Pleckstrin Homology Research Articles

Scaffold Hopping Method for Design and Development of Potential Allosteric AKT Inhibitors.

Targeting AKT is a practical strategy for cancer therapy in many cancer types. Targeted inhibitors of AKT are attractive solutions for inhibiting the interconnected signaling pathways, like PI3K/Akt/mTOR. Allosteric inhibitors are more desirable among different classes of AKT inhibitors as they could be more specific with fewer off-target proteins. In this study, a ligand/structure-based pipeline was developed to design new allosteric AKT inhibitors by employing the core hopping method. Triciribine, a traditional allosteric AKT inhibitor was used as the template, and the FDA-approved kinase inhibitors for cancer treatment were considered as the cores. The allosteric site in the crystal structure of AKT1 was used to screen the designed compounds. The results were further evaluated using molecular docking, ADME/T analysis, molecular dynamics (MD) simulation, and binding free energy calculations. The outcomes introduced 24 newly designed inhibitors, amongst which three compounds C6, C20, and C16 showed remarkable binding affinity to AKT1. While the docking scores for triciribine was around -8.6 kcal/mol, the docking scores of these compounds were about -11 to -13 kcal/mol. The MD results indicated that designed compounds target the essential residues of the PH domain and kinase domain of AKT, such as Trp80, Thr211, Tyr272, Asp274, and Asp292. Scaffold hopping is a tremendous tool for designing novel anti-cancer agents by improving already known and potential drug compounds. The designed compounds are worth to be examined by experimental investigation in vitro and in vivo.

Zinc ions activate AKT and promote prostate cancer cell proliferation via disrupting AKT intramolecular interaction.

Prostate is a zinc rich organ and the physiological function of the abundant zinc ions is relatively less understood. AKT kinase is a pivotal regulator downstream of cytokines, growth factors and other extracellular stimuli, and the attachment of its PH domain to PtdIns-3,4,5-P3 (PIP3) and the subsequent phosphorylation of its kinase domain by PDPK1 are considered important for its activation. Herein, we report a regulatory mechanism of AKT kinase by zinc ions. Mechanistically, zinc ions directly bind to AKT and facilitate AKT activation through disrupting the interaction between PH and kinase domains within AKT molecule. Consistently, AKT1-H89A/E91A mutant (zinc-binding-deficient) fails to respond to zinc ions and exhibits strong interaction between PH and kinase domains, and it is less oncogenic in orthotopic xenograft model of prostate cancer. On the other hand, the AKT1-W80L mutant with minimum intra-molecular interaction between PH and kinase domains shows strong tumor promoting capacity although it could not be further stimulated by zinc ions. Overall, this study reveals a distinctive regulatory mechanism of AKT activation and implies a tumor promoting role of the zinc ions in prostate cancer.

Two novel antimicrobial peptides P33-57 and mP168-187 from zebrafish showing potent antibacterial activities

It is known that overuse or abuse of antibiotics has undoubtedly accelerated the global antibiotic resistance crisis, and long-time use of antibiotics may have adverse effects on the health of animal, human, and ecosystem. Therefore, it is necessary to find antibiotic alternatives that can be used in aquaculture and are non-toxic to the human. Here we clearly demonstrated that both the PH and FYVE domain of Plekhf2 in zebrafish have antibacterial properties and can interact with PGN in this study. Therefore, we screened four candidate peptides from the two domains. It was demonstrated that P152-172 and P168-187 had no obvious antibacterial activities, while P33-57 and mP168-187 had strong antibacterial activities, which may be used as antimicrobial peptides. Additionally, transmission electron microscopy experiment revealed that P33-57 and mP168-187 can destroy the cell wall of bacteria, thereby directly killing bacteria. Importantly, it was found that P33-57 and mP168-187 had no hemolysis to red blood cells and lacked cytotoxicity. In summary, P33-57 and mP168-187could be seen as antibacterial activity centers of PLEKHF2 and may be promising antimicrobial peptides to combat bacterial infections facing an antibiotic-resistance crisis.

Effect of Pathogenic Mutations on the Formation of High-Order Dynamin 2 Assemblies in Living Cells.

Mutations in dynamin 2 (DNM2) have been associated with two distinct movement disorders: Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). Most of these mutations are clustered in the pleckstrin homology domain (PHD), which engages in intramolecular interactions that limit dynamin self-assembly and GTPase activation. CNM mutations interfere with these intramolecular interactions and suppress the formation of the autoinhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for phosphoinositide binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations causes either one disease or the other, despite their close proximity within the PHD sequence. We previously reported that at least one CMT-causing mutant, lacking residues 555DEE557 (ΔDEE), displays the same inability to undergo autoinhibition as observed in CNM-linked mutants. Here, we show that both the DNM2ΔDEE and CNM-linked DNM2A618T mutants form larger and more stable structures on the plasma membrane than that of wild-type DNM2 (DNM2WT). However, DNM2A618T forms cytoplasmic inclusions at concentrations lower than those of either DNM2WT or DNM2ΔDEE, suggesting that CNM-linked mutations confer more severe gain-of-function properties than the ΔDEE mutation.

PHD-BAH Domain in ASH1L Could Recognize H3K4 Methylation and Regulate the Malignant Behavior of Cholangiocarcinoma.

Histone methyltransferase absent, small, or homeotic discs1-like (ASH1L) is composed of su(var)3-9, enhancer of zeste, trithorax (SET) domain, pleckstrin homology domain (PHD) domain, middle (MID) domain, and bromo adjacent homology (BAH) domain. The SET domain of ASH1L is known to mediate mediate H3K36 dimethylation (H3K36me2) modification. However, the specific functions of the PHD-BAH domain remain largely unexplored. This study aimed to explore the biological function of the PHD-BAH domain in ASH1L. We employed a range of techniques, including a prokaryotic fusion protein expression purification system, pull-down assay, Isothermal Titration Calorimetry (ITC), polymerase chain reaction (PCR), and sitedirected mutagenesis, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) gene editing, cell culture experiment, western blot, cell proliferation assay, and cell apoptosis test. The PHD-BAH domain in ASH1L preferentially binds to the H3K4me2 peptide over H3K4 monomethylation (H3K4me1) and H3K4 trimethylation (H3K4me3) peptide. Notably, the W2603A mutation within the PHD-BAH domain could disrupt the interaction with H3K4me2 in vitro. Compared with wild-type Cholangiocarcinoma (CHOL) cells, deletion of the PHD-BAH domain in ASH1L led to increased CHOL cell apoptosis and reduced cell proliferation (P < 0.001). Additionally, the W2603A mutation affected the regulation of the proteasome 20S subunit beta (PSMB) family gene set. W2603A mutation was crucial for the interaction between the PHD-BAH domain and the H3K4me2 peptide. ASH1L regulated CHOL cell survival and proliferation through its PHD-BAH domain by modulating the expression of the PSMB family gene set.

Patient Experience With Efanesoctocog Alfa for Severe Hemophilia A: Results From the XTEND-1 Phase 3 Clinical Study Exit Interviews

PurposeHemophilia A is a rare bleeding disorder that leads to recurrent hemarthrosis, which can ultimately result in reduced mobility and poor quality of life. Qualitative exit interviews provide insights into patient perspectives and support the interpretation of quantitative trial data, such as patient-reported outcome measures. In the Phase 3 XTEND-1 study (NCT04161495) of efanesoctocog alfa in participants with severe hemophilia A, exit interviews were conducted to understand pre- and post-study experiences with pain and physical functioning and to evaluate participants’ treatment experiences. MethodsIn XTEND-1, participants (≥12 years old) received once-weekly efanesoctocog alfa prophylaxis 50 IU/kg for 52 weeks (Arm A) or on-demand efanesoctocog alfa 50 IU/kg for 26 weeks followed by 26 weeks once-weekly prophylaxis (50 IU/kg; Arm B). Optional qualitative exit interviews were conducted using a semi-structured guide in a subset of participants following study completion. Interviews included open-ended questions about participants’ pre- and post-study experiences with hemophilia A and targeted questions relating to improvements in patient-reported outcomes assessed during XTEND-1, including the Haemophilia Quality of Life Questionnaire for Adults Physical Health subscale (Haem-A-QoL PH). Content validity of the Patient-Reported Outcomes Measurement Information System (PROMIS) Pain Intensity 3a measure was also assessed, particularly the worst pain item. FindingsExit interviews were conducted with 29 of 159 patients enrolled in XTEND-1 (mean [range] age 40 [16−73] years). Of 17 participants enrolled in Arm A, 13 (76.5%) reported a “wearing off” feeling with pre-study treatment, including more aches/pain, breakthrough bleeds, and limited physical activities. Joint pain was the most reported pre-study symptom (96.6%; n = 28/29), followed by a reduced ability to move without pain (89.7%, n = 26/29). Improvements following efanesoctocog alfa prophylaxis in ≥1 Haem-A-QoL PH domain were reported by 89.7% (n = 26/29) of participants, with improvements in joint pain, the ability to move without pain, and painful swellings reported by at least 21 (84%) participants. Participants reported that the PROMIS Pain Intensity 3a items were relevant, clear, and easy to answer. Most participants (96.6%) were “quite satisfied” or “very satisfied” with efanesoctocog alfa prophylaxis. All participants preferred efanesoctocog alfa over pre-study treatment. ImplicationsThe exit interviews demonstrated that once-weekly efanesoctocog alfa prophylaxis resulted in patient-relevant and meaningful improvements in pain and physical functioning, consistent with the quantitative findings from XTEND-1. These results support the validity of the Haem-A-QoL PH and PROMIS Pain Intensity 3a assessed during XTEND-1, demonstrating the potential for change with efficacious treatment. Trial RegistryClinicalTrials.gov Trial Registration NumberNCT04161495 Registry URLhttps://clinicaltrials.gov/study/NCT04161495

Capturing Autoinhibited PDK1 Reveals the Linker's Regulatory Role, Informing Innovative Inhibitor Design.

PDK1 is crucial for PI3K/AKT/mTOR and Ras/MAPK cancer signaling. It phosphorylates AKT in a PIP3-dependent but S6K, SGK, and RSK kinases in a PIP3-independent manner. Unlike its substrates, its autoinhibited monomeric state has been unclear, likely due to its low population time, and phosphorylation in the absence of PIP3 has been puzzling too. Here, guided by experimental data, we constructed models and performed all-atom molecular dynamics simulations. In the autoinhibited PDK1 conformation that resembles autoinhibited AKT, binding of the linker between the kinase and PH domains to the PIF-binding pocket promotes the formation of the Glu130-Lys111 salt bridge and weakens the association of the kinase domain with the PH domain, shifting the population from the autoinhibited state to states accessible to the membrane and its kinase substrates. The interaction of the substrates' hydrophobic motif and the PDK1 PIF-binding pocket facilitates the release of the autoinhibition even in the absence of PIP3. Phosphorylation of the serine-rich motif within the linker further attenuates the association of the PH domain with the kinase domain. These suggest that while the monomeric autoinhibited state is relatively stable, it can readily shift to its active, catalysis-prone state to phosphorylate its diverse substrates. Our findings reveal the PDK1 activation mechanism and discover the regulatory role of PDK1's linker, which lead to two innovative linker-based inhibitor strategies: (i) locking the autoinhibited PDK1 through optimization of the interactions of AKT inhibitors with the PH domain of PDK1 and (ii) analogs (small molecules or peptidomimetics) that mimic the linker interactions with the PIF-binding pocket.

Prognostic value of PLEKHA4 and its correlation with tumor-infiltrating immune cells in breast cancer: a comprehensive study based on bioinformatics and clinical analysis validation.

Pleckstrin homology containing family A, number 4 (PLEKHA4) plays a role in a number of biological processes in human cells, including cell polarization, growth, and proliferation. However, the relationship between PLEKHA4 expression and survival in breast cancer (BC) remains unclear. The aim of this study is to investigate the potential of PLEKHA4 as a prognostic indicator in BC. We obtained gene expression profiles of BC and normal tissues from the Tumor Immune Estimation Resource (TIMER), UALCAN web. Immunohistochemistry (IHC) staining was performed to investigate the protein expression and prognostic value of PLEKHA4 in BC patients. The prognostic value was analyzed using Kaplan-Meier curve analysis and Cox regression analysis in R software after downloading The Cancer Genome Atlas (TCGA) databases. The correlations between PLEKHA4 and tumor immune infiltrates were investigated via gene set variation analysis (GSVA). Signaling pathways related to PLEKHA4 expression were identified by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Both bioinformatics and IHC results showed that PLEKHA4 was expressed at low levels in BC tissues compared with the adjacent tissues. Furthermore, the expression of PLEKHA4 was negatively correlated with ages (χ2=6.394, P=0.01), molecular subtype (χ2=15.606, P=0.001), lymph node metastasis (χ2=13.753, P=0.004), tumor-node-metastasis (TNM) stage (χ2=22.616, P<0.001). Kaplan-Meier curves implicated low expression of PLEKHA4 was associated with worse survival of BC patients [hazard ratio (HR) =0.46, P=0.01]. Cox regression models showed that low PLEKHA4 expression could be an independent risk factor for BC (HR =0.911, P=0.006). The results of gene set enrichment analysis (GSEA) showed that cell cycle, Notch signaling pathway, nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway, and Rho GTPases were highly enriched in the low PLEKHA4 expression group, as identified by GO and KEGG. Additionally, in BC, PLEKHA4 expression displayed a positive correlation with the infiltration of natural killer (NK) cells (P<0.001), CD8+ T cells (P<0.001), B cells (P<0.001), neutrophils (P<0.001), and dendritic cells (DCs) (P<0.001). The findings indicate that PLEKHA4 is an independent prognostic biomarker associated with key signaling pathways and immune infiltration in BC. Targeting PLEKHA4 may contribute to improving immunotherapy for BC.

Dilution Effect of Protein Drop to Adjust the Crystal Size and the Nucleation Rate of PDK1 PH Domain

Dilution Effect of Protein Drop to Adjust the Crystal Size and the Nucleation Rate of PDK1 PH Domain

Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2−/− mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2−/− (double knockout) mice resembled Phlpp1−/− mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1−/− and Phlpp1/2−/− chondrocytes had similarities and were significantly different from WT and Phlpp2−/− chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.

A single particle analysis method for detecting membrane remodelling and curvature sensing.

In biology, shape and function are related. Therefore, it is important to understand how membrane shape is generated, stabilised and sensed by proteins and how this relates to organelle function. Here we present an assay that can detect curvature preference and membrane remodelling using free-floating liposomes using protein concentrations in a physiologically relevant ranges. The assay reproduced known curvature preferences of BAR domains and allowed the discovery of high curvature preference for the PH domain of AKT and the FYVE domain of HRS. In addition, our method reproduced the membrane vesiculation activity of the ENTH domain of Epsin1 and showed similar activity for the ANTH domains of PiCALM and Hip1R. Finally, we found that the curvature sensitivity of the N-BAR domain of Endophilin inversely correlates to membrane charge and that deletion of its N-terminal amphipathic helix increased its curvature specificity. Thus, our method is a generally applicable qualitative method for assessing membrane curvature sensing and remodelling by proteins.

Cryo-EM Structure of Phospholipase Cε Defines N-terminal Domains and their Roles in Activity.

Phospholipase Cε (PLCε) increases intracellular Ca 2+ and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLCε to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has also limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 Å reconstruction of the largest fragment of PLCε to date in complex with an antigen binding fragment (Fab). The structure reveals that PLCε contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain form a continuous surface that could engage cytoplasmic leaflets of the plasma and perinuclear membranes, contributing to activity. Functional characterization of this surface confirm it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLCε and the first mechanistic insights into how it engages cellular membranes for activity.

Novel Mutations in AKT1 Gene in Prostate Cancer Patients in Jordan.

The AKT1 oncogene is related to various cancers due to its critical role in the PIC3CA/AKT1 pathway; however, most of the studies screened the hotspot mutation AKT1 (E17K) with various incidences. Low frequency or lack of AKT1 (E17K) mutation was reported in prostate cancer (PC) patients. This study aims to explore genetic alterations in the AKT1 PH domain by extending the sequencing to include AKT1 gene exons 3 and 4. Genomic DNA was extracted from 84 Formalin-Fixed Paraffin-Embedded samples of PC patients in Jordan, and then subjected to PCR and sequencing for the targeted exons. This study revealed the presence of two novel mutations (N53Y and Q59K) and a high frequency of mutations in exon 4, with a lack of mutations in the E17K hotspot. Nine missense and two synonymous mutations were detected in exon 4 (Phe27Tyr, Phe27Leu, Ala58Thr, Ser56Phe, Arg41Trp, Phe35Leu, Asp32Glu, Phe35Tyr, and Gln43Lys) and (Ser56 and Glu40), respectively. Two synonymous mutations were detected in exon 3 (Leu12 and Ser2). It is concluded that there is a high frequency of AKT1 mutation in PC patients in Jordan with two novel missense mutations in the Pleckstrin homology (PH) domain. E17K hotspot mutation was not detected in any tested samples, which underlined the significant role of mutations in other AKT1 exons in PC development.

The RhoGEF protein Plekhg5 self-associates via its PH domain to regulate apical cell constriction.

RhoGEFs are critical activators of Rho family small GTPases and regulate diverse biological processes, such as cell division and tissue morphogenesis. We reported previously that the RhoGEF gene plekhg5 controls apical constriction of bottle cells at the blastopore lip during Xenopus gastrulation, but the detailed mechanism of plekhg5 action is not understood in depth. In this study, we show that localization of Plekhg5 in the apical cortex depends on its N-terminal sequences and intact guanine nucleotide exchange activity, whereas the C-terminal sequences prevent ectopic localization of the protein to the basolateral compartment. We also reveal that Plekhg5 self-associates via its PH domain, and this interaction leads to functional rescue of two mutants that lack the N-terminal region and the guanine nucleotide exchange factor activity, respectively, in trans. A point mutation in the PH domain corresponding to a variant associated with human disease leads to loss of self-association and failure of the mutant to induce apical constriction. Taken together, our results suggest that PH-mediated self-association and N-terminal domain-mediated subcellular localization are both crucial for the function of Plekhg5 in inducing apical constriction.

I told you to stop: obscurin's role in epithelial cell migration.

The giant cytoskeletal protein obscurin contains multiple cell signaling domains that influence cell migration. Here, we follow each of these pathways, examine how these pathways modulate epithelial cell migration, and discuss the cross-talk between these pathways. Specifically, obscurin uses its PH domain to inhibit phosphoinositide-3-kinase (PI3K)-dependent migration and its RhoGEF domain to activate RhoA and slow cell migration. While obscurin's effect on the PI3K pathway agrees with the literature, obscurin's effect on the RhoA pathway runs counter to most other RhoA effectors, whose activation tends to lead to enhanced motility. Obscurin also phosphorylates cadherins, and this may also influence cell motility. When taken together, obscurin's ability to modulate three independent cell migration pathways is likely why obscurin knockout cells experience enhanced epithelial to mesenchymal transition, and why obscurin is a frequently mutated gene in several types of cancer.

PLEKHG1: New Potential Candidate Gene for Periventricular White Matter Abnormalities.

Hypoxic-ischemic brain damage presents a significant neurological challenge, often manifesting during the perinatal period. Specifically, periventricular leukomalacia (PVL) is emerging as a notable contributor to cerebral palsy and intellectual disabilities. It compromises cerebral microcirculation, resulting in insufficient oxygen or blood flow to the periventricular region of the brain. As widely documented, these pathological conditions can be caused by several factors encompassing preterm birth (4-5% of the total cases), as well single cotwin abortion and genetic variants such as those associated with GTPase pathways. Whole exome sequencing (WES) analysis identified a de novo causative variant within the pleckstrin homology domain-containing family G member 1 (PLEKHG1) gene in a patient presenting with PVL. The PLEKHG1 gene is ubiquitously expressed, showing high expression patterns in brain tissues. PLEKHG1 is part of a family of Rho guanine nucleotide exchange factors, and the protein is essential for cell division control protein 42 (CDC42) activation in the GTPase pathway. CDC42 is a key small GTPase of the Rho-subfamily, regulating various cellular functions such as cell morphology, migration, endocytosis, and cell cycle progression. The molecular mechanism involving PLEKHG1 and CDC42 has an intriguing role in the reorientation of cells in the vascular endothelium, thus suggesting that disruption responses to mechanical stress in endothelial cells may be involved in the formation of white matter lesions. Significantly, CDC42 association with white matter abnormalities is underscored by its MIM phenotype number. In contrast, although PLEKHG1 has been recently associated with patients showing white matter hyperintensities, it currently lacks a MIM phenotype number. Additionally, in silico analyses classified the identified variant as pathogenic. Although the patient was born prematurely and subsequently to dichorionic gestation, during which its cotwin died, we suggest that the variant described can strongly contribute to PVL. The aim of the current study is to establish a plausible association between the PLEKHG1 gene and PVL.

ACAP3 negatively regulated by HDAC2 inhibits the malignant development of papillary thyroid carcinoma cells

ArfGAP with coiled-coil, ankyrin repeat and PH domains 3 (ACAP3) level has been confirmed to be downregulated in papillary thyroid carcinoma (PTC). Histone deacetylase inhibitors (HDACIs) have therapeutic effects on PTC. Accordingly, this study probed into the potential relation of histone deacetylase 2 (HDAC2) and ACAP3 in PTC. Expressions of ACAP3 and HDAC2 in PTC were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between HDAC2 and ACAP3 was predicted by Pearson analysis. Cell functional assays (cell counting kit-8, transwell, wound healing and flow cytometry assays) and rescue assay were carried out to determine the effects of HDAC2/ACAP3 axis on biological behaviors of PTC cells. Expressions of apoptosis-, epithelial-mesenchymal transition-, Protein Kinase B (AKT)-, and P53-related proteins were measured by Western blot. ACAP3 level was downregulated in PTC tissues and cells. ACAP3 overexpression (oe-ACAP3) suppressed viability, proliferation, migration and invasion of PTC cells, facilitated apoptosis, downregulated the expressions of Protein Kinase B (Bcl-2) and N-cadherin, upregulated the expressions of Bcl-2 associated protein X (Bax) and E-cadherin, diminished the p-AKT/AKT ratio and elevated the p-p53/p53 ratio; however, ACAP3 silencing or HDAC2 overexpression (oe-HDAC2) did the opposite. HDAC2 negatively correlated with ACAP3. The tumor-suppressing effect of oe-ACAP3 in PTC was reversed by oe-HDAC2. Collectively, ACAP3 negatively regulated by HDAC2 suppresses the proliferation and metastasis while facilitating apoptosis of PTC cells.

Control of germinal center B cell survival and IgE production by an adaptor molecule containing PH and SH2 domains, Aps/Sh2b2

The germinal centers (GCs) are structure found within secondary lymphoid organs and are important for the antibody-producing response against foreign antigens. In GCs, antigen-specific B cells proliferate intensely, inducing immunoglobulin class switching. Recent studies have shown that GCs are also an important site for class switching to IgE, which is implicated in allergy. However, the mechanisms by which IgE production is regulated in GCs remain unclear. Here, we found impairment in IgE-specific production and a reduction of GC B cells after immunization in mice deficient in the Aps/Sh2b2 gene encoding the Lnk/Sh2b family adaptor protein Aps. GC B cells express higher levels of the Aps gene than non-GC B cells, and cell death of Aps-/- GC B cells is enhanced compared to wild-type GC B cells. An in vitro culture system with purified Aps-/- B cells induced the same level of IgE production and frequencies of IgE+ B cells as wild-type B cells. We found that Aps deficiency in B cells resulted in augmented depletion of IgE+ blasts by B cell receptor crosslinking with anti-CD79b antibodies compared to wild-type IgE+ cells. These results suggest that Aps regulates IgE production by controlling the survival of GC B cells and IgE+ plasma cells and may serve as a potential therapeutic target to control IgE production.

β-H-Spectrin is a key component of an apical-medial hub of proteins during cell wedging in tube morphogenesis.

Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. We previously identified that interplay between the apical-medial actomyosin, which drives apical constriction, and the underlying longitudinal microtubule array has a key role during tube budding of salivary glands in the Drosophila embryo. At this microtubule-actomyosin interface, a hub of proteins accumulates, and we have shown before that this hub includes the microtubule-actin crosslinker Shot and the microtubule minus-end-binding protein Patronin. Here, we identify two actin-crosslinkers, β-heavy (H)-Spectrin (also known as Karst) and Filamin (also known as Cheerio), and the multi-PDZ-domain protein Big bang as components of the protein hub. We show that tissue-specific degradation of β-H-Spectrin leads to reduction of apical-medial F-actin, Shot, Patronin and Big bang, as well as concomitant defects in apical constriction, but that residual Patronin is still sufficient to assist microtubule reorganisation. We find that, unlike Patronin and Shot, neither β-H-Spectrin nor Big bang require microtubules for their localisation. β-H-Spectrin is instead recruited via binding to apical-medial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of β-H-Spectrin (β-H-33) displaces endogenous β-H-Spectrin and leads to strong morphogenetic defects. This protein hub therefore requires the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining apical constriction during tubulogenesis.

Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4

PIP3-dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP3 and heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP3, we discovered that Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH–DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH–DEP1 and PH–4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.