptosis in kidney tissues.     

Structural basis for the recognition between the regulatory particles Nas6 and Rpt3 of the yeast 26S proteasome

The 26S proteasome-dependent protein degradation is an evolutionarily conserved process. The mammalian oncoprotein gankyrin, which associates with S6 of the proteasome, facilitates the degradation of pRb, and thus possibly acts as a bridging factor between the proteasome and its substrates. However, the mechanism of the proteasome-dependent protein degradation in yeast is poorly understood. Here, we report the tertiary structure of the complex between Nas6 and a C-terminal domain of Rpt3, which are the yeast orthologues of gankyrin and S6, respectively. The concave region of Nas6 bound to the alpha-helical domain of Rpt3. The stable interaction between Nas6 and Rpt3 was mediated by intermolecular interactions composed of complementary charged patches. The recognition of Rpt3 by Nas6 in the crystal suggests that Nas6 is indeed a subunit of the 26S proteasome. These results provide a structural basis for the association between Nas6 and the heterohexameric ATPase ring of the proteasome through Rpt3.     

Molecular basis of BACH1/FANCJ recognition by TopBP1 in DNA replication checkpoint control

The diverse roles of TopBP1 in DNA replication and checkpoint signaling are associated with the scaffolding ability of TopBP1 to initiate various protein-protein interactions. The recognition of the BACH1/FANCJ helicase by TopBP1 is critical for the activation of the DNA replication checkpoint at stalled replication forks and is facilitated by the C-terminal tandem BRCT7/8 domains of TopBP1 and a phosphorylated Thr(1133) binding motif in BACH1. Here we provide the structural basis for this interaction through analysis of the x-ray crystal structures of TopBP1 BRCT7/8 both free and in complex with a BACH1 phospho-peptide. In contrast to canonical BRCT-phospho-peptide recognition, TopBP1 BRCT7/8 undergoes a dramatic conformational change upon BACH1 binding such that the two BRCT repeats pivot about the central BRCT-BRCT interface to provide an extensive and deep peptide-binding cleft. Additionally, we provide the first structural mechanism for Thr(P) recognition among BRCT domains. Together with systematic mutagenesis studies, we highlight the role of key contacts in governing the unique specificity of the TopBP1-BACH1 interaction.     

Downregulation of tripartite motif protein 11 attenuates cardiomyocyte apoptosis after ischemia/reperfusion injury via DUSP1-JNK1/2

Currently, the prevention of ischemic diseases such as myocardial infarction associated with ischemia/reperfusion (I/R) injury remains to be a challenge. Thus, this study was designed to explore the effects of tripartite motif protein 11 (TRIM11) on cardiomyocytes I/R injury and its underlying mechanism. Cardiomyocytes AC16 were used to establish an I/R injury cell model. After TRIM11 downregulation in I/R cells, cell proliferation (0, 12, 24, and 48 h) and apoptosis at 48 h as well as the related molecular changes in oxidative stress-related pathways was detected. Further, after the treatment of TRIM11 overexpression, SP600125, or DUSP1 overexpression, cell proliferation, apoptosis, and related genes were detected again. As per our findings, it was determined that TRIM11 was highly expressed in the cardiomyocytes AC16 after I/R injury. Downregulation of TRIM11 was determined to have significantly reduced I/R-induced proliferation suppression and apoptosis. Besides, I/R-activated c-Jun N-terminal kinase (JNK) signaling and cleaved caspase 3 and Bax expression were significantly inhibited by TRIM11 downregulation. In addition, the overexpression of TRIM11 significantly promoted apoptosis in AC16 cells, and JNK1/2 inhibition and DUSP1 overexpression potently counteracted the induction of TRIM11 overexpression in AC16 cells. These suggested that the downregulation of TRIM11 attenuates apoptosis in AC16 cells after I/R injury probably through the DUSP1-JNK1/2 pathways.     

Lysine 263 residue of NPM/B23 is essential for regulating ATP binding and B23 stability

Here, we show that Nucleophsomin/B23 provides lysine 263 as a critical binding site for ATP. Mutagenesis of lysine 263 to asparagine (K263N) disrupts B23 from ATP binding. While B23 WT exclusively localizes to the nucleolus, the B23-K263N is redistributed from the nucleolus to the nucleoplam. Notably, the K263N mutant is unstable, and displayed rapid degradation. Alteration of K263 induced B23 instability through increased ubiquitination and proteaosomal degradation. Moreover, mutation of K263 impedes the mitogenic effect of B23 in PC12 cells. Thus, K263 is a critical site for ATP binding and required for B23 stability, confining B23 in the nucleolus.