P53 Binding Site Research Articles

Phosphoinositide Regulation of TRP Channels: A Functional Overview in the Structural Era.

Transient receptor potential (TRP) ion channels have diverse activation mechanisms including physical stimuli, such as high or low temperatures, and a variety of intracellular signaling molecules. Regulation by phosphoinositides and their derivatives is their only known common regulatory feature. For most TRP channels, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] serves as a cofactor required for activity. Such dependence on PI(4,5)P2 has been demonstrated for members of the TRPM subfamily and for the epithelial TRPV5 and TRPV6 channels. Intracellular TRPML channels show specific activation by PI(3,5)P2. Structural studies uncovered the PI(4,5)P2 and PI(3,5)P2 binding sites for these channels and shed light on the mechanism of channel opening. PI(4,5)P2 regulation of TRPV1-4 as well as some TRPC channels is more complex, involving both positive and negative effects. This review discusses the functional roles of phosphoinositides in TRP channel regulation and molecular insights gained from recent cryo-electron microscopy structures.

P10.34.B ONCOGENIC ASPP2KAPPA(K), A DOMINANT-NEGATIVE ISOFORM OF THE APOPTOSIS STIMULATING PROTEIN OF P53-2 (ASPP2) PROMOTES CLASSICAL HALLMARKS OF CANCER IN GLIOBLASTOMA

Abstract BACKGROUND GB is the most common, most aggressive primary brain tumor with a deleterious prognosis. ASPP2 is a tumor suppressor, directly interacting with p53. We recently identified a dominant-negative isoform of ASPP2, named ASPP2κ, lacking the important p53 binding sites and show that ASPP2κ is highly expressed and functionally active in glioblastoma, affecting all classical hallmarks of cancer. MATERIAL AND METHODS Grade IV glioma (n=46) were assessed for ASPP2κ expression. Isogenic GB models, stably suppressing or overexpressing ASPP2κ, were established in A-172, U-251 and T98g cell lines and from resected GB tissue. The role of ASPP2κ in induction of apoptosis, cellular proliferation, migration and invasion was assessed in vitro and in vivo. Proteome arrays allowed to study pathways involved in ASPP2κ signaling. Two ASPP2κ knock-down (KD) NOD/SCID xenotransplant mouse models were established to study ASPP2κ in tumor engraftment and progression. RESULTS Frequent overexpression of ASPP2κ was confirmed in virtually all patients analyzed compared to tumor-free tissue. Expression thereby varied widely in between patients (2-40 fold). Functional analyses confirm that ASPP2κ directly affects multiple cancer hallmarks in GB, resulting in a more aggressive phenotype with impaired induction of apoptosis (avg. -25 %), higher proliferation (avg. +25%), migration (avg. +65%) and invasion (avg. +70%) rates. Silencing of ASPP2κ rescues the ASPP2 wt tumor suppressor phenotype, rendering cells more susceptible towards temozolomide (TMZ, avg. +25%), γ-irradiation (avg. +40%) or combinational approaches. Upregulation of p53 phosphorylation sites (S15: 2,5x/S46: 1,5x) and cell cycle regulators (p21: 2x, p27: 3x and pRAD: 2,5x) thereby confirms restoration of important p53-mediated pathways. GB mouse models confirm attenuated tumor engraftment and progression of ASPP2κ KD tumors in vivo. Mice carrying ASPP2κ-KD tumors presented with statistically significant increased overall survival in both models (p= 0.03 and p=0.005) and attenuated tumor growth (p=0.0001). Importantly, neo-angiogenesis was strongly regulated in relation to ASPP2κ expression, impairing tumor growth and metastasis in the ASPP2κ KD models. Downregulation of pro-angiogenic (VEGF 20x, angiogenin 4x) and upregulation of anti-angiogenic (endostatin 2x, LAP 2,5x) players in ASPP2κ KD models were confirmed. CONCLUSION ASPP2κ is highly expressed in GB, affecting all classical hallmarks of cancer, including regulation of apoptosis, cell cycle, invasion/metastasis and angiogenesis. ASPP2κ contributes to aggressiveness of disease and resistance to therapy and silencing of ASPP2κ re-activates crucial p53-mediated tumorsuppressor pathways. Studies evaluating ASPP2κ as a target for therapy are ongoing.

The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.

Structures of p53/BCL-2 complex suggest a mechanism for p53 to antagonize BCL-2 activity

Mitochondrial apoptosis is strictly controlled by BCL-2 family proteins through a subtle network of protein interactions. The tumor suppressor protein p53 triggers transcription-independent apoptosis through direct interactions with BCL-2 family proteins, but the molecular mechanism is not well understood. In this study, we present three crystal structures of p53-DBD in complex with the anti-apoptotic protein BCL-2 at resolutions of 2.3–2.7 Å. The structures show that two loops of p53-DBD penetrate directly into the BH3-binding pocket of BCL-2. Structure-based mutations at the interface impair the p53/BCL-2 interaction. Specifically, the binding sites for p53 and the pro-apoptotic protein Bax in the BCL-2 pocket are mostly identical. In addition, formation of the p53/BCL-2 complex is negatively correlated with the formation of BCL-2 complexes with pro-apoptotic BCL-2 family members. Defects in the p53/BCL-2 interaction attenuate p53-mediated cell apoptosis. Overall, our study provides a structural basis for the interaction between p53 and BCL-2, and suggests a molecular mechanism by which p53 regulates transcription-independent apoptosis by antagonizing the interaction of BCL-2 with pro-apoptotic BCL-2 family members.

E2F7 drives autotaxin/Enpp2 transcription via chromosome looping: Repression by p53 in murine but not in human carcinomas.

Dysregulation of the autotaxin (ATX, Enpp2)-lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53-KO mice compared to wild-type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53-KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one-hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region -1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7-mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7-mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.

Abstract 2535: Inhibition of oncogenic ASPP2kappa(k), a dominant-negative isoform of the tumorsuppressor ASPP2, results in reactivation of p53 in glioblastoma

Abstract GB is the most common, most aggressive primary brain tumor with a deleterious prognosis. Novel approaches to improve therapy are urgently needed. ASPP2 is a tumor suppressor, directly interacting with p53 to induce apoptosis. We recently identified a dominant-negative isoform of ASPP2, named ASPP2kappa(k), lacking the important p53 binding sites. We now show that ASPP2k is highly expressed and functionally active in high grade glioma and that silencing of ASPP2k reactivates induction of apoptosis via p53. Freshly resected glioma (n=76) were assessed for ASPP2k expression. Isogenic, p53wt cell models, stably suppressing or overexpressing ASPP2k were established in A-172 and U-251 cells and ex vivo from GB samples and the role of ASPP2k in induction of apoptosis, cellular proliferation, migration and invasion was assessed. A proteome array allowed to study pathways involved in ASPP2k signaling. An ASPP2k knock-down, NOD/SCID xenotransplant mouse model was established to study the role of ASPP2k in tumor engraftment and progression. Frequent overexpression of ASPP2k in glioma was confirmed in virtually all patients analyzed. Expression patterns varied widely (up to 40-fold compared to tumor-free tissue). Expression of ASPP2k thereby correlates with tumor biology - with highest levels found in aggressive grade IV GB. We further demonstrate that expression of ASPP2k results in impaired induction of apoptosis in all GB models - and knock down of ASPP2k renders cells more susceptible towards temozolamide (TMZ), γ-irradiation (avg. 25% TMZ, 40% irradiation) or combinational approaches compared to controls. Vice versa, overexpression of ASPP2κ further attenuates induction of apoptosis. Even more, ASPP2k directly affects other cellular functions in GB, rendering cells to a more aggressive phenotype with increased proliferation (approx. 25%), migration (approx. 65%) and invasion rates compared to the knock down strains. In line, silencing of ASPP2k resulted in strong upregulation of crucial p53 phosphorylation sites (S15/S46), as well as cell cycle regulators (p21, p27 and pRAD) in a proteome array, arguing for re-activation of crucial p53-mediated pathways in the ASPP2k silenced cells. Results were confirmed by western immunoblotting and FACS analyses. Ongoing xenotransplant mouse models confirm that silencing of ASPP2k in GB models attenuates tumor engraftment, development and progression in vivo. In summary, we here show that ASPP2k is highly expressed in GB and directly contributes to aggressiveness of the disease as well as resistance towards therapy. Silencing of ASPP2k thereby re-activates crucial p53 pathways, resulting in re-activation of p53-mediated apoptosis pathways as well as re-installation of cell cycle checkpoints. Future studies evaluating ASPP2k as a prognostic/predictive model and potential target for therapy are warranted. Citation Format: Marlon Hafner, Alessia Ruiba, Leonie Kampa, Vincent Kälin, Marian Neidert, Konrad Buchauer, Guido Henke, Ludwig Plasswilm, Marcus M. Schittenhelmn, Kerstin M. Kampa-Schittenhelm. Inhibition of oncogenic ASPP2kappa(k), a dominant-negative isoform of the tumorsuppressor ASPP2, results in reactivation of p53 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2535.

The C2 and PH domains of CAPS constitute an effective PI(4,5)P2-binding unit essential for Ca2+-regulated exocytosis

Ca2+-dependent activator proteins for secretion (CAPSs) are required for Ca2+-regulated exocytosis in neurons and neuroendocrine cells. CAPSs contain a pleckstrin homology (PH) domain that binds PI(4,5)P2-membrane. There is also a C2 domain residing adjacent to the PH domain, but its function remains unclear. In this study, we solved the crystal structure of the CAPS-1 C2PH module. The structure showed that the C2 and PH tandem packs against one another mainly via hydrophobic residues. With this interaction, the C2PH module exhibited enhanced binding to PI(4,5)P2-membrane compared with the isolated PH domain. In addition, we identified a new PI(4,5)P2-binding site on the C2 domain. Disruption of either the tight interaction between the C2 and PH domains or the PI(4,5)P2-binding sites on both domains significantly impairs CAPS-1 function in Ca2+-regulated exocytosis at the Caenorhabditis elegans neuromuscular junction (NMJ). These results suggest that the C2 and PH domains constitute an effective unit to promote Ca2+-regulated exocytosis.

The second PI(3,5)P2 binding site in the S0 helix of KCNQ1 stabilizes PIP2-at the primary PI1 site with potential consequences on intermediate-to-open state transition.

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

Exploring the conformational dynamics of the disordered Lid region of HDM2 N-terminal domain with molecular dynamics simulation.

HDM2 is the negative regulator of the tumor suppressor p53, which plays an important role in controlling intracellular homeostasis. The p53 binding pocket of HDM2 is located at the N-terminal domain consisting of the two regions, Lid and Core. The former is a disordered region located at the N-terminus of the domain, while the latter includes the p53 binding site. Lid dynamics modulate the relative orientation of Lid and Core and thereby expose the p53 binding site. In addition, the dynamics of Lid would be affected by macromolecular crowding environments in the cell. We therefore aim to investigate the conformational dynamics of Lid in HDM2 using molecular dynamics (MD) simulations in dilute solution as well as protein-crowded solution. As the first step of this work, we performed conventional MD simulations of HDM2 for 1 μs in a dilute solution using the three force fields, namely, CHARMM36m, AMBER ff19SB, and AMBER ff99SBws-STQ. Since Lid is a highly disordered region, selecting the appropriate force field is essential for comparing the simulation results with experiments and understanding the nature of Lid conformational dynamics. All the MD simulations were performed using GENESIS software on Supercomputer HOKUSAI BigWaterfall at RIKEN in Japan. Two structures derived from the NMR structure (PDB ID: 1Z1M) were used as initial structures in the simulations. Based on the difference in the distance between Lid and Core, the two structures are named “bound” and “unbound” states, respectively. The MD simulation results showed that the secondary structure in Lid and the distance between Lid and Core are strongly dependent on the force fields used. We discuss which force field is the most reliable for studying conformational dynamics of Lid in a dilute and protein-crowded solution.

The bisphosphonate zoledronic acid is a TRPV1 channel inhibitor.

The heat and capsaicin sensor, the transient receptor potential vanilloid type 1 (TRPV1) channel, is critically involved in pain sensation. Using compounds that activate TRPV1, such as capsaicin (CAP), produces analgesic effects. Hence the channel is a promising target for the management of pain conditions. However, when TRPV1 channels are sensitized in neuropathic pain conditions, inhibitors could be a better option for pain management. We have obtained evidence supporting that the bisphosphonate compound zoledronic acid (ZOL) acts as an inhibitor of the TRPV1 channel. ZOL is typically used in the treatment of bone diseases reducing bone mass loss, deterioration in bone microarchitecture and fracture risk, having as a collateral effect a decrease in pain sensation. We have observed, expressing TRPV1 in Xenopus laevis oocytes followed by patch-clamp recordings in inside-out configuration, that ZOL at doses ranging from 1 nM up to 30 μM reduces channel conductance and inhibits the activity of the channel deeply. Only high doses of CAP (≥ 10 μM) allow for recover of TRPV1 currents after ZOL treatment. Molecular modeling followed by docking analysis suggests that ZOL could potentially bind to TRPV1 at the vanilloid pocket and the PI(4,5)P2 binding site. Furthermore, like capsazepine, ZOL (100 nM) is able to eliminate the depression observed by applying CAP (1 μM) on synaptic transmission in the dentate gyrus. In addition, when ZOL is use during the induction of synaptic plasticity mediated by TRPV1, it eliminates plasticity. The compound did not affect the endocannabinoid production machinery suggesting that the ZOL is acting specifically on TRPV1 to hinder plasticity. Together, our evidence strongly supports that ZOL can inhibit TRPV1 activity.

CircRNA_0067717 promotes paclitaxel resistance in nasopharyngeal carcinoma by acting as a scaffold for TRIM41 and p53.

Circular RNAs (circRNAs) play important roles in tumour progression. This study aimed to explore the mechanism of hsa_circ_0067717 (termed circRNA_0067717) promoting paclitaxel resistance in nasopharyngeal carcinoma (NPC). We assayed CNE-1 and HNE-2 parental cell lines and the corresponding paclitaxel-resistant NPC cell lines using circRNA microarrays. RNA pull-down assay, RNA immunoprecipitation, and RNA fluorescence in situ hybridization were used to identify the molecular mechanisms. Here, we confirm that circRNA_0067717 is significantly upregulated in NPC paclitaxel-resistant cells and is associated with paclitaxel resistance in NPC. Mechanistically, circRNA_0067717 functions as a scaffold for TRIM41 protein (a ubiquitin E3 ligase) and p53 protein. In nasopharyngeal carcinoma paclitaxel-resistant cells, the highly expressed circRNA_0067717 can bind to more TRIM41 and p53 protein, promoting TRIM41-induced p53 ubiquitination and degradation, resulting in a decrease in p53 protein level. Moreover,the 1-176 nt area of circRNA_0067717 and the 301-425 nt region of circRNA_0067717 are the binding sites for p53 and TRIM41, respectively. The resistance of NPC cells to paclitaxel can be reduced by blocking these binding regions of circRNA_0067717. We demonstrate that circRNA_0067717 acts as a scaffold for TRIM41 and p53, enhancing paclitaxel chemoresistance in NPC by promoting TRIM41-induced p53 degradation via ubiquitination.

Phosphatidylinositol 4,5-Bisphosphate and Cholesterol Regulators of the Calcium-Activated Chloride Channels TMEM16A and TMEM16B.

Chloride fluxes through homo-dimeric calcium-activated channels TMEM16A and TMEM16B are critical to blood pressure, gastrointestinal motility, hormone, fluid and electrolyte secretion, pain sensation, sensory transduction, and neuronal and muscle excitability. Their gating depends on the voltage-dependent binding of two intracellular calcium ions to a high-affinity site formed by acidic residues from α-helices 6-8 in each monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a low-abundant lipid of the inner leaflet, supports TMEM16A function; it allows TMEM16A to evade the down-regulation induced by calcium, poly-L-lysine, or PI(4,5)P2 5-phosphatase. In stark contrast, adding or removing PI(4,5)P2 diminishes or increases TMEM16B function, respectively. PI(4,5)P2-binding sites on TMEM16A, and presumably on TMEM16B, are on the cytosolic side of α-helices 3-5, opposite the calcium-binding sites. This modular structure suggested that PI(4,5)P2 and calcium cooperate to maintain the conductive state in TMEM16A. Cholesterol, the second-largest constituent of the plasma membrane, also regulates TMEM16A though the mechanism, functional outcomes, binding site(s), and effects on TMEM16A and TMEM16B remain unknown.

Therapeutic capability of selected medicinal plants' bioactive constituents against the mutant ovarian TP53 gene; a computational approach

A mutant P53 protein plays such a crucial role in ovarian cancer, and natural compounds have been known to be effective in treating cancer. The current study was conducted to discover new mutant P53 modulators in plants used for medicinal purposes. The mutant p53 3D structure was built using homology modeling, while its active binding domain was predicted using Findsitecom2.0. Docking studies were conducted with ligands derived from bioactive components of seven different plants and mutant p53 binding sites. Autodocking programs, including Discovery Studio and PyRx, were used to obtain the docking protein and its intricate visual representation. Gemcitabine and thiotepa were the reference drugs. Acute RAT toxicity and Pharmacokinetic properties were utilized in Gusar and SWISSADME, respectively, to narrow down the hit compounds to those with the highest binding affinities. Using the density functional theory (DFT) method, the electronic properties of the bioactive constituents were determined. 15 of the 50 bioactive phytochemicals displayed superior mutant p53 binding energies compared to Gemcitabine and Thioteba (−5.4 and −3.5 binding scores, respectively). Considering acute toxicity predictions and pharmacokinetics, 10-hydroxycamptothecin, irinotecan, morusin, and rubitecan were the four major compounds with low toxicity. DFT calculations uncovered regions susceptible to nucleophilic and electrophilic assaults. The study sought to identify potential drug candidates for modulating mutant P53 in ovarian cancer treatment.

Effect of Nutlin-3a on stability of p53-MDM2 complex

<sec>P53 is well recognized to be a tumor suppressor protein. In response to the external stress or environmental perturbation, p53 can promote the transcription of various target genes downstream, thus regulating the cell cycle, apoptosis, DNA repair, and angiogenesis. However, the activation of p53 is further activated by another protein, MDM2, which negatively regulates the level of p53 inverse and thus reduces the activation of p53. This phenomenon is a novel potential and promising strategy for cancer therapy, i.e. restoring the activity of p53 pathway through the competitive inhibitors that can occupy the p53-binding site of MDM2 and thus inhibit the interaction between p53 and MDM2.</sec><sec>Recently, various kinds of the inhibitors have been designed for this purpose. The Nutlin family is a group of well investigated inhibitors, which shows high efficiency for tumor suppression. Nutlin-3a mimics the MDM2-binding site of p53 essentially, and blocks the binding of MDM2 to p53. Once getting free from MDM2, p53 rapidly accumulates in the nuclei of cancer cells, the p53 target genes and the p53 pathway are activated, thereby resulting in cell-cycle arrest and apoptosis. In our previous papers, we investigated the competition mechanism between Nutlin3 and p53 <i>in vitro</i> by using molecular dynamics simulations. We found that Nutlin3 can bind faster than p53 to prevent p53 from binding to MDM2 when Nutlin-3a and p53 have equal distance from MDM2. Nutlin-3a can also bind to the p53-MDM2 complex to disturb and weaken the interactions between p53 and MDM2. However, the underlying mechanisms of p53-MDM2 complex instability <i>in vivo</i> are still unclear. And these inhibitors also have a variety of specificities and biological toxicities <i>in vivo</i> environment.</sec><sec>In this study, we go a further step to investigate the effect of Nutlin-3a on the stability of p53-MDM2 complex in physiological environment with the aid of the molecular mechanics/generalized borne surface area (MM/GBSA) method. In our simulations, a group of Nutlin-3a molecules are randomly put around the p53 binding pocket of MDM2 in the initial stages to examine the dynamics among p53, MDM2 and the group of Nutlin-3a molecules and to analyze the underlying competition mechanism between Nutlin3 and p53 binding to pocket of MDM2.</sec><sec>We find that Nutlin-3a can induce the centroid distance between p53 and MDM2 to increase. Importantly, we show that Nutlin-3a weakens the binding affinity of p53-MDM2 complex. Consistently, Nutlin-3a breaks a hydrogen bond between Phe19-Gln72 and a salt bridge between Glu17-Lys94, which weakens the interactions between p53 and MDM2. From the systematic biology point of view, the regulation of p53 by MDM2 is extremely sensitive to the strength of the p53-MDM2 interaction. The avianization of the interactions between p53 and MDM2 by Nutlin-3a can promote p53 to restore its suppression functions on tumor development.</sec><sec>This study may be helpful in understanding the molecular mechanisms of p53-MDM2 complex instability mediated by Nutlin-3a and also in searching for the effective inhibitors of p53-MDM2 interaction.</sec>

P53 maintains gallid alpha herpesvirus 1 replication by direct regulation of nucleotide metabolism and ATP synthesis through its target genes.

P53, a well-known tumor suppressor, has been confirmed to regulate the infection of various viruses, including chicken viruses. Our previous study observed antiviral effect of p53 inhibitor Pifithrin-α (PFT-α) on the infection of avian infectious laryngotracheitis virus (ILTV), one of the major avian viruses economically significant to the poultry industry globally. However, the potential link between this antiviral effect of PFT-α and p53 remains unclear. Using chicken LMH cell line which is permissive for ILTV infection as model, we explore the effects of p53 on ILTV replication and its underlying molecular mechanism based on genome-wide transcriptome analysis of genes with p53 binding sites. The putative p53 target genes were validated by ChIP-qPCR and RT-qPCR. Results demonstrated that, consistent with the effects of PFT-α on ILTV replication we previously reported, knockdown of p53 repressed viral gene transcription and the genome replication of ILTV effectively. The production of infectious virions was also suppressed significantly by p53 knockdown. Further bioinformatic analysis of genes with p53 binding sites revealed extensive repression of these putative p53 target genes enriched in the metabolic processes, especially nucleotide metabolism and ATP synthesis, upon p53 repression by PFT-α in ILTV infected LMH cells. Among these genes, eighteen were involved in nucleotide metabolism and ATP synthesis. Then eight of the 18 genes were selected randomly for validations, all of which were successfully identified as p53 target genes. Our findings shed light on the mechanisms through which p53 controls ILTV infection, meanwhile expand our knowledge of chicken p53 target genes.

Structural Insights into the Mechanism of HIV-1 Tat Secretion from the Plasma Membrane

Human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) is a small, intrinsically disordered basic protein that plays diverse roles in the HIV-1 replication cycle, including promotion of efficient viral RNA transcription. Tat is released by infected cells and subsequently absorbed by healthy cells, thereby contributing to HIV-1 pathogenesis including HIV-associated neurocognitive disorder. It has been shown that, in HIV-1-infected primary CD4 T-cells, Tat accumulates at the plasma membrane (PM) for secretion, a mechanism mediated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural basis for Tat interaction with the PM and thereby secretion is lacking. Herein, we employed NMR and biophysical methods to characterize Tat86 (86 amino acids) interactions with PI(4,5)P2 and lipid nanodiscs (NDs). Our data revealed that Arg49, Lys50 and Lys51 (RKK motif) constitute the PI(4,5)P2 binding site, that Tat86 interaction with lipid NDs is dependent on PI(4,5)P2 and phosphatidylserine (PS), and that the arginine-rich motif (RRQRRR) preferentially interacts with PS. Furthermore, we show that Trp11, previously implicated in Tat secretion, penetrates deeply in the membrane; substitution of Trp11 severely reduced Tat86 interaction with membranes. Deletion of the entire highly basic region and Trp11 completely abolished Tat86 binding to lipid NDs. Our data support a mechanism by which HIV-1 Tat secretion from the PM is mediated by a tripartite signal consisting of binding of the RKK motif to PI(4,5)P2, arginine-rich motif to PS, and penetration of Trp11 in the membrane. Altogether, these findings provide new insights into the molecular requirements for Tat binding to membranes during secretion.

The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends onthe membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but the molecular mechanism of activation by PI(4,5)P2 is not known. As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PI(4,5)P2via molecular modeling. We identified putative contact residues for PI(4,5)P2 in the pre-S1 segment, the S4-S5 linker, and the proximal C-terminal TRP domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PI(4,5)P2 levels. Changes in ligand-binding affinities via molecular mechanics/generalized Born surface area (MM/GBSA) showed reduced PI(4,5)P2 affinity for the mutants. Mutating PI(4,5)P2-interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate, pointing to an allosteric interaction between PI(4,5)P2 and pregnenolone sulfate. Similarly, mutating residues in the PI(4,5)P2 binding site in TRPM8 resulted in increased sensitivity to PI(4,5)P2 depletion and reduced sensitivity to menthol. Mutations of most PI(4,5)P2-interacting residues in TRPM3 also increased sensitivity to inhibition byGβγ, indicating allosteric interaction between Gβγ and PI(4,5)P2 regulation. Disease-associated gain-of-function TRPM3 mutations on the other hand resulted in no change of PI(4,5)P2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P2, its relationship to endogenous activators and inhibitors, as well as identify similarities and differences between PI(4,5)P2 regulation of TRPM3 and TRPM8.

Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein Represses Cellular MDM2 Expression via Suppressing the Sp1- and p53-Mediated Transactivation.

The Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded ORF50 protein is a potent transcriptional activator essential for triggering KSHV lytic reactivation. Despite extensive studies, little is known about whether ORF50 possesses the ability to repress gene expression or has an antagonistic action to cellular transcription factors. Previously, we demonstrated that human oncoprotein MDM2 can promote the degradation of ORF50 protein. Herein, we show that abundant ORF50 expression in cells can conversely downregulate MDM2 expression via repressing both the upstream (P1) and internal (P2) promoters of the MDM2 gene. Deletion analysis of the MDM2 P1 promoter revealed that there were two ORF50-dependent negative response elements located from −102 to −63 and from −39 to +1, which contain Sp1-binding sites. For the MDM2 P2 promoter, the ORF50-dependent negative response element was identified in the region from −110 to −25, which is coincident with the location of two known p53-binding sites. Importantly, we further demonstrated that overexpression of Sp1 or p53 in cells indeed upregulated MDM2 expression; however, coexpression with ORF50 protein remarkably reduced the Sp1- or p53-mediated MDM2 upregulation. Collectively, our findings propose a reciprocal negative regulation between ORF50 and MDM2 and uncover that ORF50 decreases MDM2 expression through repressing Sp1- and p53-mediated transactivation.

P53 Binding Sites in Long Terminal Repeat 5Hs (LTR5Hs) of Human Endogenous Retrovirus K Family (HML-2 Subgroup) Play Important Roles in the Regulation of LTR5Hs Transcriptional Activity.

ABSTRACTThe long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) are distributed throughout the human genome and provide favorable conditions to regulate the expression of their adjacent genes. HML-2 is the most biologically active subgroup of the HERV-K family, and expression of its members has been associated with many cancer types. The LTRs of HML-2 have been classified into three subgroups (LTR5A, LTR5B, and LTR5Hs) based on phylogenetic analyses. The current study aimed to explore the LTR transcriptional activity differences among the three subtypes and further explore the underlying factors. A total of 43 LTR5A elements, 62 LTR5B elements, and 194 LTR5Hs elements were selected. A phylogenetic tree showed that the LTR5Hs group was clearly separated from the LTR5A and LTR5B groups. A luciferase reporter assay indicated that LTR5Hs had the strongest promoter activity, followed by LTR5A and LTR5B. To investigate the underlying factors, LTR5Hs was divided into 4 sections, and the homologous fragments in LTR5B were replaced successively. Replacement of the third section (−263 to 0) significantly increased LTR5B activity. Subsequent mutation experiments revealed that the increased transcriptional activity was induced by the TATA box and the two p53 binding sites within the section. Further interference with TP53 significantly decreased LTR5Hs transcriptional activity. Chromatin immunoprecipitation (ChIP) and CUT&Tag experiments finally confirmed the direct binding of the p53 protein with the two LTR5Hs p53 binding sites. Overall, the two p53 binding sites in the third section (−263 to 0) of LTR5Hs were revealed to play critical roles in the difference in transcriptional activity among the three subtypes.IMPORTANCE Human endogenous retroviruses (HERVs) were integrated into the human genome in ancient times and have been coevolving with the host. Since the Human Genome Project, HERVs have attracted increasing attention. Many studies have focused on their characterization, evolution, and biological function. In particular, the expression of HERV-K has been associated with many diseases, such as germ cell tumors, neurotoxicity, ovarian cancer, prostate cancer, and melanoma. Indeed, two HML-2-produced proteins, Np9 and Rec, are associated with certain cancers. However, their roles in these disease associations remain unclear. The current work focused on subgroup HML-2 of HERV-K, which is recognized as the most biologically active subgroup, and aimed to explore the mechanistic basis of transcriptional activity. The results revealed that p53 deeply determined the activity of HML-2 LTR5Hs. p53 is a rather important tumor suppressor protein. It can regulate the expression of genes related to cell cycle arrest, organic processes, and apoptosis in response to cellular stress and is critical for the control of homeostasis. Previous ChIP and expression studies of individual genes suggested that p53 sites in HERV LTRs may be part of the p53 transcription program and directly regulate p53 target genes in a species-specific manner. However, the exact function of p53 in the regulation of HERV LTR expression is largely elusive. Our results clearly demonstrated the interaction between LTR5Hs of HML-2 and p53. They are of great significance for the future comprehensive study of the physiological and pathological functions of LTRs of HERVs.