Non-canonical mTORC2 Signaling Regulates Brown Adipocyte Lipid Catabolism through SIRT6-FoxO1.

mTORC2 controls glucose and lipid metabolism, but the mechanisms are unclear. Here, we show that conditionally deleting the essential mTORC2 subunit Rictor in murine brown adipocytes inhibits de novo lipid synthesis, promotes lipid catabolism and thermogenesis, and protects against diet-induced obesity and hepatic steatosis. AKT kinases are the canonical mTORC2 substrates; however, deleting Rictor in brown adipocytes appears to drive lipid catabolism by promoting FoxO1 deacetylation independently of AKT, and in a pathway distinct from its positive role in anabolic lipid synthesis. This facilitates FoxO1 nuclear retention, enhances lipid uptake and lipolysis, and potentiates UCP1 expression. We provide evidence that SIRT6 is the FoxO1 deacetylase suppressed by mTORC2 and show an endogenous interaction between SIRT6 and mTORC2 in both mouse and human cells. Our findings suggest a new paradigm of mTORC2 function filling an important gap in our understanding of this more mysterious mTOR complex.

HMGB1-mediated restriction of EPO signaling contributes to anemia of inflammation.

Anemia of inflammation, also known as anemia of chronic disease, is refractory to erythropoietin (EPO) treatment, but the mechanisms underlying the EPO refractory state are unclear. Here, we demonstrate that high mobility group box-1 protein (HMGB1), a damage-associated molecular pattern molecule recently implicated in anemia development during sepsis, leads to reduced expansion and increased death of EPO-sensitive erythroid precursors in human models of erythropoiesis. HMGB1 significantly attenuates EPO-mediated phosphorylation of the Janus kinase 2/STAT5 and mTOR signaling pathways. Genetic ablation of receptor for advanced glycation end products, the only known HMGB1 receptor expressed by erythroid precursors, does not rescue the deleterious effects of HMGB1 on EPO signaling, either in human or murine precursors. Furthermore, surface plasmon resonance studies highlight the ability of HMGB1 to interfere with the binding between EPO and the EPOR. Administration of a monoclonal anti-HMGB1 antibody after sepsis onset in mice partially restores EPO signaling in vivo. Thus, HMGB1-mediated restriction of EPO signaling contributes to the chronic phase of anemia of inflammation.

Recent Advances in the Synthesis of Aromatic Azo Compounds.

Aromatic azo compounds have -N=N- double bonds as well as a larger π electron conjugation system, which endows aromatic azo compounds with wide applications in the fields of functional materials. The properties of aromatic azo compounds are closely related to the substituents on their aromatic rings. However, traditional synthesis methods, such as the coupling of diazo salts, have a significant limitation with respect to the structural design of aromatic azo compounds. Therefore, many scientists have devoted their efforts to developing new synthetic methods. Moreover, recent advances in the synthesis of aromatic azo compounds have led to improvements in the design and preparation of light-response materials at the molecular level. This review summarizes the important synthetic progress of aromatic azo compounds in recent years, with an emphasis on the pioneering contribution of functional nanomaterials to the field.

Protein structure determination by combining sparse NMR data with evolutionary couplings.

Accurate determination of protein structure by NMR spectroscopy is challenging for larger proteins, for which experimental data are often incomplete and ambiguous. Evolutionary sequence information together with advances in maximum entropy statistical methods provide a rich complementary source of structural constraints. We have developed a hybrid approach (evolutionary coupling-NMR spectroscopy; EC-NMR) combining sparse NMR data with evolutionary residue-residue couplings and demonstrate accurate structure determination for several proteins 6-41 kDa in size.

Accurate protein structure modeling using sparse NMR data and homologous structure information.

While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining (1)H(N), (13)C, and (15)N backbone and (13)Cß chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2-1.9 Å relative to the conventional determined NMR ensembles and of 0.9-1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.

Notch1 activation in embryonic VE-cadherin populations selectively blocks hematopoietic stem cell generation and fetal liver hematopoiesis.

Hematopoietic stem cells (HSC) are found in several independent sites embryonically. Loss-of-function studies indicated that Notch1, but not Notch2 signaling was required for HSC emergence from the aortic-gonado-mesonephros (AGM) region. We previously showed that constitutive Notch1 activation impaired primitive erythroid differentiation, but its effects on HSC emergence from the AGM region were not studied. To further define specific roles of Notch receptors, we characterized HSC in mouse embryos expressing either Notch1 intracellular domain (ICD) or Notch4ICD in VE-cadherin or SM22α expressing populations. Although embryonic Notch1 activation in VE-cadherin populations led to lethality after E13.5, earlier defects in the fetal liver were observed. Embryos were analyzed at E12.5 to assess hematopoiesis and the phenotype of developing cells in the AGM region. We found that activation of Notch1 in the endothelial compartment in VE-cadherin expressing cells resulted in the absence of intra-aortic clusters and defects in fetal liver hematopoiesis. In contrast, although Notch4 expression is regulated during fetal hematopoiesis, activation of Notch4 in VE-cadherin expressing populations did not affect HSC phenotype, although later vascular remodeling was impaired. Likewise, activation of Notch1 in SM22α positive populations had no significant effect on hematopoiesis. Our results indicate a cell type-dependent activity and distinct features of Notch1 versus Notch4 signaling and their impact on HSC generation.

CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia.

Increased endothelial cell (EC) proliferation is a hallmark of arteriovenous malformations (AVMs) in hereditary hemorrhagic telangiectasia (HHT). The underlying mechanism and disease relevance of this abnormal cell proliferative state of the ECs remain unknown. Here, we report the identification of a CDK6-driven mechanism of cell cycle progression deregulation directly involved in EC proliferation and HHT vascular pathology. Specifically, HHT mouse liver ECs exhibited defects in their cell cycle control characterized by a G1/S checkpoint bypass and acceleration of cell cycle speed. Phosphorylated retinoblastoma (p-RB1)-a marker of G1/S transition through the restriction point-significantly accumulated in ECs of HHT mouse retinal AVMs and HHT patient skin telangiectasias. Mechanistically, ALK1 loss of function increased the expression of key restriction point mediators, and treatment with palbociclib or ribociclib, two CDK4/6 inhibitors, blocked p-RB1 increase and retinal AVMs in HHT mice. Palbociclib also improved vascular pathology in the brain and slowed down endothelial cell cycle speed and EC proliferation. Specific deletion of Cdk6 in ECs was sufficient to protect HHT mice from AVM pathology. Thus, CDK6-mediated endothelial cell cycle acceleration controls EC proliferation in AVMs and is a central determinant of HHT pathogenesis. We propose that clinically approved CDK4/6 inhibitors have repurposing potential in HHT.

Temperature dependence of water interactions with the amide carbonyls of α-helices.

Hydration is a key determinant of the folding, dynamics, and function of proteins. In this study, temperature-dependent Fourier transform infrared (FTIR) spectroscopy combined with singular value decomposition (SVD) and global fitting were used to investigate both the interaction of water with α-helical proteins and the cooperative thermal unfolding of these proteins. This methodology has been applied to an isolated α-helix (Fs peptide) and to globular α-helical proteins including the helical subdomain and full-length villin headpiece (HP36 and HP67). The results suggest a unique IR signature for the interaction of water with the helical amide carbonyl groups of the peptide backbone. The IR spectra indicate a weakening of the net hydrogen bond strength of water to the backbone carbonyls with increasing temperature. This weakening of the backbone solvation occurs as a discrete transition near the maximum of the temperature-dependent hydrophobic effect, not a continuous change with increasing temperature. Possible molecular origins of this effect are discussed with respect to previous molecular dynamics simulations of the temperature-dependent solvation of the helix backbone.

RhoA-mediated signaling in Notch-induced senescence-like growth arrest and endothelial barrier dysfunction.

OBJECTIVE: Notch signaling has a critical role in vascular development and morphogenesis. Activation of Notch in endothelial cells led to a senescence-like phenotype with loss of barrier function. Our objective was to understand the molecular pathways mediating this phenotype. METHODS AND RESULTS: Human primary endothelial cells increase expression of Notch receptors and ligands during propagation in vitro toward natural senescence. This senescence was induced at low passage with Notch activation. We characterized the pathways activated downstream of Notch signaling. Notch was activated by Delta-like 4 ligand or constitutively active Notch receptors and measured for cell proliferation, migration, and sprouting. Notch signaling triggered early senescence in low-passage cells, characterized by increased p53 and p21 expression. The senescence phenotype was associated with hyperpermeability of the monolayer, with disrupted vascular endothelial cadherin and ß-catenin levels and localization. Consistent with changes in cell shape and contact, we demonstrated that Notch activation increases myosin light chain phosphorylation by activating Rho kinase. Inhibition of Rho abrogated Notch-induced myosin light chain phosphorylation and led to enhanced barrier function by reorganizing F-actin to ß-catenin-containing cell-cell adherens junctions. CONCLUSIONS: Our findings show that RhoA/Rho kinase regulation by Notch signaling in endothelial cells triggers a senescence phenotype associated with endothelial barrier dysfunction.

Notch and transforming growth factor-beta (TGFbeta) signaling pathways cooperatively regulate vascular smooth muscle cell differentiation.

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Mechanisms of TGF-ß-induced differentiation in human vascular smooth muscle cells.

BACKGROUND: Transforming growth factor-ß (TGF-ß) plays an important role in vascular homeostasis through effects on vascular smooth muscle cells (SMC). Fine-tuning of TGF-ß signaling occurs at the level of ALK receptors or Smads, and is regulated with cell type specificity. METHODS: Our goal was to understand TGF-ß signaling in regulating SMC differentiation marker expression in human SMC. Activation of Smads was characterized, and loss- and gain-of-function reagents used to define ALK pathways. In addition, Smad-independent mechanisms were determined. RESULTS: TGF-ß type I receptors, ALK1 and ALK5, are expressed in human SMC, and TGF-ß1 phosphorylates Smad1/5/8 and Smad2/3 in a time- and dosage-dependent pattern. ALK5 activity, not bone morphogenetic protein type I receptors, is required for Smad phosphorylation. Endoglin, a TGF-ß type III receptor, is a TGF-ß1 target in SMC, yet endoglin does not modify TGF-ß1 responsiveness. ALK5, not ALK1, is required for TGF-ß1-induction of SMC differentiation markers, and ALK5 signals through an ALK5/Smad3- and MAP kinase-dependent pathway. CONCLUSION: The definition of the specific signaling downstream of TGF-ß regulating SMC differentiation markers will contribute to a better understanding of vascular disorders involving changes in SMC phenotype.

Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation.

Development of bone and adipose tissue are linked processes arising from a common progenitor cell, but having an inverse relationship in disease conditions such as osteoporosis. Cellular differentiation of both tissues relies on growth factor cues, and we focus this study on Sprouty1 (Spry1), an inhibitor of growth factor signaling. We tested whether Spry1 can modify the development of fat cells through its activity in regulating growth factors known to be important for adipogenesis. We utilized conditional expression and genetic-null mouse models of Spry1 in adipocytes using the fatty acid binding promoter (aP2). Conditional deletion of Spry1 results in 10% increased body fat and decreased bone mass. This phenotype was rescued on Spry1 expression, which results in decreased body fat and increased bone mass. Ex vivo bone marrow experiments indicate Spry1 in bone marrow and adipose progenitor cells favors differentiation of osteoblasts at the expense of adipocytes by suppressing CEBP-beta and PPARgamma while up regulating TAZ. Age and gender-matched littermates expressing only Cre recombinase were used as controls. Spry1 is a critical regulator of adipocyte differentiation and mesenchymal stem cell (MSC) lineage allocation, potentially acting through regulation of CEBP-beta and TAZ.

Fully automated high-quality NMR structure determination of small (2)H-enriched proteins.

Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 Å, an all atom RMSD value of 1.2 Å to the crystal structure for well-defined regions, and RMSD value of 1.1 Å to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with (15)N- and (13)C-edited NOESY data obtained with a perdeuterated (15)N, (13)C-enriched (13)CH(3) methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR.

The E. coli single protein production system for production and structural analysis of membrane proteins.

At present, only 0.9% of PDB-deposited structures are of membrane proteins in spite of the fact that membrane proteins constitute approximately 30% of total proteins in most genomes from bacteria to humans. Here we address some of the major bottlenecks in the structural studies of membrane proteins and discuss the ability of the new technology, the Single-Protein Production system, to help solve these bottlenecks.

Efficient condensed-phase production of perdeuterated soluble and membrane proteins.

Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated media. Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression plasmids, can be condensed prior to protein expression, thereby providing high-quality (2)H, (13)C, (15)N-enriched protein samples at 2.5-10% the cost of traditional methods. As an example of the value of such inexpensively-produced perdeuterated proteins, we produced (2)H, (13)C, (15)N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40x condensed phase media, and obtained NMR spectra suitable for 3D structure determination. The cSPP system was also used to produce (2)H, (13)C, (15)N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution. This allows a membrane protein's structural and functional properties to be characterized prior to reconstitution, or as a probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a standardized protocol for production of perdeuterated proteins using the cSPP system. The 10-40 fold reduction in costs of fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated proteins in spectroscopic and crystallographic studies.

The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells.

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2-expressing populations using Tie2-Cre;Rosa26R-EYFP mice. In Tie2-Cre;Rosa26R-EYFP mice, analysis of bone marrow cells showed Cre-mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ∼ 84% of each lineage was EYFP(+), and nearly all cells that come from Tie2-expressing lineages are CD45(+), confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2(+) origin. Our findings of high levels of Tie2-Cre recombination in the hematopoietic lineage have implications for the use of the Tie2-Cre mouse as a lineage-restricted driver strain.

Hairy-related transcription factors inhibit Notch-induced smooth muscle alpha-actin expression by interfering with Notch intracellular domain/CBF-1 complex interaction with the CBF-1-binding site.

Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. One Notch target is smooth muscle alpha-actin (SMA), a differentiated smooth muscle cell marker. The Notch intracellular domain (NotchICD) forms a complex with CBF-1 (C-promoter-binding factor-1) and directly induces SMA expression. Using primary human smooth muscle cells, we show that expression of the constitutive active ICD of human Notch1, Notch2, or Notch4 receptors increase SMA levels. NotchICD also induce expression of the transcriptional repressors HRT1 (Hairy-related transcription factor 1) and HRT2, in a CBF-1-dependent manner. However, unlike the activating effects of NotchICD, HRT1 or HRT2 represses basal SMA expression, and both are strong antagonists of NotchICD-induced SMA upregulation. This antagonism does not depend on histone deacetylase activity and occurs at the transcriptional level. Competitive coimmunoprecipitation experiments demonstrate that HRT does not disrupt the association of NotchICD and CBF-1, which form a complex in the presence or absence of HRTs. However, HRT suppresses NotchICD/CBF-1 binding to the SMA promoter, as measured by chromatin immunoprecipitation, and transactivation of an SMA promoter reporter spanning sequences -124/+32. SMA expression was regulated similarly following endogenous Notch activation in smooth muscle cells by coculture with endothelial cells, and this effect was also sensitive to HRT inhibition. Temporally defined HRT activity may constitute a negative feedback mechanism of Notch signaling. Our study presents a novel mechanism by which a balance between Notch signaling and HRT activity determines the expression of smooth muscle differentiation markers including SMA.

A novel 3D porous pseudographite/Si/Ni composite anode material fabricated by a facile method.

A novel 3D porous pseudographite/Si/Ni (PG/Si/Ni) composite was prepared by a facile low-temperature calcination method using saturable starch and NiCl2·6H2O as precursors. The pseudographite matrix of PG/Si/Ni was obtained from the reaction between starch and NiCl2·6H2O during the calcination process. Compared to the C/Si electrode, the PG/Si/Ni electrode delivers a high reversible specific capacity of 659.66 mA h g-1 at a current density of 1 A g-1 even after 2000 cycles. In addition, the PG/Si/Ni electrode shows superior rate performance and still maintains a high specific capacity of 1324.01 mA h g-1 when the cycle current density returns to 0.1 A g-1. The porous pseudographite structure is able to improve Li+ diffusion efficiency, reduce pulverization and lead to the formation of stable SEI layers during the cycling process. Therefore, these results suggest that the 3D porous pseudographite/Si/Ni composite is a promising novel anode material. Besides, the low-temperature synthesis method of the pseudographite matrix can be applied for further modification of carbon-based Si anode materials.

Enhanced electrochemical performance of LiNi0.8Co0.1Mn0.1O2 with a 3D-SiO2 framework by a new negative pressure immersion method.

LiNi0.8Co0.1Mn0.1O2 is one of the most promising cathode materials for lithium ion batteries; however, during the charge/discharge process, it suffers from capacity fading, which is considered to be due to intergranular cracking. Herein we develop an original concept to alleviate this problem via negative pressure immersion treatment. A 3D-SiO2 framework is formed in the intergranular voids and at grain boundaries (functioning as the buffer zone and transfer-bridge) and the SiO2 protective layer is completely and homogeneously coated on the surfaces of the pristine particles through a hydrolytic condensation reaction involving tetraethoxysilane (TEOS). The 3D-SiO2 framework has two advantages: firstly, acting as a buffer zone, the framework can effectively inhibit the generation and extension of intergranular cracking; secondly, like the SiO2 protective layer on the surface of the particles, the 3D-SiO2 framework can impede side reactions between primary particles (grains) and electrolyte inside the particles. As a result, the as-modified LiNi0.8Co0.1Mn0.1O2 exhibits enhanced cycling performance with 92.4% capacity retention after 100 cycles at 1 C (200 mA h·g-1), while the capacity retention values for the pristine particles and normal coating treatment particles are only 55.4% and 82.6%, respectively. Moreover, the thermal stability (60 °C) is distinctly enhanced and the rate performance is significantly improved at high rates (2, 3 and 5 C).