Structural basis for the intracellular regulation of ferritin degradation.

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.

The evolutionary conserved TLDc domain defines a new class of (H+)V-ATPase interacting proteins.

We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved "TLDc" domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells.

Multiple functions and essential roles of nuclear receptor coactivators of bHLH-PAS family.

Classical non-peptide hormones, such as steroids, retinoids, thyroid hormones, vitamin D3 and their derivatives including prostaglandins, benzoates, oxysterols, and bile acids, are collectively designated as small lipophilic ligands, acting via binding to the nuclear receptors (NRs). The NRs form a large superfamily of transcription factors that participate virtually in every key biological process. They control various aspects of animal development, fertility, gametogenesis, and numerous metabolic pathways, and can be misregulated in many types of cancers. Their enormous functional plasticity, as transcription factors, relates in part to NR-mediated interactions with plethora of coregulatory proteins upon ligand binding to their ligand binding domains (LBD), or following covalent modification. Here, we review some general views of a specific group of NR coregulators, so-called nuclear receptor coactivators (NRCs) or steroid receptor coactivators (SRCs) and highlight some of their unique functions/roles, which are less extensively mentioned and discussed in other reviews. We also try to pinpoint few neglected moments in the cooperative action of SRCs, which may also indicate their variable roles in the hormone-independent signaling pathways.

NCOA4 Deficiency Impairs Systemic Iron Homeostasis.

The cargo receptor NCOA4 mediates autophagic ferritin degradation. Here we show that NCOA4 deficiency in a knockout mouse model causes iron accumulation in the liver and spleen, increased levels of transferrin saturation, serum ferritin, and liver hepcidin, and decreased levels of duodenal ferroportin. Despite signs of iron overload, NCOA4-null mice had mild microcytic hypochromic anemia. Under an iron-deprived diet (2-3 mg/kg), mice failed to release iron from ferritin storage and developed severe microcytic hypochromic anemia and ineffective erythropoiesis associated with increased erythropoietin levels. When fed an iron-enriched diet (2 g/kg), mice died prematurely and showed signs of liver damage. Ferritin accumulated in primary embryonic fibroblasts from NCOA4-null mice consequent to impaired autophagic targeting. Adoptive expression of the NCOA4 COOH terminus (aa 239-614) restored this function. In conclusion, NCOA4 prevents iron accumulation and ensures efficient erythropoiesis, playing a central role in balancing iron levels in vivo.

Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells.

RNA splicing is a fundamental process for protein synthesis. Recent studies have reported that drugs that inhibit splicing have cytotoxic effects on various tumor cell lines. In this report, we demonstrate that depletion of SNW1, a component of the spliceosome, induces apoptosis in breast cancer cells. Proteomics and biochemical analyses revealed that SNW1 directly associates with other spliceosome components, including EFTUD2 (Snu114) and SNRNP200 (Brr2). The SKIP region of SNW1 interacted with the N-terminus of EFTUD2 as well as two independent regions in the C-terminus of SNRNP200. Similar to SNW1 depletion, knockdown of EFTUD2 increased the numbers of apoptotic cells. Furthermore, we demonstrate that exogenous expression of either the SKIP region of SNW1 or the N-terminus region of EFTUD2 significantly promoted cellular apoptosis. Our results suggest that the inhibition of SNW1 or its associating proteins may be a novel therapeutic strategy for cancer treatment.

Subtype-specific modulation of estrogen receptor-coactivator interaction by phosphorylation.

The estrogen receptor (ER) is the number one target for the treatment of endocrine responsive breast cancer and remains a highly attractive target for new drug development. Despite considerable efforts to understand the role of ER post-translational modifications (PTMs), the complexity of these modifications and their impact, at the molecular level, are poorly understood. Using a chemical biology approach, fundamentally rooted in an efficient protein semisynthesis of tyrosine phosphorylated ER constructs, the complex role of the ER tyrosine phosphorylation is addressed here for the first time on a molecular level. The semisynthetic approach allows for the site-specific introduction of PTMs as well as biophysical probes. A combination of biophysical techniques, including NMR, with molecular dynamics studies reveals the role of the phosphorylation of the clinically relevant tyrosine 537 (Y537) in ERα and the analogous tyrosine (Y488) in ERß. Phosphorylation has important effects on the dynamics of the ER Helix 12, which is centrally involved in receptor activity regulation, and on its interplay with ligand and cofactor binding, but with differential regulatory effects of the analogous PTMs on the two ER subtypes. Combined, the results bring forward a novel molecular model of a phosphorylation-induced subtype specific ER modulatory mechanism, alternative to the widely accepted ligand-induced activation mechanism.

Yorkie promotes transcription by recruiting a histone methyltransferase complex.

Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. We report that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. We show that Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr) methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie's ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie's mammalian homolog YAP. Our results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification.

Transcriptional coregulators: fine-tuning metabolism.

Metabolic homeostasis requires that cellular energy levels are adapted to environmental cues. This adaptation is largely regulated at the transcriptional level, through the interaction between transcription factors, coregulators, and the basal transcriptional machinery. Coregulators, which function as both metabolic sensors and transcriptional effectors, are ideally positioned to synchronize metabolic pathways to environmental stimuli. The balance between inhibitory actions of corepressors and stimulatory effects of coactivators enables the fine-tuning of metabolic processes. This tight regulation opens therapeutic opportunities to manage metabolic dysfunction by directing the activity of cofactors toward specific transcription factors, pathways, or cells/tissues, thereby restoring whole-body metabolic homeostasis.

4,4'-Unsymmetrically substituted 3,3'-biphenyl alpha helical proteomimetics as potential coactivator binding inhibitors.

A series of unsymmetrically substituted biphenyl compounds was designed as alpha helical proteomimetics with the aim of inhibiting the binding of coactivator proteins to the nuclear hormone receptor coactivator binding domain. These compounds were synthesized in good overall yields in seven steps starting from 2-bromoanisole. The final products were evaluated using cotransfection reporter gene assays and mammalian two-hybrid competitive inhibition assays to demonstrate their effectiveness as competitive binding inhibitors. The results from this study indicate that these proteomimetics possess the ability to inhibit coactivator-receptor interactions, but via a mixed mode of inhibition.

Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.

Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.

PRIP (phospholipase C-related but catalytically inactive protein) inhibits exocytosis by direct interactions with syntaxin 1 and SNAP-25 through its C2 domain.

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K(+)-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.

Targeted disruption of the p160 coactivator interface of androgen receptor (AR) selectively inhibits AR activity in both androgen-dependent and castration-resistant AR-expressing prostate cancer cells.

The evidence that androgen blockade-resistant prostate cancer, termed castration resistant, remains androgen receptor (AR) dependent is compelling. AR is re-activated through multiple mechanisms including expression of constitutively active splice variants that lack hormone binding domains (HBDs). This highlights the need to develop therapies that target regions other than the HBD. Because the p160 coactivators interact most strongly with the amino-terminus of AR, we examined the consequences of disrupting this interaction. We identified two overlapping SRC-1 peptides that interact with AR, but not with progesterone receptor. These peptides reduce AR and AR variant AR-V7 dependent induction of an AR responsive reporter. Using mammalian two hybrid assays, we found that the peptides interrupt the AR/SRC-1, AR/SRC-2 and AR N/C interactions, but not SRC-1/CARM-1 interactions. Consistent with the SRC-1 dependence of induced, but not repressed genes, in LNCaP cells, the peptides inhibited hormone dependent induction of endogenous target genes including PSA and TMPRSS2, but did not block AR dependent repression of UGT2B17 or inhibit vitamin D receptor activity. Simultaneous detection of SRC-1 peptides and PSA by double immunofluorescence in transfected LNCaP cells clearly demonstrated a strong reduction in PSA levels in cells expressing the peptides. The peptides also inhibited the AR dependent expression of PSA in castration resistant C4-2 cells. Moreover they inhibited androgen dependent proliferation of LNCaP cells and proliferation of C4-2 cells in androgen depleted medium without affecting AR negative PC-3 cells. Thus, the p160 coactivator binding site is a novel potential therapeutic target to inhibit AR activity.

Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish.

The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel ß-sheets form a central ß-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

Computational studies of LXR molecular interactions reveal an allosteric communication pathway.

The liver X receptor, LXRα, is an important regulator of genes involved in metabolism and inflammation. The mechanism of communication between the cofactor peptide and the ligand in the ligand-binding pocket is a crucial and often discussed issue for the nuclear receptors (NRs), but such allosteric signaling pathways are difficult to detect and the transmission mechanism remains elusive. Here, we apply the anisotropic thermal diffusion method to the LXRα with bound coactivator and ligand. We detected a possible communication pathway between the coactivator peptide and the ligand. The signal is transmitted both through the receptor backbone and side chains. A key signaling residue is the first leucine in the cofactor peptide recognition motif LXXLL, which is conserved within the NR cofactors, suggesting a general mechanism for allosteric signaling. Furthermore, we studied the LXR receptor and cofactor molecular interactions in detail using molecular dynamics simulations. The protein-protein interaction patterns in the complexes of nine different cofactor peptides and holo-LXRα were characterized, revealing the importance of the receptor-cofactor charge clamp interaction. Specific, but infrequently occurring interactions were observed toward the cofactor peptide C-terminal residues. Thus, additional specificity between LXRα and its cofactors is likely to be found in molecular interactions outside the cofactor peptide or in other biological factors.

Quasi-anharmonic analysis reveals intermediate states in the nuclear co-activator receptor binding domain ensemble.

The molten globule nuclear receptor co-activator binding domain (NCBD) of CREB binding protein (CBP) selectively recruits transcription co-activators (TCAs) during the formation of the transcription preinitiation complex. NCBD:TCA interactions have been implicated in several cancers, however, the mechanisms of NCBD:TCA recognition remain uncharacterized. NCBD:TCA intermolecular recognition has challenged traditional investigation as both NCBD and several of its corresponding TCAs are intrinsically disordered. Using 40µs of explicit solvent molecular dynamics simulations, we relate the conformational diversity of ligand-free NCBD to its bound configurations. We introduce two novel techniques to quantify the conformational heterogeneity of ligand-free NCBD, dihedral quasi-anharmonic analysis (dQAA) and hierarchical graph-based diffusive clustering. With this integrated approach we find that three of four ligand-bound states are natively accessible to the ligand-free NCBD simulations with root-mean squared deviation (RMSD) less than 2Å These conformations are accessible via diverse pathways while a rate-limiting barrier must be crossed in order to access the fourth bound state.

Dynamics of coregulator-induced conformational perturbations in androgen receptor ligand binding domain.

Androgen receptor (AR) coregulators modulate ligand-induced gene expression in a tissue specific manner. The molecular events that follow coactivator binding to AR and the mechanisms that govern the sequence-specific effects of AR coregulators are poorly understood. Using consensus coactivator sequence D11-FxxLF and biophysical techniques, we show that coactivator association is followed by conformational rearrangement in AR ligand binding domain (AR-LBD) that is enthalpically and entropically favorable with activation energy of 29.8±4.2 kJ/mol. Further characterization of ARA70 and SRC3-1 based consensus sequences reveal that each coactivator induces a distinct conformational state in the dihydrotestosterone:AR-LBD:coactivator complex. Complementary computational modeling revealed that coactivator induced specific alterations in the backbone flexibility of AR-LBD distant from the site of coactivator binding and that the intramolecular rearrangements in AR-LBD backbone induced by the two coactivator peptides were different. These data suggest that coactivators may impart specificity in the transcriptional machinery by changing the steady-state conformation of AR-LBD. These data provide direct evidence that even in the presence of same ligand, AR-LBD can occupy distinct conformational states depending on its interactions with specific coactivators in the tissues. We posit that this coactivator-specific conformational gating may then dictate subsequent binding partners and interaction/affinity for the DNA-response elements.

Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities.

The estrogen receptors, ERα and ERß, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERß. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERß were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity.

Evaluation of in vitro PXR-based assays and in silico modeling approaches for understanding the binding of a structurally diverse set of drugs to PXR.

The pregnane X-receptor (PXR) is a promiscuous nuclear receptor primarily responsible for the induction of genes from the cytochrome P450 3A family. In this study, we used a previously described PXR/SRC tethered protein to establish two in vitro assays for identifying PXR ligands: automated ligand identification system (ALIS) and temperature-dependent circular dichroism (TdCD). Kd values determined by ALIS and TdCD showed good correlations with the EC50 values determined by a PXR luciferase reporter-gene assay for 37 marketed drugs. The same set of compounds was modeled into the PXR ligand-binding domain that takes into consideration the structural variations of five published X-ray structures of PXR-ligand complexes. Major findings from our in silico analysis are as follows. First, the primary determinants for non-binders of PXR are molecular size and shape of the compounds. Low molecular weight (MW<300) compounds were in general found to be non-binders, and those molecules that do not match the shape of the PXR ligand-binding site may also act as a non-binder. Secondly, the favorable hydrophobic interactions, mostly through aromatic π-π interactions, and the presence of suitable hydrogen bond(s) between the compounds and PXR are attributes of strong binders. Thirdly, the structures of the PXR binding domain possess the flexibility that accommodates structurally diverse compounds, while some of the strong binders may also adapt flexible conformations for fitting into the binding site. The results from this study provide a molecular basis for future efforts in reducing/abolishing the PXR-dependent CYP3A4 induction liability.

Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein.

The activity and stability of the tumor suppressor p53 are regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased level of dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and from Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1, residues 2066-2075; Cα2, residues 2081-2092; and Cα3, residues 2095-2105) with a hydrophobic groove into which p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.