Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

An expanded universe of cancer targets.

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer's disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Nicardipine is a putative EED inhibitor and has high selectivity and potency against chemoresistant prostate cancer in preclinical models.

BACKGROUND: It is imperative to develop novel therapeutics to overcome chemoresistance, a significant obstacle in the clinical management of prostate cancer (PCa) and other cancers. METHODS: A phenotypic screen was performed to identify novel inhibitors of chemoresistant PCa cells. The mechanism of action of potential candidate(s) was investigated using in silico docking, and molecular and cellular assays in chemoresistant PCa cells. The in vivo efficacy was evaluated in mouse xenograft models of chemoresistant PCa. RESULTS: Nicardipine exhibited high selectivity and potency against chemoresistant PCa cells via inducing apoptosis and cell cycle arrest. Computational, molecular, and cellular studies identified nicardipine as a putative inhibitor of embryonic ectoderm development (EED) protein, and the results are consistent with a proposed mechanism of action that nicardipine destabilised enhancer of zeste homologue 2 (EZH2) and inhibited key components of noncanonical EZH2 signalling, including transducer and activator of transcription 3, S-phase kinase-associated protein 2, ATP binding cassette B1, and survivin. As a monotherapy, nicardipine effectively inhibited the skeletal growth of chemoresistant C4-2B-TaxR tumours. As a combination regimen, nicardipine synergistically enhanced the in vivo efficacy of docetaxel against C4-2 xenografts. CONCLUSION: Our findings provided the first preclinical evidence supporting nicardipine as a novel EED inhibitor that has the potential to be promptly tested in PCa patients to overcome chemoresistance and improve clinical outcomes.

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

PURPOSE: Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. METHODS: The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. RESULTS: MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. CONCLUSIONS: We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

Stress induces p38 MAPK-mediated phosphorylation and inhibition of Drosha-dependent cell survival.

MicroRNAs (miRNAs) regulate the translational potential of their mRNA targets and control many cellular processes. The key step in canonical miRNA biogenesis is the cleavage of the primary transcripts by the nuclear RNase III enzyme Drosha. Emerging evidence suggests that the miRNA biogenic cascade is tightly controlled. However, little is known whether Drosha is regulated. Here, we show that Drosha is targeted by stress. Under stress, p38 MAPK directly phosphorylates Drosha at its N terminus. This reduces its interaction with DiGeorge syndrome critical region gene 8 and promotes its nuclear export and degradation by calpain. This regulatory mechanism mediates stress-induced inhibition of Drosha function. Reduction of Drosha sensitizes cells to stress and increases death. In contrast, increase in Drosha attenuates stress-induced death. These findings reveal a critical regulatory mechanism by which stress engages p38 MAPK pathway to destabilize Drosha and inhibit Drosha-mediated cellular survival.

Acquisition of taxane resistance by p53 inactivation in ovarian cancer cells.

Ovarian cancer is one of the most common gynecologic malignancies in women and has a poor prognosis. Taxanes are a class of standard first-line chemotherapeutic agents for the treatment of ovarian cancer. However, tumor-intrinsic and acquired resistance to taxanes poses major challenges to improving clinical outcomes. Hence, there is an urgent clinical need to understand the mechanisms of resistance in order to discover potential biomarkers and therapeutic strategies to increase taxane sensitivity in ovarian cancer. Here, we report the identification of an association between the TP53 status and taxane sensitivity in ovarian cancer cells through complementary experimental and informatics approaches. We found that TP53 inactivation is associated with taxane resistance in ovarian cancer cells, supported by the evidence from (i) drug sensitivity profiling with bioinformatic analysis of large-scale cancer therapeutic response and genomic datasets and (ii) gene signature identification based on experimental isogenic cell line models. Further, our studies revealed TP53-dependent gene expression patterns, such as overexpression of ACSM3, as potential predictive biomarkers of taxane resistance in ovarian cancer. The TP53-dependent hyperactivation of the WNT/ß-catenin pathway discovered herein revealed a potential vulnerability to exploit in developing combination therapeutic strategies. Identification of this genotype-phenotype relationship between the TP53 status and taxane sensitivity sheds light on TP53-directed patient stratification and therapeutic discoveries for ovarian cancer treatment.

Expression and purification of active human kinases using Pichia pastoris as a general-purpose host.

BACKGROUND: The heterologous expression of human kinases in good purity and in a monomeric, soluble and active form can be challenging. Most of the reported successful attempts are carried out in insect cells as a host. The use of E. coli for expression is limited to a few kinases and usually is facilitated by large solubility tags that can limit biophysical studies and affect protein-protein interactions. In this report, we evaluate the methylotrophic yeast Pichia pastoris (P. pastoris) as a general-purpose host for expression of human kinases. METHODS: Six diverse kinases were chosen due to their therapeutic importance in human cancers. Tested proteins include serine/threonine kinases cyclin-dependent kinases 4 and 6 (CDK4 and 6) and aurora kinase A (AurKA), receptor tyrosine kinase erbB-2 (HER2), and dual specificity kinase mitogen-activated protein kinase kinase 3 (MKK3b). Noting that positively charged kinases expressed with higher yield, we sought to improve expression of two challenging targets, CDK6 and HER2, by fusing the highly basic, N-terminal domain of the secreted tyrosine-protein kinase VLK. The standard expression procedure for P. pastoris was adopted, followed by purification using affinity chromatography. Purity and activity of the proteins were confirmed and compared to published values. RESULTS: Some kinases were purified with good yield and purity and with comparable activity to commercially available versions. Addition of the VLK domain improved expression and decreased aggregation of CDK6 and HER2.

Discovery of the first chemical tools to regulate MKK3-mediated MYC activation in cancer.

The transcription master regulator MYC plays an essential role in regulating major cellular programs and is a well-established therapeutic target in cancer. However, MYC targeting for drug discovery is challenging. New therapeutic approaches to control MYC-dependent malignancy are urgently needed. The mitogen-activated protein kinase kinase 3 (MKK3) binds and activates MYC in different cell types, and disruption of MKK3-MYC protein-protein interaction may provide a new strategy to target MYC-driven programs. However, there is no perturbagen available to interrogate and control this signaling arm. In this study, we assessed the drugability of the MKK3-MYC complex and discovered the first chemical tool to regulate MKK3-mediated MYC activation. We have designed a short 44-residue inhibitory peptide and developed a cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay to discover the first small molecule MKK3-MYC PPI inhibitor. We have optimized and miniaturized the assay into an ultra-high-throughput screening (uHTS) 1536-well plate format. The pilot screen of ~6,000 compounds of a bioactive chemical library followed by multiple secondary and orthogonal assays revealed a quinoline derivative SGI-1027 as a potent inhibitor of MKK3-MYC PPI. We have shown that SGI-1027 disrupts the MKK3-MYC complex in cells and in vitro and inhibits MYC transcriptional activity in colon and breast cancer cells. In contrast, SGI-1027 does not inhibit MKK3 kinase activity and does not interfere with well-known MKK3-p38 and MYC-MAX complexes. Together, our studies demonstrate the drugability of MKK3-MYC PPI, provide the first chemical tool to interrogate its biological functions, and establish a new uHTS assay to enable future discovery of potent and selective inhibitors to regulate this oncogenic complex.

14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel.

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

High expression of MKK3 is associated with worse clinical outcomes in African American breast cancer patients.

BACKGROUND: African American women experience a twofold higher incidence of triple-negative breast cancer (TNBC) and are 40% more likely to die from breast cancer than women of other ethnicities. However, the molecular bases for the survival disparity in breast cancer remain unclear, and no race-specific therapeutic targets have been proposed. To address this knowledge gap, we performed a systematic analysis of the relationship between gene mRNA expression and clinical outcomes determined for The Cancer Genome Atlas (TCGA) breast cancer patient cohort. METHODS: The systematic differential analysis of mRNA expression integrated with the analysis of clinical outcomes was performed for 1055 samples from the breast invasive carcinoma TCGA PanCancer cohorts. A deep learning fully-convolutional model was used to determine the association between gene expression and tumor features based on breast cancer patient histopathological images. RESULTS: We found that more than 30% of all protein-coding genes are differentially expressed in White and African American breast cancer patients. We have determined a set of 32 genes whose overexpression in African American patients strongly correlates with decreased survival of African American but not White breast cancer patients. Among those genes, the overexpression of mitogen-activated protein kinase kinase 3 (MKK3) has one of the most dramatic and race-specific negative impacts on the survival of African American patients, specifically with triple-negative breast cancer. We found that MKK3 can promote the TNBC tumorigenesis in African American patients in part by activating of the epithelial-to-mesenchymal transition induced by master regulator MYC. CONCLUSIONS: The poor clinical outcomes in African American women with breast cancer can be associated with the abnormal elevation of individual gene expression. Such genes, including those identified and prioritized in this study, could represent new targets for therapeutic intervention. A strong correlation between MKK3 overexpression, activation of its binding partner and major oncogene MYC, and worsened clinical outcomes suggests the MKK3-MYC protein-protein interaction as a new promising target to reduce racial disparity in breast cancer survival.

The OncoPPi Portal: an integrative resource to explore and prioritize protein-protein interactions for cancer target discovery.

Motivation: As cancer genomics initiatives move toward comprehensive identification of genetic alterations in cancer, attention is now turning to understanding how interactions among these genes lead to the acquisition of tumor hallmarks. Emerging pharmacological and clinical data suggest a highly promising role of cancer-specific protein-protein interactions (PPIs) as druggable cancer targets. However, large-scale experimental identification of cancer-related PPIs remains challenging, and currently available resources to explore oncogenic PPI networks are limited. Results: Recently, we have developed a PPI high-throughput screening platform to detect PPIs between cancer-associated proteins in the context of cancer cells. Here, we present the OncoPPi Portal, an interactive web resource that allows investigators to access, manipulate and interpret a high-quality cancer-focused network of PPIs experimentally detected in cancer cell lines. To facilitate prioritization of PPIs for further biological studies, this resource combines network connectivity analysis, mutual exclusivity analysis of genomic alterations, cellular co-localization of interacting proteins and domain-domain interactions. Estimates of PPI essentiality allow users to evaluate the functional impact of PPI disruption on cancer cell proliferation. Furthermore, connecting the OncoPPi network with the approved drugs and compounds in clinical trials enables discovery of new tumor dependencies to inform strategies to interrogate undruggable targets like tumor suppressors. The OncoPPi Portal serves as a resource for the cancer research community to facilitate discovery of cancer targets and therapeutic development. Availability and implementation: The OncoPPi Portal is available at http://oncoppi.emory.edu. Contact: andrey.ivanov@emory.edu or hfu@emory.edu.

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism.

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.

Identification of higenamine as a novel α1 -adrenergic receptor antagonist.

The α1 -adrenoceptor (α1 -AR) antagonists are potential candidates for the treatment of blood pressure. Higenamine (HG) is a novel α1 -AR antagonist. In this study, we investigated the effects of HG in HEK293A cells transfected with α1A -, α1B -, and α1D -AR in vitro, rat mesenteric artery ex vivo, Wistar-Kyoto rats and spontaneously hypertensive rats in vivo. The radioligand binding assay showed that HG competitively inhibited the binding of [3 H]-prazosin to α1 -AR in a concentration-dependent manner. The affinities (pKi) of HG for the cloned α1A -, α1B -, and α1D -AR were 6.57, 6.48, and 6.35, respectively, indicating that HG displayed no selectivity for the three α1 -AR subtypes. In in vitro studies, HG was able to blunt inositol monophosphate production. It also displayed an inhibitory effect on the influx and entry of calcium ions and phosphorylation of extracellular signal-regulated kinase 1 and 2 induced by phenylephrine (PE). In ex vivo studies, PE caused a dose-dependent inotropic response curve, and the pA2 value for HG was 6.86 ± 0.29. In addition, the in vivo results showed that HG could decrease the blood pressure in normotension, spontaneous hypertension, and PE-induced hypertension models. These results indicate that HG can directly bind to α1 -AR and it appears to be a novel antagonist for α1 -AR, which may contribute to its hypotensive effect.

Polyethylene-glycol-coated gold nanoparticles improve cardiac function after myocardial infarction in mice.

Gold nanoparticles (AuNPs) are widely used for drug delivery because of their unique biological properties, such as their safety and ability to prolong drug action. Some studies have demonstrated that AuNPs accumulate in the heart, especially during pathological processes. Therefore, it is very important to understand the effect of AuNPs on the heart. Myocardial infarction (MI) is a major cause of morbidity and mortality; however, the effect of AuNPs on MI remains unclear. In the present study, we carried out a comprehensive evaluation of AuNPs on acute MI. The results showed that AuNPs accumulated in infarcted hearts, decreased infarction size, improved systolic function, and inhibited cardiac fibrosis and TNF-α accumulation. Our work indicated that AuNPs have cardioprotective effects and can be used in drug delivery systems for the treatment of cardiac diseases.

AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay.

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

Characterization of a Linked Jumonji Domain of the KDM5/JARID1 Family of Histone H3 Lysine 4 Demethylases.

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the design of KDM5 demethylase inhibitors with improved potency and selectivity.

Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis.

TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.

Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor.

Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.

Downregulation of urea transporter UT-A1 activity by 14-3-3 protein.

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione-S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.