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Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers.

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of β-catenin, a key transcription factor in the Wnt pathway. At the protein level, β-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of β-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with β-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and β-catenin interact as a potential new component of the Wnt signaling pathway.

Sterile tissue injury is thought to locally activate innate immune responses via damage associated molecular patterns (DAMPs). Whether innate immune pathways are remotely activated remains relatively unexplored. Here, by analyzing ~145,000 single cell transcriptomes at steady state and after myocardial infarction (MI) in mice and humans, we show that the type I interferon (IFN) response, characterized by expression of interferon-stimulated genes (ISGs), begins far from the site of injury, in neutrophil and monocyte progenitors within the bone marrow. In the peripheral blood of patients, we observed defined subsets of ISG-expressing neutrophils and monocytes. In the bone marrow and blood of mice, ISG expression was detected in neutrophils and monocytes and their progenitors; intensified with maturation at steady-state and after MI; and was controlled by Tet2 and Irf3 transcriptional regulators. Within the infarcted heart, ISG-expressing cells were negatively regulated by Nrf2 activation in Ccr2(−) steady-state cardiac macrophages. Our results show that IFN signaling begins in the bone marrow, implicate multiple transcriptional regulators (Tet2, Irf3, Nrf2) in governing ISG expression, and provide a clinical biomarker (ISG score) for studying IFN signaling in patients.

Pannexin 1 (Panx1) is a channel-forming glycoprotein important in paracrine signaling and cellular development. In this study, we discovered that mice globally lacking Panx1 (KO) have significantly greater total fat mass and reduced lean mass compared to wild type (WT) mice under a normal diet. Despite having higher fat content, Panx1 KO mice on a high fat diet exhibited no differences in weight gain and blood markers of obesity as compared to WT controls, except for an increase in glucose and insulin levels. However, metabolic cage data revealed that these Panx1 KO mice display significantly increased activity levels, higher ambulatory activity, and reduced sleep duration relative to their WT littermates on a high-fat diet. To uncover the cellular mechanism responsible for the increased fat content in the KO, we isolated primary cultures of adipose-derived stromal cells (ASCs) from WT and KO fat pads. In WT ASCs we observed that Panx1 protein levels increase upon induction into an adipogenic lineage. ASCs isolated from Panx1 KO mice proliferate less but demonstrate enhanced adipogenic differentiation with increased intracellular lipid accumulation, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and adipokine secretion, as compared to WT ASCs. This was consistent with the increased adipocyte size and decreased adipocyte numbers observed in subcutaneous fat of the Panx1 KO mice compared to WT. We concluded that Panx1 plays a key role in adipose stromal cells during the early stages of adipogenic proliferation and differentiation, regulating fat accumulation in vivo.

Abnormal expression of sialylated Thomsen–Friedenreich antigen (Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Thr, sialyl-T) has a strong relationship with various types of human cancers and many other diseases. However, the size and structural complexity, and relatively lower abundance of sialyl-T have posed a significant challenge to its detection. Therefore, details about the role of sialyl-T in a variety of physiological and pathological processes are still poorly understood. Here, a one-step chemoenzymatic labeling strategy to probe sialyl-T is described. This approach enables the sensitive, selective, and rapid detection of sialyl-T, and global profiling and identification of unknown sialyl-T-attached glycoproteins, which are potential therapeutic targets or biomarkers. The use of one-step labeling strategy not only has a higher sensitivity than a typical two-step reporter strategy but also avoids undergoing an additional chemical reaction step to introduce a reporter group after the labeling reaction, making it particularly useful for detecting low-abundance glycan epitopes on living cells., A one-step chemoenzymatic labeling strategy is described for the selective and rapid detection of sialyl-T, and global profiling and identification of unknown sialyl-T-attached glycoproteins.

Previous investigations of sequence-specific DNA binding by model minor groove-binding compounds showed that the ligand/DNA complex was destabilized in the presence of compatible co-solutes. Inhibition was interpreted in terms of osmotic stress theory as the uptake of significant numbers of excess water molecules from bulk solvent upon complex formation. Here, we interrogated the AT-specific DNA complex formed with the symmetric heterocyclic diamidine DB1976 as a model for minor groove DNA recognition using both ionic (NaCl) and non-ionic cosolutes (ethylene glycol, glycine betaine, maltose, nicotinamide, urea). While the non-ionic cosolutes all destabilized the ligand/DNA complex, their quantitative effects were heterogeneous in a cosolute- and salt-dependent manner. Perturbation with NaCl in the absence of non-ionic cosolute showed that preferential hydration water was released upon formation of the DB1976/DNA complex. As salt probes counter-ion release from charged groups such as the DNA backbone, we propose that the preferential hydration uptake in DB1976/DNA binding observed in the presence of osmolytes reflects the exchange of preferentially bound cosolute with hydration water in the environs of the bound DNA, rather than a net uptake of hydration waters by the complex.

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. Unlike other ETS homologues, such as Ets-1, DNA recognition by PU.1 is highly sensitive to its osmotic environment due to excess interfacial hydration in the complex. To investigate interfacial hydration in the two homologues, we mutated a conserved tyrosine residue, which is exclusively engaged in coordinating a well-defined water contact between the protein and DNA among ETS proteins, to phenylalanine. The loss of this water-mediated contact blunted the osmotic sensitivity of PU.1/DNA binding, but did not alter binding under normo-osmotic conditions, suggesting that PU.1 has evolved to maximize osmotic sensitivity. The homologous mutation in Ets-1, which was minimally sensitive to osmotic stress due to a sparsely hydrated interface, reduced DNA-binding affinity at normal osmolality but the complex became stabilized by osmotic stress. Molecular dynamics simulations of wildtype and mutant PU.1 and Ets-1 in their free and DNA-bound states, which recapitulated experimental features of the proteins, showed that abrogation of this tyrosine-mediated water contact perturbed the Ets-1/DNA complex not through disruption of interfacial hydration, but by inhibiting local dynamics induced specifically in the bound state. Thus, a configurationally identical water-mediated contact plays mechanistically distinct roles in mediating DNA recognition by structurally homologous ETS transcription factors.

Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/&beta, catenin pathway, because &beta, catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac34dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resist...

Rare sugars offer a plethora of applications in the pharmaceutical, medicinal, and industries, as well as in synthetic chemistry. However, studies of rare sugars have been hampered by their relative scarcity. In this work, we describe a two-step strategy to efficiently and conveniently prepare 6-deoxy-L-psicose from L-rhamnose. In the first reaction step, the isomerization of L-rhamnose (6-deoxy-L-mannose) to L-rhamnulose (6-deoxy-L-fructose) catalyzed by L-rhamnose isomerase (RhaI), and the epimerization of L-rhamnulose to 6-deoxy-L-psicose catalyzed by D-tagatose 3-epimerase (DTE) were coupled with selective phosphorylation reaction by fructose kinase from human (HK), which selectively phosphorylate 6-deoxy-L-psicose at C-1 position. 6-deoxy-L-psicose 1-phosphate was purified by a silver nitrate precipitation method. In the second step, the phosphate group of the 6-deoxy-L-sorbose 1-phosphate was hydrolyzed with acid phosphatase (AphA) to produce 6-deoxy-L-psicose in 81% yield with respect to L-rhamnose. This method allows that the 6-deoxy-L-psicose to be obtained from readily available starting materials with high purity and without having to undergo isomer separation.

An efficient and convenient platform for the facile synthesis of phosphorylated ketoses is described. All eight phosphorylated ketopentoses were produced using this platform starting from two common and inexpensive aldoses (d-xylose and l-arabinose) in more than 84% isolated yield (gram scale). In this method, reversible conversions (isomerization or epimerization) were accurately controlled toward the formation of desired ketose phosphates by targeted phosphorylation reactions catalyzed by substrate-specific kinases. The byproducts were selectively removed by silver nitrate precipitation avoiding the tedious and time-consuming separation of sugar phosphate from adenosine phosphates (ATP and ADP). Moreover, the described strategy can be expanded for the synthesis of other sugar phosphates.

L-Rhamnulose (6-deoxy-L-arabino-2-hexulose) and L-fuculose (6-deoxy-L-lyxo-2-hexulose) were prepared from L-rhamnose and L-fucose by a two-step strategy. In the first reaction step, isomerization of L-rhamnose to L-rhamnulose, or L-fucose to L-fuculose was combined with a targeted phosphorylation reaction catalyzed by L-rhamnulose kinase (RhaB). The by-products (ATP and ADP) were selectively removed by silver nitrate precipitation method. In the second step, the phosphate group was hydrolyzed to produce L-rhamnulose or L-fuculose with purity exceeding 99% in more than 80% yield (gram scale).

Metal ions play crucial roles in numerous biological processes, facilitating biochemical reactions by binding to various proteins. An increasing body of evidence suggests that neurotoxicity associated with exposure to nonessential metals (e.g., Pb(2+)) involves disruption of synaptic activity, and these observed effects are associated with the ability of Pb(2+) to interfere with Zn(2+) and Ca(2+)-dependent functions. However, the molecular mechanism behind Pb(2+) toxicity remains a topic of debate. In this review, we first discuss potential neuronal Ca(2+) binding protein (CaBP) targets for Pb(2+) such as calmodulin (CaM), synaptotagmin, neuronal calcium sensor-1 (NCS-1), N-methyl-d-aspartate receptor (NMDAR) and family C of G-protein coupled receptors (cGPCRs), and their involvement in Ca(2+)-signalling pathways. We then compare metal binding properties between Ca(2+) and Pb(2+) to understand the structural implications of Pb(2+) binding to CaBPs. Statistical and biophysical studies (e.g., NMR and fluorescence spectroscopy) of Pb(2+) binding are discussed to investigate the molecular mechanism behind Pb(2+) toxicity. These studies identify an opportunistic, allosteric binding of Pb(2+) to CaM, which is distinct from ionic displacement. Together, these data suggest three potential modes of Pb(2+) activity related to molecular and/or neural toxicity: (i) Pb(2+) can occupy Ca(2+)-binding sites, inhibiting the activity of the protein by structural modulation, (ii) Pb(2+) can mimic Ca(2+) in the binding sites, falsely activating the protein and perturbing downstream activities, or (iii) Pb(2+) can bind outside of the Ca(2+)-binding sites, resulting in the allosteric modulation of the protein activity. Moreover, the data further suggest that even low concentrations of Pb(2+) can interfere at multiple points within the neuronal Ca(2+) signalling pathways to cause neurotoxicity.

// Lanlan Zang 1, * , Shukkoor M. Kondengaden 2, * , Qing Zhang 2, * , Xiaobo Li 5 , Dilep K. Sigalapalli 3 , Shameer M. Kondengadan 4 , Kenneth Huang 2 , Keqin Kathy Li 2 , Shanshan Li 2 , Zhongying Xiao 2 , Liuqing Wen 2 , Hailiang Zhu 2 , Bathini N. Babu 3 , Lijuan Wang 1 , Fengyuan Che 1 and Peng George Wang 2 1 Central Laboratory, Linyi People's Hospital, Linyi, Shandong 276003, P.R. China 2 Chemistry Department and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA 3 Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, Telangana 500037, India 4 Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Mohali, Punjab 160062, India 5 Department of Immunology, Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin 300070, P.R. China * These authors have contributed equally to this work Correspondence to: Peng George Wang, email: pwang11@gsu.edu Fengyuan Che, email: che1971@126.com Lijuan Wang, email: lijian.wang730@outlook.com Keywords: epigenetics, pharmacophore, dual inhibitors, G9a inhibitors, HDAC inhibitors Received: April 14, 2017 Accepted: May 23, 2017 Published: June 28, 2017 ABSTRACT Aberrant enzymatic activities or expression profiles of epigenetic regulations are therapeutic targets for cancers. Among these, histone 3 lysine 9 methylation (H3K9Me2) and global de-acetylation on histone proteins are associated with multiple cancer phenotypes including leukemia, prostatic carcinoma, hepatocellular carcinoma and pulmonary carcinoma. Here, we report the discovery of the first small molecule capable of acting as a dual inhibitor targeting both G9a and HDAC. Our structure based design, synthesis, and screening for the dual activity of the small molecules led to the discovery of compound 14 which displays promising inhibition of both G9a and HDAC in low micro-molar range in cell based assays.

Ca2+-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca2+]o) homeostasis and many (patho)physiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids, and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT) domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR's cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs) in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics.

Sialic acids are typically linked α(2-3) or α(2-6) to the galactose that located at the non-reducing terminal end of glycans, playing important but distinct roles in a variety of biological and pathological processes. However, details about their respective roles are still largely unknown due to the lack of an effective analytical technique. Herein, a two-step chemoenzymatic approach for the rapid and sensitive detection of N-acetylneuraminic acid−α(2-3)-galactose glycans is described.

Structural and functional characterization of the extracellular domain of the human CaSR with bound Mg2+ and a tryptophan derivative., Ca2+-sensing receptors (CaSRs) modulate calcium and magnesium homeostasis and many (patho)physiological processes by responding to extracellular stimuli, including divalent cations and amino acids. We report the first crystal structure of the extracellular domain (ECD) of human CaSR bound with Mg2+ and a tryptophan derivative ligand at 2.1 Å. The structure reveals key determinants for cooperative activation by metal ions and aromatic amino acids. The unexpected tryptophan derivative was bound in the hinge region between two globular ECD subdomains, and represents a novel high-affinity co-agonist of CaSR. The dissection of structure-function relations by mutagenesis, biochemical, and functional studies provides insights into the molecular basis of human diseases arising from CaSR mutations. The data also provide a novel paradigm for understanding the mechanism of CaSR-mediated signaling that is likely shared by the other family C GPCR [G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor] members and can facilitate the development of novel CaSR-based therapeutics.

Lysine methyltransferase G9a regulates the transcription of multiple genes by primarily catalyzing mono- and di-methylation of histone H3 lysine 9, as well as several non-histone lysine sites. An attractive therapeutic target in treating leukemia, knockout studies of G9a in mice have found dramatically slowed proliferation and self-renewal of acute myeloid leukemia (AML) cells due to the attenuation of HoxA9-dependent transcription. In this study, a series of compounds were identified as potential inhibitors through structure-based virtual screening. Among these compounds, a new G9a inhibitor, DCG066, was confirmed by in vitro biochemical, and cell based enzyme assays. DCG066 has a novel molecular scaffold unlike other G9a inhibitors presently available. Similar to G9a’s histone substrate, DCG066 can bind directly to G9a and inhibit methyltransferase activity in vitro. In addition to suppressing G9a methyltransferase activity and reducing histone H3 methylation levels, DCG066 displays low cytotoxicity in leukemia cell lines with high levels of G9a expression, including K562. This work presents DCG066 as an inhibitor of G9a with a novel structure, providing both a lead in G9a inhibitor design and a means for probing the functionality of G9a.