Loss of skeletal muscle estrogen-related receptors leads to severe exercise intolerance.

OBJECTIVE: Skeletal muscle oxidative capacity is central to physical activity, exercise capacity and whole-body metabolism. The three estrogen-related receptors (ERRs) are regulators of oxidative metabolism in many cell types, yet their roles in skeletal muscle remain unclear. The main aim of this study was to compare the relative contributions of ERRs to oxidative capacity in glycolytic and oxidative muscle, and to determine defects associated with loss of skeletal muscle ERR function. METHODS: We assessed ERR expression, generated mice lacking one or two ERRs specifically in skeletal muscle and compared the effects of ERR loss on the transcriptomes of EDL (predominantly glycolytic) and soleus (oxidative) muscles. We also determined the consequences of the loss of ERRs for exercise capacity and energy metabolism in mice with the most severe loss of ERR activity. RESULTS: ERRs were induced in human skeletal muscle in response to an exercise bout. Mice lacking both ERRα and ERRγ (ERRα/γ dmKO) had the broadest and most dramatic disruption in skeletal muscle gene expression. The most affected pathway was "mitochondrial function", in particular Oxphos and TCA cycle genes, and transcriptional defects were more pronounced in the glycolytic EDL than the oxidative soleus. Mice lacking ERRß and ERRγ, the two isoforms expressed highly in oxidative muscles, also exhibited defects in lipid and branch chain amino acid metabolism genes, specifically in the soleus. The pronounced disruption of oxidative metabolism in ERRα/γ dmKO mice led to pale muscles, decreased oxidative capacity, histochemical patterns reminiscent of minicore myopathies, and severe exercise intolerance, with the dmKO mice unable to switch to lipid utilization upon running. ERRα/γ dmKO mice showed no defects in whole-body glucose and energy homeostasis. CONCLUSIONS: Our findings define gene expression programs in skeletal muscle that depend on different combinations of ERRs, and establish a central role for ERRs in skeletal muscle oxidative metabolism and exercise capacity. Our data reveal a high degree of functional redundancy among muscle ERR isoforms for the protection of oxidative capacity, and show that ERR isoform-specific phenotypes are driven in part, but not exclusively, by their relative levels in different muscles.

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs.

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12-186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target "degradability."

TGF-ß Induced CTGF Expression in Human Lung Epithelial Cells through ERK, ADAM17, RSK1, and C/EBPß Pathways.

BACKGROUND: Lung epithelial cells play critical roles in idiopathic pulmonary fibrosis. METHODS: In the present study, we investigated whether transforming growth factor-ß (TGF-ß)-induced expression of connective tissue growth factor (CTGF) was regulated by the extracellular signal-regulated kinase (ERK)/a disintegrin and metalloproteinase 17 (ADAM17)/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein ß (C/EBPß) signaling pathway in human lung epithelial cells (A549). RESULTS: Our results revealed that TGF-ß-induced CTGF expression was weakened by ADAM17 small interfering RNA (ADAM17 siRNA), TNF-α processing inhibitor-0 (TAPI-0, an ADAM17 inhibitor), U0126 (an ERK inhibitor), RSK1 siRNA, and C/EBPß siRNA. TGF-ß-induced ERK phosphorylation as well as ADAM17 phosphorylation was attenuated by U0126. The TGF-ß-induced increase in RSK1 phosphorylation was inhibited by TAPI-0 and U0126. TGF-ß-induced C/EBPß phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. In addition, TGF-ß increased the recruitment of C/EBPß to the CTGF promoter. Furthermore, TGF-ß enhanced fibronectin (FN), an epithelial-mesenchymal transition (EMT) marker, and CTGF mRNA levels and reduced E-cadherin mRNA levels. Moreover, TGF-ß-stimulated FN protein expression was reduced by ADAM17 siRNA and CTGF siRNA. CONCLUSION: The results suggested that TGF-ß induces CTGF expression through the ERK/ADAM17/RSK1/C/EBPß signaling pathway. Moreover, ADAM17 and CTGF participate in TGF-ß-induced FN expression in human lung epithelial cells.

Interactions between identical DNA double helices.

The molecular mechanism of specific interactions between double stranded DNA molecules has been investigated for many years. Problems remain in how confinement, ions, and condensing agents change the interactions. We consider how the orientational alignment of DNAs contributes to the interactions via free energy simulations. Here we report on the effective interactions between two parallel DNA double helices in 150-mM NaCl solution using all atom models. We calculate the potential of mean force (PMF) of DNA-DNA interactions as a function of two coordinates, interhelical separation of parallel double helices and relative rotation of a DNA molecule with respect to the other about the helical axis. We generate the two-dimensional PMF to better understand the effective interactions when a DNA molecule is in juxtaposition with another. The analysis of the ion and solvent distributions around the DNA and particularly in the interface region shows that certain alignments of the DNA pair enhance the interactions. At local free energy minima in distance and alignment, water molecules and Na^{+} ions form a hydrogen bonded network with the phosphates from each DNA. This network contributes an attractive energy component to the DNA-DNA interactions. Our results provide a molecular mechanism whereby local DNA-DNA interactions, depending on the helical orientation, give a potential mechanism for stabilizing pairing of much larger lengths of homologous DNA that have been seen experimentally. The study suggests an atomically detailed local picture of relevance to certain aspects of DNA condensation or aggregation.

Free Energy Calculations Based on Coupling Proximal Distribution Functions and Thermodynamic Cycles.

Techniques to calculate the free energy changes of a system are very useful in the study of biophysical and biochemical properties. In practice, free energy changes can be described with thermodynamic cycles, and the free energy change of an individual process can be computed by sufficiently sampling the corresponding configurations. However, this is still time-consuming especially for large biomolecular systems. Previously, we have shown that by utilizing precomputed solute-solvent correlations, so-called proximal distribution functions (pDF), we are capable of reconstructing the solvent environment near solute atoms, thus estimating the solute-solvent interactions and solvation free energies of molecules. In this contribution, we apply the technique of pDF-reconstructions to calculate chemical potentials and use this information in thermodynamic cycles. This illustrates how free energy changes of nontrivial chemical processes in aqueous solution systems can be rapidly estimated.

Nonpolar Solvation Free Energy from Proximal Distribution Functions.

Using precomputed near neighbor or proximal distribution functions (pDFs) that approximate solvent density about atoms in a chemically bonded context one can estimate the solvation structures around complex solutes and the corresponding solute-solvent energetics. In this contribution, we extend this technique to calculate the solvation free energies (ΔG) of a variety of solutes. In particular we use pDFs computed for small peptide molecules to estimate ΔG for larger peptide systems. We separately compute the non polar (ΔGvdW) and electrostatic (ΔGelec) components of the underlying potential model. Here we show how the former can be estimated by thermodynamic integration using pDF-reconstructed solute-solvent interaction energy. The electrostatic component can be approximated with Linear Response theory as half of the electrostatic solute-solvent interaction energy. We test the method by calculating the solvation free energies of butane, propanol, polyalanine, and polyglycine and by comparing with traditional free energy simulations. Results indicate that the pDF-reconstruction algorithm approximately reproduces ΔGvdW calculated by benchmark free energy simulations to within ∼ kcal/mol accuracy. The use of transferable pDFs for each solute atom allows for a rapid estimation of ΔG for arbitrary molecular systems.

Molecular modeling of ions at interfaces: exploring similarities to hydrophobic solvation through the lens of induced aqueous interfacial fluctuations.

Ion specific effects are ubiquitous in chemistry and biology. While ion specific effects in molecular simulations are generally explained as the result of an interplay between ionic hydration, non-electrostatic potentials and ionic polarizability, this perspective considers an alternative discussion of the coupling of ionic hydration and an interfacial solvent as a contributing component leading to differing interfacial stabilities amongst ions. Interfacially stable ions, characterized as such by minima in free energy profiles, induce larger interfacial fluctuations compared to non-interfacial active species, conferring more covariance entropy approaching the interface. Larger anions, particularly those that are modeled classically with low charge-density, behave as canonical hydrophobic solutes with respect to their solvent-mediated interactions with soft interfaces; whereas smaller anions and cations show no interfacially stable states, nor enhanced interfacial fluctuations. Underlying this phenomenon is the fundamental nature of the hydration shell structure, dynamics, and rigidity around the solutes.

Solute-Solvent Energetics Based on Proximal Distribution Functions.

We consider the hydration structure and thermodynamic energetics of solutes in aqueous solution. On the basis of the dominant local correlation between the solvent and the chemical nature of the solute atoms, proximal distribution functions (pDF) can be used to quantitatively estimate the hydration pattern of the macromolecules. We extended this technique to study the solute-solvent energetics including the van der Waals terms representing excluded volume and tested the method with butane and propanol. Our results indicate that the pDF-reconstruction algorithm can reproduce van der Waals solute-solvent interaction energies to useful kilocalorie per mole accuracy. We subsequently computed polyalanine-water interaction energies for a variety of conformers, which also showed agreement with the simulated values.

Free energetics of carbon nanotube association in aqueous inorganic NaI salt solutions: Temperature effects using all-atom molecular dynamics simulations.

In this study, we examine the temperature dependence of free energetics of nanotube association using graphical processing unit-enabled all-atom molecular dynamics simulations (FEN ZI) with two (10,10) single-walled carbon nanotubes in 3 m NaI aqueous salt solution. Results suggest that the free energy, enthalpy and entropy changes for the association process are all reduced at the high temperature, in agreement with previous investigations using other hydrophobes. Via the decomposition of free energy into individual components, we found that solvent contribution (including water, anion, and cation contributions) is correlated with the spatial distribution of the corresponding species and is influenced distinctly by the temperature. We studied the spatial distribution and the structure of the solvent in different regions: intertube, intratube and the bulk solvent. By calculating the fluctuation of coarse-grained tube-solvent surfaces, we found that tube-water interfacial fluctuation exhibits the strongest temperature dependence. By taking ions to be a solvent-like medium in the absence of water, tube-anion interfacial fluctuation shows similar but weaker dependence on temperature, while tube-cation interfacial fluctuation shows no dependence in general. These characteristics are discussed via the malleability of their corresponding solvation shells relative to the nanotube surface. Hydrogen bonding profiles and tetrahedrality of water arrangement are also computed to compare the structure of solvent in the solvent bulk and intertube region. The hydrophobic confinement induces a relatively lower concentration environment in the intertube region, therefore causing different intertube solvent structures which depend on the tube separation. This study is relevant in the continuing discourse on hydrophobic interactions (as they impact generally a broad class of phenomena in biology, biochemistry, and materials science and soft condensed matter research), and interpretations of hydrophobicity in terms of alternative but parallel signatures such as interfacial fluctuations, dewetting transitions, and enhanced fluctuation probabilities at interfaces.

Protein denaturants at aqueous-hydrophobic interfaces: self-consistent correlation between induced interfacial fluctuations and denaturant stability at the interface.

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous-hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm(+)) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein-water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid-vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous-hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A 2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss 2013, 160, 89).

Association of alkanes with the aqueous liquid-vapor interface: a reference system for interpreting hydrophobicity generally through interfacial fluctuations.

We report free energy calculations and fluctuation profiles of single alkanes (from methane to pentane) along the direction normal to the air-water interface. The induced fluctuations and the interfacial stabilities of alkanes are found to be correlated and similar to the results of inorganic monovalent ions (Ou et al., J. Phys. Chem. B, 2013, 117, 11732). This suggests that hydrophobic solvation of solutes and ions is important in determining the adsorption behavior.

Electrostatic contribution from solvent in modulating single-walled carbon nanotube association.

We perform all-atom molecular dynamics simulations to compute the potential of mean force (PMF) between two (10,10) single-walled carbon nanotubes solvated in pure nonpolarizable SPC/E and polarizable TIP4P-FQ water, at various temperatures. In general, the reversible work required to bring two nanotubes from a dissociated state (free energy reference) to contact state (free energy minimum) is more favorable and less temperature-dependent in TIP4P-FQ than in SPC/E water models. In contrast, molecular properties and behavior of water such as the spatially-resolved water number density (intertube, intratube, or outer regions), for TIP4P-FQ are more sensitive to temperature than SPC/E. Decomposition of the solvent-induced PMF into different spatial regions suggests that TIP4P-FQ has stronger temperature dependence; the opposing destabilizing/stabilizing contributions from intertube water and more distal water balance each other and suppress the temperature dependence of total association free energy. Further investigation of hydrogen bonding network in intertube water reveals that TIP4P-FQ retains fewer hydrogen bonds than SPC/E, which correlates with the lower water number density in this region. This reduction of hydrogen bonds affects the intertube water dipoles. As the intertube volume decreases, TIP4P-FQ dipole moment approaches the gas phase value; the distribution of dipole magnitude also becomes narrower due to less average polarization/perturbation from other water molecules. Our results imply that the reduction of water under confinement may seem trivial, but underlying effects to structure and free energetics are non-negligible.