Aurora A-mediated pyruvate kinase M2 phosphorylation promotes biosynthesis with glycolytic metabolites and tumor cell cycle progression.

Cancer cells have distinctive demands for intermediates from glucose metabolism for biosynthesis and energy in different cell cycle phases. However, how cell cycle regulators and glycolytic enzymes coordinate to orchestrate the essential metabolic processes are still poorly characterized. Here, we report a novel interaction between the mitotic kinase, Aurora A, and the glycolytic enzyme, pyruvate kinase M2 (PKM2), in the interphase of the cell cycle. We found Aurora A-mediated phosphorylation of PKM2 at threonine 45. This phosphorylation significantly attenuated PKM2 enzymatic activity by reducing its tetramerization and also promoted glycolytic flux and the branching anabolic pathways. Replacing the endogenous PKM2 with a nonphosphorylated PKM2 T45A mutant inhibited glycolysis, glycolytic branching pathways, and tumor growth in both in vitro and in vivo models. Together, our study revealed a new protumor function of Aurora A through modulating a rate-limiting glycolytic enzyme, PKM2, mainly during the S phase of the cell cycle. Our findings also showed that although both Aurora A and Aurora B kinase phosphorylate PKM2 at the same residue, the spatial and temporal regulations of the specific kinase and PKM2 interaction are context dependent, indicating intricate interconnectivity between cell cycle and glycolytic regulators.

Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis.

Spindle position control is essential for cell fate determination and organogenesis. Early studies indicate the essential role of the evolutionarily conserved Gαi/LGN/NuMA network in spindle positioning. However, the regulatory mechanisms that couple astral microtubules dynamics to the spindle orientation remain elusive. Here we delineated a new mitosis-specific crotonylation-regulated astral microtubule-EB1-NuMA interaction in mitosis. EB1 is a substrate of TIP60, and TIP60-dependent crotonylation of EB1 tunes accurate spindle positioning in mitosis. Mechanistically, TIP60 crotonylation of EB1 at Lys66 forms a dynamic link between accurate attachment of astral microtubules to the lateral cell cortex defined by NuMA-LGN and fine tune of spindle positioning. Real-time imaging of chromosome movements in HeLa cells expressing genetically encoded crotonylated EB1 revealed the importance of crotonylation dynamics for accurate control of spindle orientation during metaphase-anaphase transition. These findings delineate a general signaling cascade that integrates protein crotonylation with accurate spindle positioning for chromosome stability in mitosis.

Inhibitors against Two PDZ Domains of MDA-9 Suppressed Migration of Breast Cancer Cells.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

Fragment-Based Discovery of AF9 YEATS Domain Inhibitors.

YEATS (YAF9, ENL, AF9, TAF14, SAS5) family proteins recognize acylated histones and in turn regulate chromatin structure, gene transcription, and stress signaling. The chromosomal translocations of ENL and mixed lineage leukemia are considered oncogenic drivers in acute myeloid leukemia and acute lymphoid leukemia. However, known ENL YEATS domain inhibitors have failed to suppress the proliferation of 60 tested cancer cell lines. Herein, we identified four hits from the NMR fragment-based screening against the AF9 YEATS domain. Ten inhibitors of new chemotypes were then designed and synthesized guided by two complex structures and affinity assays. The complex structures revealed that these inhibitors formed an extra hydrogen bond to AF9, with respect to known ENL inhibitors. Furthermore, these inhibitors demonstrated antiproliferation activities in AF9-sensitive HGC-27 cells, which recapitulated the phenotype of the CRISPR studies against AF9. Our work will provide the basis for further structured-based optimization and reignite the campaign for potent AF9 YEATS inhibitors as a precise treatment for AF9-sensitive cancers.

Phase separation drives decision making in cell division.

Liquid-liquid phase separation (LLPS) of biomolecules drives the formation of subcellular compartments with distinct physicochemical properties. These compartments, free of lipid bilayers and therefore called membraneless organelles, include nucleoli, centrosomes, heterochromatin, and centromeres. These have emerged as a new paradigm to account for subcellular organization and cell fate decisions. Here we summarize recent studies linking LLPS to mitotic spindle, heterochromatin, and centromere assembly and their plasticity controls in the context of the cell division cycle, highlighting a functional role for phase behavior and material properties of proteins assembled onto heterochromatin, centromeres, and central spindles via LLPS. The techniques and tools for visualizing and harnessing membraneless organelle dynamics and plasticity in mitosis are also discussed, as is the potential for these discoveries to promote new research directions for investigating chromosome dynamics, plasticity, and interchromosome interactions in the decision-making process during mitosis.

Dynamic acetylation of the kinetochore-associated protein HEC1 ensures accurate microtubule-kinetochore attachment.

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.

Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.

Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate important physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with arrestin allosterically regulates the specific conformational states and functional outcomes of remote ß-arrestin 1 proline regions (PRs). The observed NMR chemical shifts of arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment, which was controlled by defined propagation pathways. Moreover, in vitro reconstitution experiments and biophysical results showed that the receptor-arrestin complex promoted SRC kinase activity through an allosteric mechanism. Thus, allosteric regulation of the conformational states of ß-arrestin 1 PRs by GPCRs and the allosteric activation of downstream effectors by arrestin are two important mechanisms underlying GPCR-to-SH3-CP signaling.

Acute changes in colonic PGE2 levels as a biomarker of efficacy after treatment of the Pirc (F344/NTac-Apc am1137) rat with celecoxib.

OBJECTIVE: This study sought to evaluate short-term treatment with COX-2 inhibitors and acute changes in colonic PGE2 levels as predictors of long-term efficacy in a genetic model of colorectal cancer. METHODS: Celecoxib oral suspension (40 mg/kg BID) was dosed to Apc-mutant Pirc (F344/NTac-Apcam1137) rats for 4 days (short-term group), or the equivalent dose of 1500 ppm celecoxib was administered in the diet for 4 months (long-term group). Percent inhibition of colonic PGE2 was calculated, and the reduction in colonic PGE2 was assessed in relation to suppression of adenomatous colon polyps. RESULTS: Colonic mucosa PGE2 was fourfold higher in Pirc than in F344 wild-type rats (21 vs. 5.6 pg/mg epithelial tissue), due at least in part to higher COX-2 expression, and this was confirmed by elevated PGE2-d11 levels in Pirc colonic S9 incubations. In the 4-day study, dose-dependent reductions in PGE2 were observed in colonic epithelium (-33% (P>0.05) and -57% (P=0.0012)), after low- and high-dose celecoxib treatments of 4 mg/kg and 40 mg/kg (bid), respectively. In the 4-month study, 1500 ppm celecoxib suppressed colonic epithelium PGE2 by 43.5%, and tumor multiplicity by 80% (P<0.0015). Suppression of plasma 6-keto PGF1α also was corroborated following long-term treatment with 1500 ppm celecoxib (P<0.05). CONCLUSIONS: Acute changes in colonic mucosa PGE2 provided a rapid means of predicting long-term chemopreventive effects from celecoxib, and might be useful for screening of new COX-2 inhibitor compounds.

A novel single-chain enzyme complex with chain reaction properties rapidly producing thromboxane A2 and exhibiting powerful anti-bleeding functions.

Uncontrollable bleeding is still a worldwide killer. In this study, we aimed to investigate a novel approach to exhibit effective haemostatic properties, which could possibly save lives in various bleeding emergencies. According to the structure-based enzymatic design, we have engineered a novel single-chain hybrid enzyme complex (SCHEC), COX-1-10aa-TXAS. We linked the C-terminus of cyclooxygenase-1 (COX-1) to the N-terminus of the thromboxane A2 (TXA2 ) synthase (TXAS), through a 10-amino acid residue linker. This recombinant COX-1-10aa-TXAS can effectively pass COX-1-derived intermediate prostaglandin (PG) H2 (PGH2 ) to the active site of TXAS, resulting in an effective chain reaction property to produce the haemostatic prostanoid, TXA2 , rapidly. Advantageously, COX-1-10aa-TXAS constrains the production of other pro-bleeding prostanoids, such as prostacyclin (PGI2 ) and prostaglandin E2 (PGE2 ), through reducing the common substrate, PGH2 being passed to synthases which produce aforementioned prostanoids. Therefore, based on these multiple properties, this novel COX-1-10aa-TXAS indicated a powerful anti-bleeding ability, which could be used to treat a variety of bleeding situations and could even be useful for bleeding prone situations, including nonsteroidal anti-inflammatory drugs (NSAIDs)-resulted TXA2 -deficient and PGI2 -mediated bleeding disorders. This novel SCHEC has a great potential to be developed into a biological haemostatic agent to treat severe haemorrhage emergencies, which will prevent the complications of blood loss and save lives.

NMR Fragment-Based Screening against Tandem RNA Recognition Motifs of TDP-43.

The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the 15N-labeled tandem RRM and RRM2, respectively, and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing (HADDOCK). These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.

Giant spontaneous exchange bias obtained by tuning magnetic compensation in samarium ferrite single crystals.

Spontaneous exchange bias (SEB) under zero field cooling (ZFC) has recently attracted lots of attention due to its underlying physics and potential applications. Here we report the giant SEB (GSEB) of SmFeO3 single crystals by tuning magnetic compensation by temperature, which is rather convenient. A SEB field of up to 1 T at 3.9 K after ZFC (-1.4 T at 3.9 K after field cooling) was obtained. The SEB shows reciprocal relationship with remnant magnetization.

The key residue within the second extracellular loop of human EP3 involved in selectively turning down PGE2- and retaining PGE1-mediated signaling in live cells.

Key residues and binding mechanisms of PGE1 and PGE2 on prostanoid receptors are poorly understood due to the lack of X-ray structures for the receptors. We constructed a human EP3 (hEP3) model through integrative homology modeling using the X-ray structure of the ß2-adrenergic receptor transmembrane domain and NMR structures of the thromboxane A2 receptor extracellular loops. PGE1 and PGE2 docking into the hEP3 model showed differing configurations within the extracellular ligand recognition site. While PGE2 could form possible binding contact with S211, PGE1 is unable to form similar contacts. Therefore, S211 could be the critical residue for PGE2 recognition, but is not a significant for PGE1. This prediction was confirmed using HEK293 cells transfected with hEP3 S211L cDNA. The S211L cells lost PGE2 binding and signaling. Interestingly, the S211L cells retained PGE1-mediated signaling. It indicates that S211 within the second extracellular loop is a key residue involved in turning down PGE2 signaling. Our study provided information that S211L within EP3 is the key residue to distinguish PGE1 and PGE2 binding to mediate diverse biological functions at the initial recognition step. The S211L mutant could be used as a model for studying the binding mechanism and signaling pathway specifically mediated by PGE1.

NMR characterization of weak interactions between RhoGDI2 and fragment screening hits.

BACKGROUND: The delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine-nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis. METHODS: Protein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits. RESULTS: We identified three hits of RhoGDI2 using streamlined NMR fragment-based screening. The binding site residues were assigned using non-uniformly sampled Cα- and Hα-based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis. CONCLUSIONS: Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach. GENERAL INTERESTS: Binders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques.

Fluorine Pseudocontact Shifts Used for Characterizing the Protein-Ligand Interaction Mode in the Limit of NMR Intermediate Exchange.

The characterization of protein-ligand interaction modes becomes recalcitrant in the NMR intermediate exchange regime as the interface resonances are broadened beyond detection. Here, we determined the 19 F low-populated bound-state pseudocontact shifts (PCSs) of mono- and di-fluorinated inhibitors of the BRM bromodomain using a highly skewed protein/ligand ratio. The bound-state 19 F PCSs were retrieved from 19 F chemical exchange saturation transfer (CEST) in the presence of the lanthanide-labeled protein, which was termed the 19 F PCS-CEST approach. These PCSs enriched in spatial information enabled the identification of best-fitting poses, which agree well with the crystal structure of a more soluble analog in complex with the BRM bromodomain. This approach fills the gap of the NMR structural characterization of lead-like inhibitors with moderate affinities to target proteins, which are essential for structure-guided hit-to-lead evolution.

NMR Fragment Screening Hit Induces Plasticity of BRD7/9 Bromodomains.

The complex biology associated with inhibition of bromodomain and extra-terminal (BET) domains by chemical probes has attracted increasing attention, and there is a need to identify non-BET bromodomain (BD) inhibitors. Several potent inhibitors of the BRD9 BD have recently been discovered, with anticancer and anti-inflammation activity. However, its paralogue, BRD7 BD, remains unexploited. Here, we identified new chemotypes targeting BRD7 BD by using NMR fragment-based screening. BRD7/9 BDs exhibit similar patterns of chemical-shift perturbation upon the titration of hit compound 1. The crystal structure revealed that 1 repels the Y222 group of BRD9 BD in a similar way to that for butyryllysine, but not acetyllysine and known inhibitors. Hit 1 induced less rearrangement of residue F161 of BRD9 BD than acetyllysine, butyryllysine, and crotonyllysine. Our study provides structural insight into a new generation of butyryllysine mimics for probing the function of BRD7/9 BD.

NMR Backbone Assignment of Large Proteins by Using (13) Cα -Only Triple-Resonance Experiments.

Nuclear magnetic resonance (NMR) is a powerful tool to interrogate protein structure and dynamics residue by residue. However, the prerequisite chemical-shift assignment remains a bottleneck for large proteins due to the fast relaxation and the frequency degeneracy of the (13) Cα nuclei. Herein, we present a covariance NMR strategy to assign the backbone chemical shifts by using only HN(CO)CA and HNCA spectra that has a high sensitivity even for large proteins. By using the peak linear correlation coefficient (LCC), which is a sensitive probe even for tiny chemical-shift displacements, we correctly identify the fidelity of approximately 92 % cross-peaks in the covariance spectrum, which is thus a significant improvement on the approach developed by Snyder and Brüschweiler (66 %) and the use of spectral derivatives (50 %). Thus, we calculate the 4D covariance spectrum from HN(CO)CA and HNCA experiments, in which cross-peaks with LCCs above a universal threshold are considered as true correlations. This 4D covariance spectrum enables the sequential assignment of a 42 kDa maltose binding protein (MBP), in which about 95 % residues are successfully assigned with a high accuracy of 98 %. Our LCC approach, therefore, paves the way for a residue-by-residue study of the backbone structure and dynamics of large proteins.

Identification of the two-phase mechanism of arachidonic acid regulating inflammatory prostaglandin E2 biosynthesis by targeting COX-2 and mPGES-1.

Through linking inducible cyclooxygenase (COX)-2 with microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), a Single-Chain Enzyme Complex (SCEC, COX-2-10aa-mPGES-1) was engineered to mimic a specific inflammatory PGE2 biosynthesis from omega-6 fatty acid, arachidonic acid (AA), by eliminating involvements of non-inducible COX-1 and other PGE2 synthases. Using the SCEC, we characterized coupling reactions between COX-2 and mPGES-1 at 1:1 ratio of inflammatory PGE2 production. AA demonstrated two phase activities to regulate inflammatory PGE2 production. In the first phase (<2 µM), AA was a COX-2 substrate and converted to increasing production of PGE2. In the second phase with a further increased AA level (2-10 µM), AA bound to mPGES-1 and inhibited the PGE2 production. The SCEC study was identical to the co-expression of COX-2 and mPGES-1. This was further confirmed by using mPGES-1 and PGH2 as a direct enzyme target and substrate, respectively. Furthermore, the carboxylic acid group of AA binding to R67 and R70 of mPGES-1 was identified by X-ray structure-based docking and mutagenesis. mPGES-1 mutants, R70A, R70K, R67A and R67K, lost 40-100% binding to [(14)C]-AA. To conclude, a cellular model, in which AA is involved in self-controlling initial initiating and later resolving inflammation by its two phase activities, was discussed.