Search

Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here, we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg 2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short β-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics., Competing Interests: Conflicts of interests The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brown, Jenkins, Crooks, Surman, Mazur, Xu, Arimilli, Yang, Lane, Fan, Schrump, Linehan, Ripley and Appella.)

The wild-type p53 induced phosphatase 1 (Wip1), a member of the serine/threonine-specific PP2C family, is overexpressed in numerous human cancers. Wip1 dephosphorylates p53 as well as several kinases (such as p38 MAPK, ATM, Chk1, and Chk2) in the DNA damage response pathway that are responsible for maintaining genomic stability and preventing oncogenic transformation. As a result, Wip1 is an attractive target for synthetic inhibitors that could be further developed into therapeutics to treat some cancers. In this study, we report a series of alkyl-substituted N-methylaryl-N'-aryl-4-aminobenzamides and their inhibitory activity of the Wip1 phosphatase. A straightforward synthetic route was developed to synthesize the target compounds from commercially available starting materials. Three different portions (R 1 , R 2 , R 3 ) of the core scaffold were extensively modified to examine structure-activity relationships. This study revealed interesting trends about a new molecular scaffold to inhibit Wip1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Masson SAS.)

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator., Competing Interests: The authors declare no competing financial interest., (Not subject to U.S. Copyright. Published 2022 by American Chemical Society.)

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC-γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC-γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC-γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC-γ1 activation are regulated both independently and additively by recruitment of PLC-γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC-γ1 tetramer, and by tyrosine phosphorylation of PLC-γ1. The recently solved structure of PLC-γ1 indicates that, in the resting state, several PLC-γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC-γ1, enabling contact with its membrane substrate, disinhibition of PLC-γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022. Published by Elsevier Inc.)

PPM1D/Wip1 is a negative regulator of the tumor suppressor p53 and is overexpressed in several human solid tumors. Recent reports associate gain-of-function mutations of PPM1D in immune cells with worse outcomes for several human cancers. Here we show that mice with genetic knockout of Ppm1d or with conditional knockout of Ppm1d in the hematopoietic system, in myeloid cells, or in neutrophils all display significantly reduced growth of syngeneic melanoma or lung carcinoma tumors. Ppm1d knockout neutrophils infiltrate tumors extensively. Chemical inhibition of Wip1 in human or mouse neutrophils increases anti-tumor phenotypes, p53-dependent expression of co-stimulatory ligands, and proliferation of co-cultured cytotoxic T cells. These results suggest that inhibition of Wip1 in neutrophils enhances immune anti-tumor responses.

Because of microbicide noncompliance and lack of a durable, highly effective vaccine, a combined approach might improve HIV prophylaxis. We tested whether a vaccine-microbicide combination would enhance protection against SIV infection in rhesus macaques. Four macaque groups included vaccine only, vaccine-microbicide, microbicide only, and controls. Vaccine groups were primed twice mucosally with replicating adenovirus type 5 host range mutant SIV env/rev, gag, and nef recombinants and boosted twice i.m. with SIV gp120 proteins in alum. Controls and the microbicide-only group received adenovirus type 5 host range mutant empty vector and alum. The microbicide was SAMT-247, a 2-mercaptobenzamide thioester that targets the viral nucleocapsid protein NCp7, causing zinc ejection and preventing RNA encapsidation. Following vaccination, macaques were challenged intravaginally with repeated weekly low doses of SIV mac251 administered 3 h after application of 0.8% SAMT-247 gel (vaccine-microbicide and microbicide groups) or placebo gel (vaccine-only and control groups). The microbicide-only group exhibited potent protection; 10 of 12 macaques remained uninfected following 15 SIV challenges. The vaccine-only group developed strong mucosal and systemic humoral and cellular immunity but did not exhibit delayed acquisition compared with adjuvant controls. However, the vaccine-microbicide group exhibited significant acquisition delay compared with both control and vaccine-only groups, indicating further exploration of the combination strategy is warranted. Impaired protection in the vaccine-microbicide group compared with the microbicide-only group was not attributed to a vaccine-induced increase in SIV target cells. Possible Ab-dependent enhancement will be further investigated. The potent protection provided by SAMT-247 encourages its movement into human clinical trials.

This year marks the 40th anniversary of the initial identification of p53 as a transformation-related Ag, which was the result of our effort to identify an antigenically distinct tumor Ag of a chemically induced mouse tumor and develop a cancer vaccine. Many researchers at the time viewed this effort as folly. Since then, its characterization has progressed from being an attractive cancer vaccine candidate to recognition as a key player in regulating critical pathways controlling the cell cycle and oncogenesis. Advances in molecular immunology and oncology have enhanced the role of p53 in both fields. It is now apparent that p53 plays a critical role in controlling immune recognition and responses in normal tissues as well as the tumor microenvironment. Together with the advances in clinical implementation of p53-based cancer immunotherapy, they highlight the importance of p53 in many areas of basic and translational cancer research., (Copyright © 2020 by The American Association of Immunologists, Inc.)

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, P i We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens., (© 2019 Clausse et al.)

Dysregulated immune responses are essential underlying causes of a plethora of pathologies including cancer, autoimmunity, and immunodeficiency. We here investigated 4 patients from unrelated families presenting with immunodeficiency, autoimmunity, and malignancy. We identified 4 distinct homozygous mutations in TNFRSF9 encoding the tumor necrosis factor receptor superfamily member CD137/4-1BB, leading to reduced, or loss of, protein expression. Lymphocytic responses crucial for immune surveillance, including activation, proliferation, and differentiation, were impaired. Genetic reconstitution of CD137 reversed these defects. CD137 deficiency is a novel inborn error of human immunity characterized by lymphocytic defects with early-onset Epstein-Barr virus (EBV)-associated lymphoma. Our findings elucidate a functional role and relevance of CD137 in human immune homeostasis and antitumor responses., (© 2019 by The American Society of Hematology.)

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited., Competing Interests: The authors declare no conflict of interest.

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg 2+ or Mn 2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn 2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1A cat , complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1A cat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1A cat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

The wild-type p53 induced phosphatase 1, Wip1 (PP2Cδ), is a protein phosphatase 2C (PP2C) family serine/threonine phosphatase that negatively regulates the function of the tumor suppressor p53 and several of its positive regulators such as ATM, Chk1, Chk2, Mdm2, and p38 MAPK. Wip1 dephosphorylates and inactivates its protein targets, which are critical for cellular stress responses. Additionally, Wip1 is frequently amplified and overexpressed in several human cancer types. Because of its negative role in regulating the function of tumor suppressor proteins, Wip1 has been identified as a potential therapeutic target in various types of cancers. Based on a recently reported Wip1 inhibitor (G-1), we performed an extensive structure-activity relationship (SAR) analysis. This led us to interesting findings in SAR trends and to the discovery of new chemical analogues with good specificity and bioavailability., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70 T293A ) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70 T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex., Competing Interests: The authors declare no conflict of interest.

TP53 mutants (mutp53) are involved in the pathogenesis of most human cancers. Specific mutp53 proteins gain oncogenic functions (GOFs) distinct from the tumor suppressor activity of the wild-type protein. Tumor-associated macrophages (TAMs), a hallmark of solid tumors, are typically correlated with poor prognosis. Here, we report a non-cell-autonomous mechanism, whereby human mutp53 cancer cells reprogram macrophages to a tumor supportive and anti-inflammatory state. The colon cancer cells harboring GOF mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their miR-1246-dependent reprogramming into a cancer-promoting state. Mutp53-reprogammed TAMs favor anti-inflammatory immunosuppression with increased activity of TGF-β. These findings, associated with poor survival in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis.

Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions.

The strategy of simultaneously attacking multiple targets is worthy of exploration in the field of microbicide development to combat HIV-1 sequence diversity and minimize the transmission of resistant variants. A combination of S-acyl-2-mercaptobenzamide thioester-10 (SAMT10), an inhibitor of the HIV-1 nucleocapsid protein (NCp7), and the fusion inhibitor sifuvirtide (SFT) may exert synergistic effects, since SFT can block viral fusion at an early stage of the viral cycle and SAMT10 can disrupt viral particles at a later stage. In this study, we investigated the effect of the combination of SAMT10 and SFT on HIV-1 infection using in vitro cell culture and ex vivo mucosal explant models. A range of doses for each compound was tested at 10-fold serial dilutions based on their 50% effective concentrations (EC 50 ). We observed a synergistic effect of SAMT10 and SFT in vitro against both the laboratory-adapted HIV-1 strain HIV-1 IIIB (subtype B, X4) and three pseudotyped viruses prevalent in Chinese sexually transmitted populations (SVPB16 (subtype B, R5), SVPC12 (subtype C, R5) and SH1.81 (CRF01_AE, R5)). In the ex vivo study, the EC 50 values of the inhibitor combinations were reduced 1.5- to 2-fold in colorectal mucosal explants compared to treatment with SAMT10 or SFT alone by using with HIV-1 IIIB . These results may provide a novel strategy for microbicide development against HIV-1 sexual transmission.

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn 2+ or Mg 2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn 2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg 2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange-mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg 2+ , metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30-300 s. Submillimolar Mg 2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192-200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg 2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg 2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.

Given the paucity of druggable mutations in high-risk neuroblastoma (NB), we undertook chromatin-focused small interfering RNA and chemical screens to uncover epigenetic regulators critical for the differentiation block in high-risk NB. High-content Opera imaging identified 53 genes whose loss of expression led to a decrease in NB cell proliferation and 16 also induced differentiation. From these, the secondary chemical screen identified SETD8, the H4 K20me1 methyltransferase, as a druggable NB target. Functional studies revealed that SETD8 ablation rescued the pro-apoptotic and cell-cycle arrest functions of p53 by decreasing p53 K382me1 , leading to activation of the p53 canonical pathway. In pre-clinical xenograft NB models, genetic or pharmacological (UNC0379) SETD8 inhibition conferred a significant survival advantage, providing evidence for SETD8 as a therapeutic target in NB., (Published by Elsevier Inc.)

Cancer stem cells (CSC) typically over-express aldehyde dehydrogenase (ALDH). Thus, ALDHbright tumor cells represent targets for developing novel cancer prevention/treatment interventions. Loss of p53 function is a common genetic event during cancer development wherein small molecular weight compounds (SMWC) that restore p53 function and reverse tumor growth have been identified. Here, we focused on two widely studied p53 SMWC, CP-31398 and PRIMA-1, to target ALDHbright CSC in human breast, endometrial and pancreas carcinoma cell lines expressing mutant or wild type (WT) p53. CP-31398 and PRIMA-1 significantly reduced CSC content and sphere formation by these cell lines in vitro. In addition, these agents were more effective in vitro against CSC compared to cisplatin and gemcitabine, two often-used chemotherapeutic agents. We also tested a combinatorial treatment in methylcholantrene (MCA)-treated mice consisting of p53 SMWC and p53-based vaccines. Yet using survival end-point analysis, no increased efficacy in the presence of either p53 SMWC alone or with vaccine compared to vaccine alone was observed. These results may be due, in part, to the presence of immune cells, such as activated lymphocytes expressing WT p53 at levels comparable to some tumor cells, wherein further increase of p53 expression by p53 SMWC may alter survival of these immune cells and negatively impact an effective immune response. Continuous exposure of mice to MCA may have also interfered with the action of these p53 SMWC, including potential direct interaction with MCA. Nonetheless, the effect of p53 SMWC on CSC and cancer treatment remains of great interest., Competing Interests: The authors do not have any potential conflicts of interest related to this research.

Protein interactions are fundamental to the proper functioning of cells, and aberrant formation or regulation of protein interactions is at the heart of many diseases, including cancer. The advancement of methods to study the identity, function, and regulation of protein complexes makes possible the understanding of how those complexes malfunction in human diseases. New methodologies in mass spectrometry, microscopy, and protein structural analysis are rapidly advancing the amount and quality of the data, as well as the level of detail that can be obtained from experiments. With this progress, the questions that can be addressed and the biological landscape are changing. This series of minireviews highlights methodological advances and how they have been applied in novel ways to explore the function and regulation of pathways and dynamic networks in cells., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells., (© 2015 Di Donato et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The p53 tumor suppressor is a critical mediator of the cellular response to stress. The N-terminal transactivation domain of p53 makes protein interactions that promote its function as a transcription factor. Among those cofactors is the histone acetyltransferase p300, which both stabilizes p53 and promotes local chromatin unwinding. Here, we report the nuclear magnetic resonance solution structure of the Taz2 domain of p300 bound to the second transactivation subdomain of p53. In the complex, p53 forms an α-helix between residues 47 and 55 that interacts with the α1-α2-α3 face of Taz2. Mutational analysis indicated several residues in both p53 and Taz2 that are critical for stabilizing the interaction. Finally, further characterization of the complex by isothermal titration calorimetry revealed that complex formation is pH-dependent and releases a bound chloride ion. This study highlights differences in the structures of complexes formed by the two transactivation subdomains of p53 that may be broadly observed and play critical roles in p53 transcriptional activity.

p53 is dynamically regulated through various posttranslational modifications (PTMs), which differentially modulate its function and stability. The dimethylated marks p53K370me2 and p53K382me2 are associated with p53 activation or stabilization and both are recognized by the tandem Tudor domain (TTD) of 53BP1, a p53 cofactor. Here we detail the molecular mechanisms for the recognition of p53K370me2 and p53K382me2 by 53BP1. The solution structures of TTD in complex with the p53K370me2 and p53K382me2 peptides show a remarkable plasticity of 53BP1 in accommodating these diverse dimethyllysine-containing sequences. We demonstrate that dimeric TTDs are capable of interacting with the two PTMs on a single p53K370me2K382me2 peptide, greatly strengthening the 53BP1-p53 interaction. Analysis of binding affinities of TTD toward methylated p53 and histones reveals strong preference of 53BP1 for p53K382me2, H4K20me2, and H3K36me2 and suggests a possible role of multivalent contacts of 53BP1 in p53 targeting to and accumulation at the sites of DNA damage., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Individual posttranslational modifications (PTMs) of p53 mediate diverse p53-dependent responses; however, much less is known about the combinatorial action of adjacent modifications. Here, we describe crosstalk between the early DNA damage response mark p53K382me2 and the surrounding PTMs that modulate binding of p53 cofactors, including 53BP1 and p300. The 1.8 Å resolution crystal structure of the tandem Tudor domain (TTD) of 53BP1 in complex with p53 peptide acetylated at K381 and dimethylated at K382 (p53K381acK382me2) reveals that the dual PTM induces a conformational change in p53. The α-helical fold of p53K381acK382me2 positions the side chains of R379, K381ac, and K382me2 to interact with TTD concurrently, reinforcing a modular design of double PTM mimetics. Biochemical and nuclear magnetic resonance analyses show that other surrounding PTMs, including phosphorylation of serine/threonine residues of p53, affect association with TTD. Our findings suggest a novel PTM-driven conformation switch-like mechanism that may regulate p53 interactions with binding partners., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

SIRT1, the mammalian homolog of yeast Sir2, is a founding member of a family of 7 protein and histone deacetylases that are involved in numerous biological functions. Previous studies revealed that SIRT1 deficiency results in genome instability, which eventually leads to cancer formation, yet the underlying mechanism is unclear. To investigate this, we conducted a proteomics study and found that SIRT1 interacted with many proteins involved in replication fork protection and origin firing. We demonstrated that loss of SIRT1 resulted in increased replication origin firing, asymmetric fork progression, defective intra-S-phase checkpoint, and chromosome damage. Mechanistically, SIRT1 deacetylates and affects the activity of TopBP1, which plays an essential role in DNA replication fork protection and replication origin firing. Our study demonstrated that ectopic over-expression of the deacetylated form of TopBP1 in SIRT1 mutant cells repressed replication origin firing, while the acetylated form of TopBP1 lost this function. Thus, SIRT1 acts upstream of TopBP1 and plays an essential role in maintaining genome stability by modulating DNA replication fork initiation and the intra-S-phase cell cycle checkpoint.