Structural Basis for Regulation of Phosphoinositide Kinases and Their Involvement in Human Disease.

Lipid phosphoinositides play fundamental roles in virtually all pathways that control a cell's decision to grow, move, divide, and die. Because of this, kinases that phosphorylate phosphoinositide lipids are critically involved in myriad essential functions including growth, development, and membrane trafficking. The misregulation of phosphoinositide kinases is critical in human diseases, including cancer, primary immunodeficiencies, and developmental disorders. Phosphoinositide kinases also play a role in mediating bacterial and viral infections for many potent human pathogens. Furthermore, inhibitors of parasite phosphoinositide kinases are in development as therapies for both malaria and cryptosporidiosis. Therefore, understanding how phosphoinositide kinases are regulated has implications for the treatment of many devastating human diseases. Recent structures of phosphoinositide kinases have revealed unique molecular insight into their regulation. This review will summarize our current molecular knowledge on phosphoinositide kinase regulation, and how this information is being used to generate novel small molecule inhibitors as potential therapeutics.

Structural insights into the interaction of three Y-shaped ligands with PI3Kα.

Class IA phosphoinositide 3-kinase alpha (PI3Kα) is an important drug target because it is one of the most frequently mutated proteins in human cancers. However, small molecule inhibitors currently on the market or under development have safety concerns due to a lack of selectivity. Therefore, other chemical scaffolds or unique mechanisms of catalytic kinase inhibition are needed. Here, we report the cryo-electron microscopy structures of wild-type PI3Kα, the dimer of p110α and p85α, in complex with three Y-shaped ligands [cpd16 (compound 16), cpd17 (compound 17), and cpd18 (compound 18)] of different affinities and no inhibitory effect on the kinase activity. Unlike ATP-competitive inhibitors, cpd17 adopts a Y-shaped conformation with one arm inserted into a binding pocket formed by R770 and W780 and the other arm lodged in the ATP-binding pocket at an angle that is different from that of the ATP phosphate tail. Such a special interaction induces a conformation of PI3Kα resembling that of the unliganded protein. These observations were confirmed with two isomers (cpd16 and cpd18). Further analysis of these Y-shaped ligands revealed the structural basis of differential binding affinities caused by stereo- or regiochemical modifications. Our results may offer a different direction toward the design of therapeutic agents against PI3Kα.

PI3KCIIα-Dependent Autophagy Program Protects From Endothelial Dysfunction and Atherosclerosis in Response to Low Shear Stress in Mice.

BACKGROUND: The ability to respond to mechanical forces is a basic requirement for maintaining endothelial cell (ECs) homeostasis, which is continuously subjected to low shear stress (LSS) and high shear stress (HSS). In arteries, LSS and HSS have a differential impact on EC autophagy processes. However, it is still unclear whether LSS and HSS differently tune unique autophagic machinery or trigger specific autophagic responses in ECs. METHODS: Using fluid flow system to generate forces on EC and multiscale imaging analyses on ApoE-/- mice whole arteries, we studied the cellular and molecular mechanism involved in autophagic response to LSS or HSS on the endothelium. RESULTS: We found that LSS and HSS trigger autophagy activation by mobilizing specific autophagic signaling modules. Indeed, LSS-induced autophagy in endothelium was independent of the class III PI3K (phosphoinositide 3-kinase) VPS34 (vacuolar sorting protein 34) but controlled by the α isoform of class II PI3K (phosphoinositide 3-kinase class II α [PI3KCIIα]). Accordingly, reduced PI3KCIIα expression in ApoE-/- mice (ApoE-/-PI3KCIIα+/-) led to EC dysfunctions associated with increased plaque deposition in the LSS regions. Mechanistically, we revealed that PI3KCIIα inhibits mTORC1 (mammalian target of rapamycin complex 1) activation and that rapamycin treatment in ApoE-/-PI3KCIIα+/- mice specifically rescue autophagy in arterial LSS regions. Finally, we demonstrated that absence of PI3KCIIα led to decreased endothelial primary cilium biogenesis in response to LSS and that ablation of primary cilium mimics PI3KCIIα-decreased expression in EC dysfunction, suggesting that this organelle could be the mechanosensor linking PI3KCIIα and EC homeostasis. CONCLUSIONS: Our data reveal that mechanical forces variability within the arterial system determines EC autophagic response and supports a central role of PI3KCIIα/mTORC1 axis to prevent EC dysfunction in LSS regions.

ATP-competitive inhibitors of PI3K enzymes demonstrate an isoform selective dual action by controlling membrane binding.

PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and Biolayer Interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites.

Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.

Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα.

Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.

Fibroblast growth factor 2 enhances BMSC stemness through ITGA2-dependent PI3K/AKT pathway activation.

Bone marrow-derived mesenchymal stem cells (BMSC) are promising cellular reservoirs for treating degenerative diseases, tissue injuries, and immune system disorders. However, the stemness of BMSCs tends to decrease during in vitro cultivation, thereby restricting their efficacy in clinical applications. Consequently, investigating strategies that bolster the preservation of BMSC stemness and maximize therapeutic potential is necessary. Transcriptomic and single-cell sequencing methodologies were used to perform a comprehensive examination of BMSCs with the objective of substantiating the pivotal involvement of fibroblast growth factor 2 (FGF2) and integrin alpha 2 (ITGA2) in stemness regulation. To investigate the impact of these genes on the BMSC stemness in vitro, experimental approaches involving loss and gain of function were implemented. These approaches encompassed the modulation of FGF2 and ITGA2 expression levels via small interfering RNA and overexpression plasmids. Furthermore, we examined their influence on the proliferation and differentiation capacities of BMSCs, along with the expression of stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, and sex determining region Y-box 2. Transcriptomic analyzes successfully identified FGF2 and ITGA2 as pivotal genes responsible for regulating the stemness of BMSCs. Subsequent single-cell sequencing revealed that elevated FGF2 and ITGA2 expression levels within specific stem cell subpopulations are closely associated with stemness maintenance. Moreover, additional in vitro experiments have convincingly demonstrated that FGF2 effectively enhances the BMSC stemness by upregulating ITGA2 expression, a process mediated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. This conclusion was supported by the observed upregulation of stemness markers following the induction of FGF2 and ITGA2. Moreover, administration of the BEZ235 pathway inhibitor resulted in the repression of stemness transcription factors, suggesting the substantial involvement of the PI3K/AKT pathway in stemness preservation facilitated by FGF2 and ITGA2. This study elucidates the involvement of FGF2 in augmenting BMSC stemness by modulating ITGA2 and activating the PI3K/AKT pathway. These findings offer valuable contributions to stem cell biology and emphasize the potential of manipulating FGF2 and ITGA2 to optimize BMSCs for therapeutic purposes.

Transmembrane protein 176B regulates amino acid metabolism through the PI3K-Akt-mTOR signaling pathway and promotes gastric cancer progression.

BACKGROUND: The present study aimed to investigate the expression level, biological function, and underlying mechanism of transmembrane protein 176B (TMEM176B) in gastric cancer (GC). METHODS: TMEM176B expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). The function of TMEM176B was determined by various in vitro assays including colony formation, 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and flow cytometry. Bioinformatics techniques were then used to elucidate the signaling pathways associated with TMEM176B activity. Tumor formation experiments were conducted on nude mice for in vivo validation of the preceding findings. TMEM176B expression was cross-referenced to clinicopathological parameters and survival outcomes. RESULTS: It was observed that TMEM176B was overexpressed in GC cells and tissues. Targeted TMEM176B abrogation inhibited colony formation, proliferation, migration, and invasion but promoted apoptosis in GC cell lines while TMEM176B overexpression had the opposite effects. Subsequent experimental validation disclosed an association between TMEM176B and the phosphatidylinositol 3-carboxykinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling axis. Moreover, TMEM176B affects GC cancer progression by regulating asparagine synthetase (ASNS). The in vivo assays confirmed that TMEM176B is oncogenic and the clinical data revealed a connection between TMEM176B expression and the clinicopathological determinants of GC. CONCLUSION: The foregoing results suggest that TMEM176B significantly promotes the development of gastric cancer and is an independent prognostic factor of it.

PI3Kγδ inhibition suppresses key disease features in a rat model of asthma.

BACKGROUND: Two isoforms of Phosphoinositide 3-kinase (PI3K), p110γ and p110δ, are predominantly expressed in leukocytes and represent attractive therapeutic targets for the treatment of allergic asthma. The study aim was to assess the impact of administration of an inhaled PI3Kγδ inhibitor (AZD8154) in a rat model of asthma. METHODS: Firstly, we checked that the tool compound, AZD8154, inhibited rat PI3K γ & δ kinases using rat cell-based assays. Subsequently, a time-course study was conducted in a rat model of asthma to assess PI3K activity in the lung and how it is temporally associated with other key transcription pathways and asthma like features of the model. Finally, the impact on lung dosed AZD8154 on target engagement, pathway specificity, airway inflammation and lung function changes was assessed. RESULTS: Data showed that AZD8154 could inhibit rat PI3K γ & δ isoforms and, in a rat model of allergic asthma the PI3K pathway was activated in the lung. Intratracheal administration of AZD8154 caused a dose related suppression PI3K pathway activation (reduction in pAkt) and unlike after budesonide treatment, STAT and NF-κB pathways were not affected by AZD8154. The suppression of the PI3K pathway led to a marked inhibition of airway inflammation and reduction in changes in lung function. CONCLUSION: These data show that a dual PI3Kγδ inhibitor suppress key features of disease in a rat model of asthma to a similar degree as budesonide and indicate that dual PI3Kγδ inhibition may be an effective treatment for people suffering from allergic asthma.

Fluoxetine exerts anti-inflammatory effects on human epidermal keratinocytes and suppresses their endothelin release.

Fluoxetine is a safe antidepressant with remarkable anti-inflammatory actions; therefore, we aimed to investigate its effects on immortalized (HaCaT) as well as primary human epidermal keratinocytes in a polyinosinic-polycytidylic acid (p(I:C))-induced inflammatory model. We found that a non-cytotoxic concentration (MTT-assay, CyQUANT-assay) of fluoxetine significantly suppressed p(I:C)-induced expression and release of several pro-inflammatory cytokines (Q-PCR, cytokine array, ELISA), and it decreased the release of the itch mediator endothelins (ELISA). These effects were not mediated by the inhibition of the NF-κB or p38 MAPK pathways (western blot), or by the suppression of the p(I:C)-induced elevation of mitochondrial ROS production (MitoSOX Red labeling). Instead, unbiased activity profiling revealed that they were most likely mediated via the inhibition of the phosphoinositide 3-kinase (PI3K) pathway. Importantly, the PI3K-inhibitor GDC0941 fully mimicked the effects of fluoxetine (Q-PCR, ELISA). Although fluoxetine was able to occupy the binding site of GDC0941 (in silico molecular docking), and exerted direct inhibitory effect on PI3K (cell-free PI3K activity assay), it exhibited much lower potency and efficacy as compared to GDC0941. Finally, RNA-Seq analysis revealed that fluoxetine deeply influenced the transcriptional alterations induced by p(I:C)-treatment, and exerted an overall anti-inflammatory activity. Collectively, our findings demonstrate that fluoxetine exerts potent anti-inflammatory effects, and suppresses the release of the endogenous itch mediator endothelins in human keratinocytes, most likely via interfering with the PI3K pathway. Thus, clinical studies are encouraged to explore whether the currently reported beneficial effects translate in vivo following its topical administration in inflammatory and pruritic dermatoses.

Ilicicolin C suppresses the progression of prostate cancer by inhibiting PI3K/AKT/mTOR pathway.

Aberrant activation of the PI3K/AKT pathway is a driving factor in the development of prostate cancer. Therefore, inhibiting the function of the PI3K/AKT signaling pathway is a strategy for the treatment of prostate cancer. Ilicicolin C is an ascochlorin derivative isolated from the coral-derived fungus Acremonium sclerotigenum GXIMD 02501. Which has anti-inflammatory activity, but its activity against prostate cancer has not yet been elucidated. MTT assay, plate clone-formation assay, flow cytometry and real-time cell analysis technology were used to detect the effects of ilicicolin C on cell viability, proliferation, apoptosis and migration of prostate cancer cells. Molecular docking software and surface plasmon resonance technology were used to analyze the interaction between ilicicolin C and PI3K/AKT proteins. Western blot assay was performed to examine the changes in protein expression. Finally, QikProp software was used to simulate the process of ilicicolin C in vivo, and a zebrafish xenograft model was used to further verify the anti-prostate cancer activity of ilicicolin C in vivo. Ilicicolin C showed cytotoxic effects on prostate cancer cells, with the most significant effect on PC-3 cells. Ilicicolin C inhibited proliferation and migration of PC-3 cells. It could also block the cell cycle and induce apoptosis in PC-3 cells. In addition, ilicicolin C could bind to PI3K/AKT proteins. Furthermore, ilicicolin C inhibited the expression of PI3K, AKT and mTOR proteins and could also regulate the expression of downstream proteins in the PI3K/AKT/mTOR signaling pathway. Moreover, the calculations speculated that ilicicolin C was well absorbed orally, and the zebrafish xenograft model confirmed the in vivo anti-prostate cancer effect of ilicicolin C. Ilicicolin C emerges as a promising marine compound capable of inducing apoptosis of prostate cancer cells by counteracting the aberrant activation of PI3K/AKT/mTOR, suggesting that ilicicolin C may be a viable candidate for anti-prostate cancer drug development. These findings highlight the potential of ilicicolin C against prostate cancer and shed light on its mechanism of action.

The PI3K/Akt signaling axis and type 2 diabetes mellitus (T2DM): From mechanistic insights into possible therapeutic targets.

Type 2 diabetes mellitus (T2DM) is an immensely debilitating chronic disease that progressively undermines the well-being of various bodily organs and, indeed, most patients succumb to the disease due to post-T2DM complications. Although there is evidence supporting the activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway by insulin, which is essential in regulating glucose metabolism and insulin resistance, the significance of this pathway in T2DM has only been explored in a few studies. The current review aims to unravel the mechanisms by which different classes of PI3Ks control the metabolism of glucose; and also to discuss the original data obtained from international research laboratories on this topic. We also summarized the role of the PI3K/Akt signaling axis in target tissues spanning from the skeletal muscle to the adipose tissue and liver. Furthermore, inquiries regarding the impact of disrupting this axis on insulin function and the development of insulin resistance have been addressed. We also provide a general overview of the association of impaired PI3K/Akt signaling pathways in the pathogenesis of the most prevalent diabetes-related complications. The last section provides a special focus on the therapeutic potential of this axis by outlining the latest advances in active compounds that alleviate diabetes via modulation of the PI3K/Akt pathway. Finally, we comment on the future research aspects in which the field of T2DM therapies using PI3K modulators might be developed.

Inhibition of phosphoinositide 3-kinase activity attenuates neutrophilic airway inflammation and inhibits pyrin domain-containing 3 inflammasome activation in an ovalbumin-lipopolysaccharide-induced asthma murine model.

BACKGROUND: Existing investigations suggest that the blockade of phosphoinositide 3-kinase (PI3K) activity contributes to inflammatory solution in allergic asthma, but whether this inhibition directly attenuates neutrophilic airway inflammation in vivo is still unclear. We explored the pharmacological effects of LY294002, a specific inhibitor of PI3K on the progression of neutrophilic airway inflammation and investigated the underlying mechanism. METHODS AND RESULTS: Female C57BL/6 mice were intranasally sensitized with ovalbumin (OVA) together with lipopolysaccharide (LPS) on days 0 and 6, and challenged with OVA on days 14-17 to establish a neutrophilic airway disease model. In the challenge phase, a subset of mice was treated intratracheally with LY294002. We found that treatment of LY294002 attenuates clinic symptoms of inflammatory mice. Histological studies showed that LY294002 significantly inhibited inflammatory cell infiltration and mucus production. The treatment also significantly inhibited OVA-LPS induced increases in inflammatory cell counts, especially neutrophil counts, and IL-17 levels in bronchoalveolar lavage fluid (BALF). LY294002 treated mice exhibited significantly increased IL-10 levels in BALF compared to the untreated mice. In addition, LY294002 reduced the plasma concentrations of IL-6 and IL-17. The anti-inflammatory effects of LY29402 were correlated with the downregulation of NLRP3 inflammasome. CONCLUSIONS: Our findings suggested that LY294002 as a potential pharmacological target for neutrophilic airway inflammation.

Design, synthesis and bioevaluation of dual EGFR-PI3Kα inhibitors for potential treatment of NSCLC.

Aberrant activation or mutation of the EGFR-PI3K-Akt-mTOR signaling pathway has been implicated in a wide range of human cancers, especially non-small-cell lung cancer (NSCLC). Thus, dual inhibition of EGFR and PI3K has been investigated as a promising strategy to address acquired drug resistance resulting from the use of tyrosine kinase inhibitors. A series of dual EGFR/PI3Kα inhibitors was synthesized using pharmacophore hybridization of the third-generation EGFR inhibitor olmutinib and the PI3Kα selective inhibitor TAK-117. The optimal compound 30k showed potent kinase inhibitory activities with IC50 values of 3.6 and 30.0 nM against EGFRL858R/T790M and PI3Kα, respectively. Compound 30k exhibited a significant antiproliferative effect in NCI-H1975 cells with a higher selectivity profile than olmutinib. The potential antitumor mechanism, molecular binding modes, and in vitro metabolic stability of compound 30k were also clarified.

Chemical analogue based drug design for cancer treatment targeting PI3K: integrating machine learning and molecular modeling.

Cancer is a generic term for a group of disorders defined by uncontrolled cell growth and the potential to invade or spread to other parts of the body. Gene and epigenetic alterations disrupt normal cellular control, leading to abnormal cell proliferation, resistance to cell death, blood vessel development, and metastasis (spread to other organs). One of the several routes that play an important role in the development and progression of cancer is the phosphoinositide 3-kinase (PI3K) signaling pathway. Moreover, the gene PIK3CG encodes the catalytic subunit gamma (p110γ) of phosphoinositide 3-kinase (PI3Kγ), a member of the PI3K family. Therefore, in this study, PIK3CG was targeted to inhibit cancer by identifying a novel inhibitor through computational methods. The study screened 1015 chemical fragments against PIK3CG using machine learning-based binding estimation and docking to select the potential compounds. Later, the analogues were generated from the selected hits, and 414 analogues were selected, which were further screened, and as most potential candidates, three compounds were obtained: (a) 84,332, 190,213, and 885,387. The protein-ligand complex's stability and flexibility were then investigated by dynamic modeling. The 100 ns simulation revealed that 885,387 exhibited the steadiest deviation and constant creation of hydrogen bonds. Compared to the other compounds, 885,387 demonstrated a superior binding free energy (ΔG = -18.80 kcal/mol) with the protein when the MM/GBSA technique was used. The study determined that 885,387 showed significant therapeutic potential and justifies further experimental investigation as a possible inhibitor of the PIK3CG target implicated in cancer.

A deep learning-based theoretical protocol to identify potentially isoform-selective PI3Kα inhibitors.

Phosphoinositide 3-kinase alpha (PI3Kα) is one of the most frequently dysregulated kinases known for their pivotal role in many oncogenic diseases. While the side effects linked to existing drugs against PI3Kα-induced cancers provide an avenue for further research, the significant structural conservation among PI3Ks makes it extremely difficult to develop new isoform-selective PI3Kα inhibitors. Embracing this challenge, we herein designed a hybrid protocol by integrating machine learning (ML) with in silico drug-designing strategies. A deep learning classification model was developed and trained on the physicochemical descriptors data of known PI3Kα inhibitors and used as a screening filter for a database of small molecules. This approach led us to the prediction of 662 compounds showcasing appropriate features to be considered as PI3Kα inhibitors. Subsequently, a multiphase molecular docking was applied to further characterize the predicted hits in terms of their binding affinities and binding modes in the targeted cavity of the PI3Kα. As a result, a total of 12 compounds were identified whereas the best poses highlighted the efficiency of these ligands in maintaining interactions with the crucial residues of the protein to be targeted for the inhibition of associated activity. Notably, potential activity of compound 12 in counteracting PI3Kα function was found in a previous in vitro study. Following the drug-likeness and pharmacokinetic characterizations, six compounds (compounds 1, 2, 3, 6, 7, and 11) with suitable ADME-T profiles and promising bioavailability were selected. The mechanistic studies in dynamic mode further endorsed the potential of identified hits in blocking the ATP-binding site of the receptor with higher binding affinities than the native inhibitor, alpelisib (BYL-719), particularly the compounds 1, 2, and 11. These outcomes support the reliability of the developed classification model and the devised computational strategy for identifying new isoform-selective drug candidates for PI3Kα inhibition.

circZNF532 promotes endothelial-to-mesenchymal transition in diabetic retinopathy by recruiting TAF15 to stabilize PIK3CD.

Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.

Macropinocytosis: mechanisms and regulation.

Macropinocytosis is defined as an actin-dependent but coat- and dynamin-independent endocytic uptake process, which generates large intracellular vesicles (macropinosomes) containing a non-selective sampling of extracellular fluid. Macropinocytosis provides an important mechanism of immune surveillance by dendritic cells and macrophages, but also serves as an essential nutrient uptake pathway for unicellular organisms and tumor cells. This review examines the cell biological mechanisms that drive macropinocytosis, as well as the complex signaling pathways - GTPases, lipid and protein kinases and phosphatases, and actin regulatory proteins - that regulate macropinosome formation, internalization, and disposition.

The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.

IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitro binding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.

Cryo-EM structures of PI3Kα reveal conformational changes during inhibition and activation.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases essential for growth and metabolism. Their aberrant activation is associated with many types of cancers. Here we used single-particle cryoelectron microscopy (cryo-EM) to determine three distinct conformations of full-length PI3Kα (p110α-p85α): the unliganded heterodimer PI3Kα, PI3Kα bound to the p110α-specific inhibitor BYL-719, and PI3Kα exposed to an activating phosphopeptide. The cryo-EM structures of unbound and of BYL-719-bound PI3Kα are in general accord with published crystal structures. Local deviations are presented and discussed. BYL-719 stabilizes the structure of PI3Kα, but three regions of low-resolution extra density remain and are provisionally assigned to the cSH2, BH, and SH3 domains of p85. One of the extra density regions is in contact with the kinase domain blocking access to the catalytic site. This conformational change indicates that the effects of BYL-719 on PI3Kα activity extend beyond competition with adenosine triphosphate (ATP). In unliganded PI3Kα, the DFG motif occurs in the "in" and "out" positions. In BYL-719-bound PI3Kα, only the DFG-in position, corresponding to the active conformation of the kinase, was observed. The phosphopeptide-bound structure of PI3Kα is composed of a stable core resolved at 3.8 Å. It contains all p110α domains except the adaptor-binding domain (ABD). The p85α domains, linked to the core through the ABD, are no longer resolved, implying that the phosphopeptide activates PI3Kα by fully releasing the niSH2 domain from binding to p110α. The structures presented here show the basal form of the full-length PI3Kα dimer and document conformational changes related to the activated and inhibited states.