An AIB1 Isoform Alters Enhancer Access and Enables Progression of Early-Stage Triple-Negative Breast Cancer.

AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an "enabler" phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population. SIGNIFICANCE: A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.

Thevebioside, the active ingredient of traditional Chinese medicine, promotes ubiquitin-mediated SRC-3 degradation to induce NSCLC cells apoptosis.

Non-small cell lung cancer (NSCLC) accounts for more than 85% of lung cancer with high incidence and mortality. Accumulating studies have shown that traditional Chinese medicine (TCM) and its active ingredients have good anti-tumor activity. However, the anti-tumor effect of Thevebioside (THB), an active ingredient from TCM, is still unknown in NSCLC. In this study, to our best knowledge, it was the first time to report the underlying mechanism of its tumor-suppressive activity in NSCLC based on our previous high-throughput screening data. We further demonstrated that THB effectively inhibited the proliferation of NSCLC cells (A549 and H460) by inducing cellular apoptosis rather than cell cycle arrest. Notably, it was demonstrated that SRC-3 was significantly down-regulated after THB treatment dependent on ubiquitin-proteasome-mediated degradation, which subsequently inhibited the IGF-1R-PI3K-AKT signaling pathway and promoted apoptosis via both in vivo and in vitro experiments. Collectively, THB exerted inhibitory effect on tumor growth of NSCLC through inhibiting SRC-3 mediated IGF-1R-PI3K-AKT signaling by ubiquitination to induce cellular apoptosis with minimal toxicity no matter in vitro or vivo.

Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes.

Steroid receptors activate gene transcription by recruiting coactivators to initiate transcription of their target genes. For most nuclear receptors, the ligand-dependent activation function domain-2 (AF-2) is a primary contributor to the nuclear receptor (NR) transcriptional activity. In contrast to other steroid receptors, such as ERα, the activation function of androgen receptor (AR) is largely dependent on its ligand-independent AF-1 located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different AF domain from other receptors despite that NRs share similar domain organizations. Here, we present cryoelectron microscopy (cryo-EM) structures of DNA-bound full-length AR and its complex structure with key coactivators, SRC-3 and p300. AR dimerization follows a unique head-to-head and tail-to-tail manner. Unlike ERα, AR directly contacts a single SRC-3 and p300. The AR NTD is the primary site for coactivator recruitment. The structures provide a basis for understanding assembly of the AR:coactivator complex and its domain contributions for coactivator assembly and transcriptional regulation.

Mapping the transition state for a binding reaction between ancient intrinsically disordered proteins.

Intrinsically disordered protein domains often have multiple binding partners. It is plausible that the strength of pairing with specific partners evolves from an initial low affinity to a higher affinity. However, little is known about the molecular changes in the binding mechanism that would facilitate such a transition. We previously showed that the interaction between two intrinsically disordered domains, NCBD and CID, likely emerged in an ancestral deuterostome organism as a low-affinity interaction that subsequently evolved into a higher-affinity interaction before the radiation of modern vertebrate groups. Here we map native contacts in the transition states of the low-affinity ancestral and high-affinity human NCBD/CID interactions. We show that the coupled binding and folding mechanism is overall similar but with a higher degree of native hydrophobic contact formation in the transition state of the ancestral complex and more heterogeneous transient interactions, including electrostatic pairings, and an increased disorder for the human complex. Adaptation to new binding partners may be facilitated by this ability to exploit multiple alternative transient interactions while retaining the overall binding and folding pathway.

Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism.

Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.

A structurally heterogeneous transition state underlies coupled binding and folding of disordered proteins.

Many intrinsically disordered proteins (IDPs) attain a well-defined structure in a coupled folding and binding reaction with another protein. Such reactions may involve early to late formation of different native structural regions along the reaction pathway. To obtain insights into the transition state for a coupled binding and folding reaction, we performed restrained molecular dynamics simulations using previously determined experimental binding Φb values of the interaction between two IDP domains: the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors (ACTR) and the nuclear co-activator binding domain (NCBD) of CREB-binding protein, each forming three well-defined α-helices upon binding. These simulations revealed that both proteins are largely disordered in the transition state for complex formation, except for two helices, one from each domain, that display a native-like structure. The overall transition state structure was extended and largely dynamic with many weakly populated contacts. To test the transition state model, we combined site-directed mutagenesis with kinetic experiments, yielding results consistent with overall diffuse interactions and formation of native intramolecular interactions in the third NCBD helix during the binding reaction. Our findings support the view that the transition state and, by inference, any encounter complex in coupled binding and folding reactions are structurally heterogeneous and largely independent of specific interactions. Furthermore, experimental Φb values and Brønsted plots suggested that the transition state is globally robust with respect to most mutations but can display more native-like features for some highly destabilizing mutations, possibly because of Hammond behavior or ground-state effects.

Atomistic Modeling of Intrinsically Disordered Proteins Under Polyethylene Glycol Crowding: Quantitative Comparison with Experimental Data and Implication of Protein-Crowder Attraction.

The malleability of intrinsically disordered proteins (IDPs) has generated great interest in understanding how their conformations respond to crowded cellular environments. Experiments can report gross properties such as fluorescence resonance energy transfer (FRET) efficiency but cannot resolve the conformational ensembles of IDPs and their interactions with macromolecular crowders. Computation can in principle provide the latter information but in practice has been hampered by the enormous expense for realistic modeling of IDPs and crowders and for sufficient conformational sampling. Here, taking advantage of a powerful method called FMAP (fast Fourier transform-based modeling of atomistic protein-crowder interactions), we computed how the conformational ensembles of three IDPs are modified in concentrated polyethylene glycol (PEG) 6000 solutions. We represented the IDPs at the all-atom level and the PEG molecules at a coarse-grained level and calculated the experimental observable, i.e., FRET efficiency. Whereas accounting for only steric repulsion of PEG led to overestimation of crowding effects, quantitative agreement with experimental data was obtained upon including mild IDP-PEG attraction. The present work demonstrates that realistic modeling of IDPs under crowded conditions for direct comparison with experiments is now achievable.

Charge Interactions Can Dominate Coupled Folding and Binding on the Ribosome.

Interactions between emerging nascent polypeptide chains and the ribosome can modulate cotranslational protein folding. However, it has remained unclear how such interactions can affect the binding of nascent chains to their cellular targets. We thus investigated on the ribosome the interaction between two intrinsically disordered proteins of opposite charge, ACTR and NCBD, which form a high-affinity complex in a coupled folding-and-binding reaction. Using fluorescence correlation spectroscopy and arrest-peptide-mediated force measurements in vitro and in vivo, we find that the ACTR-NCBD complex can form cotranslationally but only with ACTR as the nascent chain and NCBD free in solution, not vice versa. We show that this surprising asymmetry in behavior is caused by pronounced charge interactions: attraction of the positively charged nascent chain of NCBD to the negatively charged ribosomal surface competes with complex formation and prevents ACTR binding. In contrast, the negatively charged nascent ACTR is repelled by the ribosomal surface and thus remains available for productively binding its partner. Electrostatic interactions may thus be more important for cotranslational folding and binding than previously thought.

Structural and Functional Impacts of ER Coactivator Sequential Recruitment.

Nuclear receptors recruit multiple coactivators sequentially to activate transcription. This "ordered" recruitment allows different coactivator activities to engage the nuclear receptor complex at different steps of transcription. Estrogen receptor (ER) recruits steroid receptor coactivator-3 (SRC-3) primary coactivator and secondary coactivators, p300/CBP and CARM1. CARM1 recruitment lags behind the binding of SRC-3 and p300 to ER. Combining cryo-electron microscopy (cryo-EM) structure analysis and biochemical approaches, we demonstrate that there is a close crosstalk between early- and late-recruited coactivators. The sequential recruitment of CARM1 not only adds a protein arginine methyltransferase activity to the ER-coactivator complex, it also alters the structural organization of the pre-existing ERE/ERα/SRC-3/p300 complex. It induces a p300 conformational change and significantly increases p300 HAT activity on histone H3K18 residues, which, in turn, promotes CARM1 methylation activity on H3R17 residues to enhance transcriptional activity. This study reveals a structural role for a coactivator sequential recruitment and biochemical process in ER-mediated transcription.

Quantifying Protection in Disordered Proteins Using Millisecond Hydrogen Exchange-Mass Spectrometry and Peptic Reference Peptides.

The extent and location of transient structure in intrinsically disordered proteins (IDPs) provide valuable insights into their conformational ensembles and can lead to a better understanding of coupled binding and folding. Millisecond amide hydrogen exchange (HX) can provide such information, but it is difficult to quantify the degree of transient structuring. One reason is that transiently disordered proteins undergo HX at rates only slightly slower than the rate of amide HX by an unstructured random coil, the chemical HX rate. In this work, we evaluate several different methods of obtaining an accurate model for the chemical HX rate suitable for millisecond hydrogen exchange mass spectrometry (HX-MS) analysis of disordered proteins: (1) calculations using the method of Englander [Bai, Y., et al. (1993) Proteins 17, 75-86], (2) measurement of HX in the presence of 6 M urea or 3 M guanidinium chloride, and (3) measurement of HX by peptide fragments derived directly from the proteins of interest. First, using unstructured model peptides and disordered domains of the activator for thyroid and retinoid receptors and the CREB binding protein as the model IDPs, we show that the Englander method has slight inaccuracies that lead to underestimation of the chemical exchange rate. Second, HX-MS measurements of model peptides show that HX rates are changed dramatically by high concentrations of the denaturant. Third, we find that measurements of HX by reference peptides from the proteins of interest provide the most accurate approach for quantifying the extent of transient structure in disordered proteins by millisecond HX-MS.

Steroid receptor coactivators present a unique opportunity for drug development in hormone-dependent cancers.

Steroid receptor coactivators (SRCs) are essential regulators of nuclear hormone receptor function. SRCs coactivate transcription mediated by hormone stimulation of nuclear receptors and other transcription factors and have essential functions in human physiology and health. The SRCs are over expressed in a number of cancers such as breast, prostate, endometrial and pancreatic cancers where they promote tumor growth, invasion, metastasis and chemo-resistance. With their multiple roles in cancer, the SRCs are promising targets for the development of small molecule agents that can interfere with their function. For instance, perturbing SRC function with small molecule inhibitors and stimulators has been shown to be effective in reducing tumor growth in vivo. These early studies demonstrate that targeting the SRCs might prove effective for cancer treatment and more effort should be made to realize the untapped potential of developing drugs designed to target these coactivators.

Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration.

The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well-understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L-1 and 400 g L-1 of Ficoll, a synthetic polysaccharide, using a recently-developed strong cation exchange-based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275-1282]. Transiently helical regions of ACTR exchanged faster in 300 g L-1 Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L-1 Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L-1 Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L-1 Ficoll is a semi-dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468-1479. © 2017 Wiley Periodicals, Inc.

Quantifying Protein Disorder through Measures of Excess Conformational Entropy.

Intrinsically disordered proteins (IDPs) and proteins with a large degree of disorder are abundant in the proteomes of eukaryotes and viruses, and play a vital role in cellular homeostasis and disease. One fundamental question that has been raised on IDPs is the process by which they offset the entropic penalty involved in transitioning from a heterogeneous ensemble of conformations to a much smaller collection of binding-competent states. However, this has been a difficult problem to address, as the effective entropic cost of fixing residues in a folded-like conformation from disordered amino acid neighborhoods is itself not known. Moreover, there are several examples where the sequence complexity of disordered regions is as high as well-folded regions. Disorder in such cases therefore arises from excess conformational entropy determined entirely by correlated sequence effects, an entropic code that is yet to be identified. Here, we explore these issues by exploiting the order-disorder transitions of a helix in Pbx-Homeodomain together with a dual entropy statistical mechanical model to estimate the magnitude and sign of the excess conformational entropy of residues in disordered regions. We find that a mere 2.1-fold increase in the number of allowed conformations per residue (∼0.7kBT favoring the unfolded state) relative to a well-folded sequence, or ∼2(N) additional conformations for a N-residue sequence, is sufficient to promote disorder under physiological conditions. We show that this estimate is quite robust and helps in rationalizing the thermodynamic signatures of disordered regions in important regulatory proteins, modeling the conformational folding-binding landscapes of IDPs, quantifying the stability effects characteristic of disordered protein loops and their subtle roles in determining the partitioning of folding flux in ordered domains. In effect, the dual entropy model we propose provides a statistical thermodynamic basis for the relative conformational propensities of amino acids in folded and disordered environments in proteins. Our work thus lays the foundation for understanding and quantifying protein disorder through measures of excess conformational entropy.

Estrogen receptor alpha/co-activator interaction assay: TR-FRET.

Time-resolved fluorescence resonance energy transfer, TR-FRET, is a time-gated fluorescence intensity measurement which defines the relative proximity of two biomolecules (e.g., proteins, peptides, or DNA) based on the extent of non-radiative energy transfer between two fluorophores with overlapping emission/excitation spectra. In these assays, an excited lanthanide ion acts as a "donor" that transfers energy to an "acceptor" fluorophore through dipole-dipole interactions. A FRET signal is reported as the ratio of acceptor to donor emission following donor excitation. When a donor-conjugated protein interacts with an acceptor-conjugated protein, the donor and acceptor fluorophores are brought in close proximity allowing energy transfer from the donor to the acceptor resulting in a FRET signal. Because the lanthanide donors have a long emission half-life, the energy transfer measurement can be time-gated, which dramatically reduces assay interference (due to background autofluorescence and direct acceptor excitation) and thereby increases data quality. Here, we describe a TR-FRET assay that monitors the interaction of the estrogen receptor (ER) α ligand binding domain (labeled with a terbium chelate via a streptavidin-biotin interaction) with a sequence of coactivator protein SRC3 (labeled directly with fluorescein) and the disruption of this interaction with a peptide and a small molecule inhibitor.

All in the family: a portrait of a nuclear receptor co-activator complex.

In this issue of Molecular Cell, Yi et al. (2015) use biochemical assays and cryo-EM to determine the molecular architecture of an estrogen receptor (ERα) co-activator complex bound to DNA.

Structure of a biologically active estrogen receptor-coactivator complex on DNA.

Estrogen receptor (ER/ESR1) is a transcription factor critical for development, reproduction, metabolism, and cancer. ER function hinges on its ability to recruit primary and secondary coactivators, yet structural information on the full-length receptor-coactivator complex to complement preexisting and sometimes controversial biochemical information is lacking. Here, we use cryoelectron microscopy (cryo-EM) to determine the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator 3 (SRC-3/NCOA3), and a secondary coactivator (p300/EP300). Our structural model suggests the following assembly mechanism for the complex: each of the two ligand-bound ERα monomers independently recruits one SRC-3 protein via the transactivation domain of ERα; the two SRC-3s in turn bind to different regions of one p300 protein through multiple contacts. We also present structural evidence for the location of activation function 1 (AF-1) in a full-length nuclear receptor, which supports a role for AF-1 in SRC-3 recruitment.

Lysine residues 639 and 673 of mouse Ncoa3 are ubiquitination sites for the regulation of its stability.

Ncoa3 is a transcriptional coactivator involved in a wide range of biological processes. Regulation of Ncoa3 protein stability is important to control its activity precisely. Here, we found that deleting amino acid residues 614-740 of Ncoa3 enhances the protein expression level. Replacing two lysine residues, K639 and K673, within this region by arginine, increases the stability of the luciferase fusion protein as well as Ncoa3 protein. When these two lysine residues are mutated to arginine, the overall ubiquitination level of Ncoa3 decreases, indicating that lysine 639 and 673 are its ubiquitination sites. Taken together, we identified two ubiquitination sites at lysine 639 and 673 of Ncoa3. Ubiquitination of these two lysine residues leads to proteasomal degradation of Ncoa3.

Chaperonin CCT-mediated AIB1 folding promotes the growth of ERα-positive breast cancer cells on hard substrates.

Clinical observations have revealed a strong association between estrogen receptor alpha (ERα)-positive tumors and the development of bone metastases, however, the mechanism underlying this association remains unknown. We cultured MCF-7 (ERα-positive) on different rigidity substrates. Compared with cells grown on more rigid substrates (100 kPa), cells grown on soft substrates (10 kPa) exhibited reduced spreading ability, a lower ratio of cells in the S and G2/M cell cycle phases, and a decreased proliferation rate. Using stable isotope labeling by amino acids (SILAC), we further compared the whole proteome of MCF-7 cells grown on substrates of different rigidity (10 and 100 kPa), and found that the expression of eight members of chaperonin CCT increased by at least 2-fold in the harder substrate. CCT folding activity was increased in the hard substrate compared with the soft substrates. Amplified in breast cancer 1 (AIB1), was identified in CCT immunoprecipitates. CCT folding ability of AIB1 increased on 100-kPa substrate compared with 10- and 30-kPa substrates. Moreover, using mammalian two-hybrid protein-protein interaction assays, we found that the polyglutamine repeat sequence of the AIB1 protein was essential for interaction between CCTζ and AIB1. CCTζ-mediated AIB1 folding affects the cell area spreading, growth rate, and cell cycle. The expressions of the c-myc, cyclin D1, and PgR genes were higher on hard substrates than on soft substrate in both MCF-7 and T47D cells. ERα and AIB1 could up-regulate the mRNA and protein expression levels of the c-myc, cyclin D1, and PgR genes, and that 17 ß-estradiol could enhance this effects. Conversely, 4-hydroxytamoxifen, could inhibit these effects. Taken together, our studies demonstrate that some ERα-positive breast cancer cells preferentially grow on more rigid substrates. CCT-mediated AIB1 folding appears to be involved in the rigidity response of breast cancer cells, which provides novel insight into the mechanisms of bone metastasis.

Mutation in KANK2, encoding a sequestering protein for steroid receptor coactivators, causes keratoderma and woolly hair.

BACKGROUND: The combination of palmoplantar keratoderma and woolly hair is uncommon and reported as part of Naxos and Carvajal syndromes, both caused by mutations in desmosomal proteins and associated with cardiomyopathy. We describe two large consanguineous families with autosomal-recessive palmoplantar keratoderma and woolly hair, without cardiomyopathy and with no mutations in any known culprit gene. The aim of this study was to find the mutated gene in these families. METHODS AND RESULTS: Using whole-exome sequencing, we identified a homozygous missense c.2009C>T mutation in KANK2 in the patients (p.Ala670Val). KANK2 encodes the steroid receptor coactivator (SRC)-interacting protein (SIP), an ankyrin repeat containing protein, which sequesters SRCs in the cytoplasm and controls transcription activation of steroid receptors, among others, also of the vitamin D receptor (VDR). The mutation in KANK2 is predicted to abolish the sequestering abilities of SIP. Indeed, vitamin D-induced transactivation was increased in patient's keratinocytes. Furthermore, SRC-2 and SRC-3, coactivators of VDR and important components of epidermal differentiation, are localised to the nucleus of epidermal basal cells in patients, in contrast to the cytoplasmic distribution in the heterozygous control. CONCLUSIONS: These findings provide evidence that keratoderma and woolly hair can be caused by a non-desmosomal mechanism and further underline the importance of VDR for normal hair and skin phenotypes.

A frustrated binding interface for intrinsically disordered proteins.

Intrinsically disordered proteins are very common in the eukaryotic proteome, and many of them are associated with diseases. Disordered proteins usually undergo a coupled binding and folding reaction and often interact with many different binding partners. Using double mutant cycles, we mapped the energy landscape of the binding interface for two interacting disordered domains and found it to be largely suboptimal in terms of interaction free energies, despite relatively high affinity. These data depict a frustrated energy landscape for interactions involving intrinsically disordered proteins, which is likely a result of their functional promiscuity.