Pleckstrin Homology Research Articles

TheSTX17-SNAP47-VAMP7/VAMP8 complex is the default SNARE complex mediating autophagosome-lysosome fusion.

Autophagosome-lysosome fusion mediated by SNARE complexes is an essential step in autophagy. Two SNAP29-containing SNARE complexes have been extensively studied in starvation-induced bulk autophagy, while the relevant SNARE complexes in other types of autophagy occurring under non-starvation conditions have been overlooked. Here, we found that autophagosome-lysosome fusion in selective autophagy under non-starvation conditions does not require SNAP29-containing SNARE complexes, but requires the STX17-SNAP47-VAMP7/VAMP8 SNARE complex. Further, the STX17-SNAP47-VAMP7/VAMP8 SNARE complex also functions in starvation-induced autophagy. SNAP47 is recruited to autophagosomes following concurrent detection of ATG8s and PI(4,5)P2 via its Pleckstrin homology domain. By contrast, SNAP29-containing SNAREs are excluded from selective autophagy due to inactivation by O-GlcNAcylation under non-starvation conditions. These findings depict a previously unknown, default SNARE complex responsible for autophagosome-lysosome fusion in both selective and bulk autophagy, which could guide research and therapeutic development in autophagy-related diseases.

The Abl-interactor Abi suppresses the function of the BRAG2 GEF family member Schizo.

Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Consequently, BRAG proteins have been implicated to regulate the surface levels of adhesive and signaling receptors. However, not much is known about the mechanism leading to the regulation of these surface proteins. In this study, we found that the Drosophila BRAG GEF Schizo interacts physically with the Abl-interactor (Abi). schizo mutants display severe defects in myoblast fusion during syncytial muscle formation and show increased amounts of the cell adhesion protein N-cadherin. We demonstrate that the schizo myoblast fusion phenotype can be rescued by the expression of the Schizo GEF (Sec7) and membrane-binding (pleckstrin homology) domain. Furthermore, the expression of the Sec7-PH domain in a wild-type background decreases the amounts of N-cadherin and impairs myoblast fusion. These findings support the notion that the Sec7-PH domain serves as a constitutive-active form of Schizo. Using a yeast-two hybrid assay, we show that the SH3 domain of Abi interacts with the N-terminal region of Schizo. This region is also able to bind to the cytodomain of the cell adhesion molecule N-cadherin. To shed light on the function of Schizo and Abi in N-cadherin removal, we employed epistasis experiments in different developmental contexts of Drosophila. These studies point towards a new model for the regulation of Schizo. We propose that the binding of Abi to the N-terminal part of Schizo antagonizes Schizo function to inhibit N-cadherin removal.

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, β2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of β2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

The role of Phafin proteins in cell signaling pathways and diseases.

Membrane-associated proteins are important membrane readers that mediate and facilitate the signaling and trafficking pathways in eukaryotic membrane-bound compartments. The protein members in the Phafin family are membrane readers containing two phosphoinositide recognition domains: the Pleckstrin Homology domain and the FYVE (Fab1, YOTB, Vac1, and early endosome antigen 1) domain. Phafin proteins, categorized into two subfamilies, Phafin1 and Phafin2, associate with cellular membranes through interactions involving membrane-embedded phosphoinositides and phosphoinositide-binding domains. These membrane-associated Phafin proteins play pivotal roles by recruiting binding partners and forming complexes, which contribute significantly to apoptotic, autophagic, and macropinocytotic pathways. Elevated expression levels of Phafin1 and Phafin2 are observed in various cancers. A recent study highlights a significant increase in Phafin1 protein levels in the lungs of idiopathic pulmonary fibrosis patients compared to normal subjects, suggesting a crucial role for Phafin1 in the pathogenesis of pulmonary fibrosis. Additionally, phosphatidylinositol-3-phosphate-binding 2 (Pib2), a close relative of the Phafin1 protein, functions as an amino acid sensor activating the TOCR1 pathway in yeasts. This review focuses on delineating the involvement of Phafin proteins in cellular signaling and their implications in diseases and briefly discusses the latest research findings concerning Pib2.

The roles of phosphorylation of signaling proteins in the prognosis of acute myeloid leukemia.

Signaling pathways of Retinoblastoma (Rb) protein, Akt-kinase, and Erk-kinase (extracellular signal-regulated kinase) have an important role in the pathogenesis of acute myeloid leukemia. Constitutive activation of these proteins by phosphorylation contributes to cell survival by regulation of cell cycle, proliferation and proapoptotic signaling processes. According to previous data phosphorylated forms of these proteins represent a worse outcome for cancer patients. We investigated the presence of phosphorylated Rb (P-Rb), Akt (P-Akt) and Erk (P-Erk) proteins by Western blot technique using phospho-specific antibodies in bone marrow or peripheral blood samples of 69 AML patients, 36 patients with myelodysplastic syndrome (MDS) and 10 healthy volunteers. Expression level of PTEN (Phosphatase and tensin homolog) and PHLPP (PH domain and leucine-rich repeat Protein Phosphatase) phosphatases, the negative regulators of Akt kinase pathway were also examined. We tested the effect of these proteins on survival and on the correlation with known prognostic features in AML. We found 46.3% of AML patients had detectable P-Rb, 34.7% had P-Akt and 28.9% had P-Erk protein. 66.1% of patients expressing PTEN, 38.9% PHLPP, 37.2% both PTEN and PHLPP and 32.2% neither PTEN nor PHLPP phosphatases. Compared to nucleophosmin mutation (NPMc) negative samples P-Erk was significantly less in nucleophosmin mutated patients, P-Rb was significantly less in patients' group with more than 30G/L peripheral leukocyte count by diagnosis. PHLPP was significantly present in FAB type M5. The expression of P-Rb represented significant better overall survival (OS), while P-Akt represented significantly worse event-free survival (EFS) in unfavorable cytogenetics patients. The presence of both PHLPP and PTEN phosphatases contributes to better OS and EFS, although the differences were not statistically significant. We confirmed significant positive correlation between P-Akt and PHLPP. Assessing the phosphorylation of Rb, Akt and Erk may define a subgroup of AML patients who would benefit especially from new targeted treatment options complemented the standard chemotherapy, and it may contribute to monitoring remission, relapse or progression of AML.

Adiponectin Resistance in Obesity: Adiponectin Leptin/Insulin Interaction.

The adiponectin (APN) levels in obesity are negatively correlated with chronic subclinical inflammation markers. The hypertrophic adipocytes cause obesity-linked insulin resistance and metabolic syndrome. Furthermore, macrophage polarization is a key determinant regulating adiponectin receptor (AdipoR1/R2) expression and differential adiponectin-mediated macrophage inflammatory responses in obese individuals. In addition to decrease in adiponectin concentrations, the decline in AdipoR1/R2 messenger ribonucleic acid (mRNA) expression leads to a decrement in adiponectin binding to cell membrane, and this turns into attenuation in the adiponectin effects. This is defined as APN resistance, and it is linked with insulin resistance in high-fat diet-fed subjects. The insulin-resistant group has a significantly higher leptin-to-APN ratio. The leptin-to-APN ratio is more than twofold higher in obese individuals. An increase in expression of AdipoRs restores insulin sensitivity and β-oxidation of fatty acids via triggering intracellular signal cascades. The ratio of high molecular weight to total APN is defined as the APN sensitivity index (ASI). This index is correlated to insulin sensitivity. Homeostasis model of assessment (HOMA)-APN and HOMA-estimated insulin resistance (HOMA-IR) are the most suitable methods to estimate the metabolic risk in metabolic syndrome. While morbidly obese patients display a significantly higher plasma leptin and soluble (s)E-selectin concentrations, leptin-to-APN ratio, there is a significant negative correlation between leptin-to-APN ratio and sP-selectin in obese patients. When comparing the metabolic dysregulated obese group with the metabolically healthy obese group, postprandial triglyceride clearance, insulin resistance, and leptin resistance are significantly delayed following the oral fat tolerance test in the first group. A neuropeptide, Spexin (SPX), is positively correlated with the quantitative insulin sensitivity check index (QUICKI) and APN. APN resistance together with insulin resistance forms a vicious cycle. Despite normal or high APN levels, an impaired post-receptor signaling due to adaptor protein-containing pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper motif 1 (APPL1)/APPL2 may alter APN efficiency and activity. However, APPL2 blocks adiponectin signaling through AdipoR1 and AdipoR2 because of the competitive inhibition of APPL1. APPL1, the intracellular binding partner of AdipoRs, is also an important mediator of adiponectin-dependent insulin sensitization. The elevated adiponectin levels with adiponectin resistance are compensatory responses in the condition of an unusual discordance between insulin resistance and APN unresponsiveness. Hypothalamic recombinant adeno-associated virus (rAAV)-leptin (Lep) gene therapy reduces serum APN levels, and it is a more efficient strategy for long-term weight maintenance.

ORP9-PH domain-based fluorescent reporters for visualizing phosphatidylinositol 4-phosphate dynamics in living cells

New fluorescent reporters based on the ORP9 PH domain visualize phosphatidylinositol 4-phosphate (PI4P) dynamics at multiple cellular locations with high specificity and contrast in live-cell imaging.

Molecular Analysis of Alternative Spliced Transcripts of Equine Pleckstrin Homology and RhoGEF Domain Containing G1 (PLEKHG1) gene

Since horse industry get bigger with horse racing and horse riding, athletic performance become most important trait on horses, but the molecular analysis and regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we conducted RNA-sequencing analysis with muscle and blood samples by exercise in Thoroughbreds. Through RNA-sequencing, we identified Pleckstrin Homology and RhoGEF Domain Containing G1 (PLEKHG1) gene expressed differentially by alternative spliced isoforms in skeletal muscle during exercise. In this study, we conducted reverse transcriptase polymerase chain reaction (RT-PCR) to validate two isoforms of equine PLEKHG1 transcripts (PLEKHG1a, PLEKHG1b), and cloned the transcripts to confirm the sequences. Additionally, we validated expression pattern of PLEKHG1a (long form of transcript) and PLEKHG1b (short form of transcript) in horse tissue by quantitative RT-PCR (qRT-PCR). Prediction of protein structure of these isoforms revealed two putative phosphorylation sites at the amino acid sequences encoded in exon 7, which is deleted in PLEKHG1b. Expression pattern of PLEKHG1a and PLEKHG1b shows cross expressed pattern as RNA-sequencing data. PLEKHG1a increased after exercise whereas PLEKHG1b decreased after exercise. Collectively, it is assumed that the expression patterns of PLEKHG1a and PLEKHG1b transcripts would be involved in regulation of myogenic differentiation and myogenic proliferation through the Ras signaling pathway. Further study should be necessary to uncover biological function(s) and significance of the alternative splicing isoforms in equine skeletal muscle

Potential therapeutic applications of targeting signal-transducing adaptor protein-2 in autoimmune diseases

Adaptor proteins are involved in various immune responses via the modulation of many signaling pathways. Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains typical domains such as the pleckstrin homology (PH) domain, Src homology domain, and a proline-rich region from the N-terminal region. In T cells, STAP-2 positively regulates T cell receptor (TCR)-mediated signaling by associating with CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) and lymphocyte-specific protein tyrosine kinase (LCK). Therefore, a peptide that inhibits the interaction between STAP-2 and CD3ζ ITAMs is likely to suppress TCR-mediated T cell activation, as well as T cell-mediated diseases. As expected, the peptide successfully inhibited the STAP-2/CD3ζ ITAM interaction and suppressed TCR-mediated signaling, cell proliferation, and interleukin (IL)-2 production in human/murine T cells. Furthermore, this inhibitor suppressed the pathogenesis of experimental autoimmune encephalomyelitis (EAE), which is widely recognized as a mouse model of multiple sclerosis, via the downregulation of T cell activation and infiltration of T helper (Th) 1/Th17 cells. These results suggest a new strategy for the treatment of multiple sclerosis and other immune diseases.

EXPLORING THE POTENTIAL OF PHLPP INHIBITORS FOR OSTEOARTHRITIS TREATMENT

Abstract Osteoarthritis (OA) is a degenerative joint disease characterized by pain, stiffness, and limited joint motion affecting 7% of the world’s population. PH domain leucine-rich repeat protein phosphates (PHLPPs) have been identified as modifiable targets for cartilage regeneration and joint pain. PHLPP1 and PHLPP2 are abnormally expressed in osteoarthritic cartilage and sensory neurons. PHLPP1-/- mice are protected from cartilage degradation and pain-related behaviors (allodynia and reduced mobility) after surgery that destabilizes the medial meniscus (DMM). Here, we investigate the effects of PHLPP inhibitors (i) on human chondrocytes and the effect of Phlpp1 deletion on cell populations within lumbar dorsal root ganglia (DRGs). Immunohistochemistry was performed on mouse DRGs by cryosectioning fixed L3-5 DRGs and using primary antibodies against Fabp7, Anti tubulin beta 3, and PGP9.5/UCHL1. Human chondrocytes were subjected to Cycloheximide chase experiments and treated with PHLPPi (NSC117079 or NSC45586), followed by Western blot analysis to assess protein levels including Phlpp1, Phlpp2, Sox9, p-AKT (Ser473), total AKT, and B-actin, at various time points. We found that PHLPPi prolongs the phosphorylated AKT response when compared to controls and does not affect degradation of Phlpp1 or Phlpp2; additionally, NSC117079 increased production of cartilage extracellular matrix components (glycosaminoglycans and aggrecan) in over 90% of human articular cartilage explants from OA patients. These data advance our understanding of how PHLPPi function within chondrocytes and provide a basis toward understanding their activity and safety in vivo. Our results indicate that PHLPPi NSC117079 is a novel osteoarthritis disease-modifying drug candidate that may have palliative effects.

Anillin-related Mid1 as an adaptive and multimodal contractile ring anchoring protein: A simulation study

Cytokinesis of animal and fungi cells depends crucially on the anillin scaffold proteins. Fission yeast anillin-related Mid1 anchors cytokinetic ring precursor nodes to the membrane. However, it is unclear if both of its Pleckstrin Homology (PH) and C2 C-terminal domains bind to the membrane as monomers or dimers, and if one domain plays a dominant role. We studied Mid1 membrane binding with all-atom molecular dynamics near a membrane with yeast-like lipid composition. In simulations with the full C terminal region started away from the membrane, Mid1 binds through the disordered L3 loop of C2 in a vertical orientation, with the PH away from the membrane. However, a configuration with both C2 and PH initially bound to the membrane remains associated with the membrane. Simulations of C2-PH dimers show extensive asymmetric membrane contacts. These multiple modes of binding may reflect Mid1's multiple interactions with membranes, node proteins, and ability to sustain mechanical forces.

Comparative Serum Proteome Profiling of Canine Benign Prostatic Hyperplasia before and after Castration.

BPH is the most prevalent prostatic condition in aging dogs. Nevertheless, clinical diagnosis and management remain inconsistent. This study employed in-solution digestion coupled with nano-liquid chromatography tandem mass spectrometry to assess serum proteome profiling of dogs with BPH and those dogs after castration. Male dogs were divided into two groups; control and BPH groups. In the BPH group, each dog was evaluated at two time points: Day 0 (BF subgroup) and Day 30 after castration (AT subgroup). In the BF subgroup, three proteins were significantly upregulated and associated with dihydrotestosterone: solute carrier family 5 member 5, tyrosine-protein kinase, and FRAT regulator of WNT signaling pathway 1. Additionally, the overexpression of polymeric immunoglobulin receptors in the BF subgroup hints at its potential as a novel protein linked to the BPH development process. Conversely, alpha-1-B glycoprotein (A1BG) displayed significant downregulation in the BF subgroup, suggesting A1BG's potential as a predictive protein for canine BPH. Finasteride was associated with increased proteins in the AT subgroup, including apolipoprotein C-I, apolipoprotein E, apolipoprotein A-II, TAO kinase 1, DnaJ homolog subfamily C member 16, PH domain and leucine-rich repeat protein phosphatase 1, neuregulin 1, and pseudopodium enriched atypical kinase 1. In conclusion, this pilot study highlighted alterations in various serum proteins in canine BPH, reflecting different pathological changes occurring in this condition. These proteins could be a source of potential non-invasive biomarkers for diagnosing this disease.

Calcium influx promotes PLEKHG4B localization to cell-cell junctions and regulates the integrity of junctional actin filaments.

PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.

Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health.

Intervertebral disc (IVD) degeneration is associated with chronic back pain. We previously demonstrated that the phosphatase pleckstrin homology domain and leucine-rich repeat protein phosphatase (PHLPP) 1 was positively correlated with IVD degeneration and its deficiency decelerated IVD degeneration in both mouse IVDs and human nucleus pulposus (NP) cells. Small molecule PHLPP inhibitors may offer a translatable method to alleviate IVD degeneration. In this study, we tested the effectiveness of the two PHLPP inhibitors NSC117079 and NSC45586 in promoting a healthy NP phenotype. Tail IVDs of 5-month-old wildtype mice were collected and treated with NSC117079 or NSC45586 under low serum conditions ex vivo. Hematoxylin & eosin staining was performed to examine IVD structure and NP cell morphology. The expression of KRT19 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human NP cells were obtained from patients with IVD degeneration. The gene expression of KRT19, ACAN, SOX9, and MMP13 was analyzed via real time qPCR, and AKT phosphorylation and the protein expression of FOXO1 was analyzed via immunoblot. In a mouse IVD organ culture model, NSC45586, but not NSC117079, preserved vacuolated notochordal cell morphology and KRT19 expression while suppressing cell apoptosis, counteracting the degenerative changes induced by serum deprivation, especially in males. Likewise, in degenerated human NP cells, NSC45586 increased cell viability and the expression of KRT19, ACAN, and SOX9 and reducing the expression of MMP13, while NSC117079 treatment only increased KRT19 expression. Mechanistically, NSC45586 treatment increased FOXO1 protein expression in NP cells, and inhibiting FOXO1 offset NSC45586-induced regenerative potential, especially in males. Our study indicates that NSC45586 was effective in promoting NP cell health, especially in males, suggesting that PHLPP plays a key role in NP cell homeostasis and that NSC45586 might be a potential drug candidate in treating IVD degeneration.

ARAP1 negatively regulates stress fibers formation and metastasis in lung adenocarcinoma via controlling Rho signaling

Small GTPases regulate multiple important cellular behaviors and their activities are strictly controlled by a mass of regulators. The dysfunction or abnormal expression of small GTPases or their regulators was frequently observed in various cancers. Here, we analyzed the expression and prognostic correlation of several GTPases and related regulators based on the TCGA database and found that Ankyrin Repeat and PH Domain 1 (ARAP1), a GTPase activating protein (GAP), is reduced in lung adenocarcinoma tissues compared to normal tissues and displays a positive correlation with overall survival (OS) and progression-free survival (PFS) of patients with lung adenocarcinoma. qPCR and western blot verified that ARAP1 is frequently downregulated in lung adenocarcinoma tumor tissues and cancer cells, and its downregulation might be mediated by epigenetic modification. Moreover, metastatic assays showed that overexpression of ARAP1 significantly inhibits metastasis of lung adenocarcinoma in vitro and in vivo. We further demonstrated that Rho signaling inhibition, mediated by RhoGAP activity of ARAP1, majorly contributes to suppressing migration and invasion of lung adenocarcinoma cancer cells via inhibiting stress fibers formation. In summary, this study indicates that ARAP1 may serve as a potential prognostic predictor and a metastatic suppressor in lung adenocarcinoma via its RhoGAP activity.

The lack of homology domain and leucine rich repeat protein phosphatase 2 ameliorates visual impairment in rats with diabetic retinopathy through regulation of the AKT-GSK-3β-Nrf2 signal cascade

Pleckstrin homology domain and leucine rich repeat protein phosphatase 2 (PHLPP2) is an emerging player in diverse disorders. Our previous findings have documented that reducing PHLPP2 levels in cultured retinal ganglion cells protects against cellular damage caused by high glucose, indicating a possible link between PHLPP2 and diabetic retinopathy (DR). The present work was dedicated to the investigation of PHLPP2 in DR through in vivo experiments with rat models induced by intraperitoneal injection of streptozotocin. Compared to normal rats, the retinas of rats with DR exhibited a notable increase in the level of PHLPP2. The reduction of PHLPP2 levels in the retina was achieved by the intravitreal administration of adeno-associated viruses expressing specific shRNA targeting PHLPP2. Decreasing the expression of PHLPP2 ameliorated visual function impairment and improved the pathological changes of retina in DR rats. Moreover, decreasing the expression of PHLPP2 repressed the apoptosis, oxidative stress and proinflammatory response in the retinas of rats with DR. Reduction of PHLPP2 levels led to an increase in the levels of phosphorylated AKT and glycogen synthase kinase-3β (GSK-3β). Decreasing the expression of PHLPP2 resulted in increased activation of nuclear factor erythroid 2-related factor 2 (Nrf2), which was reversed by suppressing AKT. Notably, the protective effect of reducing PHLPP2 on DR was eliminated when Nrf2 was restrained. These observations show that the down-regulation of PHLPP2 has protective effects on DR by preserving the structure and function of the retina by regulating the AKT-GSK-3β-Nrf2 signal cascade. Therefore, targeting PHLPP2 may hold promise in the treatment of DR.

Myr-Arf1 conformational flexibility at the membrane surface sheds light on the interactions with ArfGAP ASAP1

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.

CXADR promote epithelial–mesenchymal transition in endometriosis by modulating AKT/GSK-3β signaling

ABSTRACT Endometriosis is a benign high prevalent disease exhibiting malignant features. However, the underlying pathogenesis and key molecules of endometriosis remain unclear. By integrating and analysis of existing expression profile datasets, we identified coxsackie and adenovirus receptor (CXADR), as a novel key gene in endometriosis. Based on the results of immunohistochemistry (IHC), we confirmed significant down-regulation of CXADR in ectopic endometrial tissues obtained from women with endometriosis compared with healthy controls. Further in vitro investigation indicated that CXADR regulated the stability and function of the phosphatases and AKT inhibitors PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2) and PTEN (phosphatase and tensin homolog). Loss of CXADR led to phosphorylation of AKT and glycogen synthase kinase-3β (GSK-3β), which resulted in stabilization of an epithelial–mesenchymal transition (EMT) factor, SNAIL1 (snail family transcriptional repressor 1). Therefore, EMT processs was induced, and the proliferation, migration and invasion of Ishikawa cells were enhanced. Over-expression of CXADR showed opposite effects. These findings suggest a previously undefined role of AKT/GSK-3β signaling axis in regulating EMT and reveal the involvement of a CXADR-induced EMT, in pathogenic progression of endometriosis.

A novel small molecule, CU05-1189, targeting the pleckstrin homology domain of PDK1 suppresses VEGF-mediated angiogenesis and tumor growth by blocking the Akt signaling pathway.

Inhibition of angiogenesis is considered a promising therapeutic approach for cancer treatment. Our previous genetic research showed that the use of a cell-penetrating peptide to inhibit the pleckstrin homology (PH) domain of 3-phosphoinositide-dependent kinase 1 (PDK1) was a viable approach to suppress pathological angiogenesis. Herein, we synthesized and characterized a novel small molecule, CU05-1189, based on our prior study and present evidence for the first time that this compound possesses antiangiogenic properties both in vitro and in vivo. The computational analysis showed that CU05-1189 can interact with the PH domain of PDK1, and it significantly inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, invasion, and tube formation in human umbilical vein endothelial cells without apparent toxicity. Western blot analysis revealed that the Akt signaling pathway was specifically inhibited by CU05-1189 upon VEGF stimulation, without affecting other VEGF receptor 2 downstream molecules or cytosolic substrates of PDK1, by preventing translocation of PDK1 to the plasma membrane. We also found that CU05-1189 suppressed VEGF-mediated vascular network formation in a Matrigel plug assay. More importantly, CU05-1189 had a good pharmacokinetic profile with a bioavailability of 68%. These results led to the oral administration of CU05-1189, which resulted in reduced tumor microvessel density and growth in a xenograft mouse model. Taken together, our data suggest that CU05-1189 may have great potential and be a promising lead as a novel antiangiogenic agent for cancer treatment.

Membrane-bound myosin IC drives the chiral rotation of the gliding actin filament around its longitudinal axis

Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. In this study, we evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P2 concentrations revealed that the rotational pitch was longer at lower PI(4,5)P2 concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane.