Rodrigo L. Silveira

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.

Background: PPARγ agonists improve insulin sensitivity but also evoke weight gain. Results: GQ-16 is a PPARγ partial agonist that blocks receptor phosphorylation by Cdk5 and improves insulin sensitivity in diabetic mice in the absence of weight gain. Conclusion: The unique binding mode of GQ-16 appears to be responsible for the compounds advantageous pharmacological profile. Significance: Similar compounds could have promise as anti-diabetic therapeutics. The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compounds efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compounds weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby “ideal” PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.

Plant Biomass Recalcitrance: Effect of Hemicellulose Composition on Nanoscale Forces that Control Cell Wall Strength

Efficient conversion of lignocellulosic biomass to second-generation biofuels and valuable chemicals requires decomposition of resilient plant cell wall structure. Cell wall recalcitrance varies among plant species and even phenotypes, depending on the chemical composition of the noncellulosic matrix. Changing the amount and composition of branches attached to the hemicellulose backbone can significantly alter the cell wall strength and microstructure. We address the effect of hemicellulose composition on primary cell wall assembly forces by using the 3D-RISM-KH molecular theory of solvation, which provides statistical-mechanical sampling and molecular picture of hemicellulose arrangement around cellulose. We show that hemicellulose branches of arabinose, glucuronic acid, and especially glucuronate strengthen the primary cell wall by strongly coordinating to hydrogen bond donor sites on the cellulose surface. We reveal molecular forces maintaining the cell wall structure and provide directions for genetic modulation of plants and pretreatment design to render biomass more amenable to processing.

Mode of peroxisome proliferator-activated receptor gamma activation by luteolin.

The peroxisome proliferator-activated receptor γ (PPARγ) is a target for treatment of type II diabetes and other conditions. PPARγ full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPARγ but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPARγ agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPARγ target genes in 3T3-L1 cells (LPL, ORL1, and CEBPα) and PPARγ-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPARγ ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPARγ LBD conformer and occupies a space near the β-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPARγ, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Ω-loop among H2′, H3, and the β-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPARγ-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPARγ modulators.

Joint X‐ray crystallographic and molecular dynamics study of cellobiohydrolase I from Trichoderma harzianum: deciphering the structural features of cellobiohydrolase catalytic activity

Aiming to contribute toward the characterization of new, biotechnologically relevant cellulolytic enzymes, we report here the first crystal structure of the catalytic core domain of Cel7A (cellobiohydrolase I) from the filamentous fungus Trichoderma harzianum IOC 3844. Our structural studies and molecular dynamics simulations show that the flexibility of Tyr260, in comparison with Tyr247 from the homologous Trichoderma reesei Cel7A, is enhanced as a result of the short side‐chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. T. harzianum cellobiohydrolase I also has a shortened loop at the entrance of the cellulose‐binding tunnel, which has been described to interact with the substrate in T. reesei Cel7A. These structural features might explain why T. harzianum Cel7A displays higher kcat and Km values, and lower product inhibition on both glucoside and lactoside substrates, compared with T. reesei Cel7A.

Enzyme Microheterogeneous Hydration and Stabilization in Supercritical Carbon Dioxide

Supercritical carbon dioxide is a promising green-chemistry solvent for many enzyme-catalyzed chemical reactions, yet the striking stability of some enzymes in such unconventional environments is not well understood. Here, we investigate the stabilization of the Candida antarctica Lipase B (CALB) in supercritical carbon dioxide-water biphasic systems using molecular dynamics simulations. The preservation of the enzyme structure and optimal activity depend on the presence of small amounts of water in the supercritical dispersing medium. When the protein is at least partially hydrated, water molecules bind to specific sites on the enzyme surface and prevent carbon dioxide from penetrating its catalytic core. Strikingly, water and supercritical carbon dioxide cover the protein surface quite heterogeneously. In the first solvation layer, the hydrophilic residues at the surface of the protein are able to pin down patches of water, whereas carbon dioxide solvates preferentially hydrophobic surface residues. In the outer solvation shells, water molecules tend to cluster predominantly on top of the larger water patches of the first solvation layer instead of spreading evenly around the remainder of the protein surface. For CALB, this exposes the substrate-binding region of the enzyme to carbon dioxide, possibly facilitating diffusion of nonpolar substrates into the catalytic funnel. Therefore, by means of microheterogeneous solvation, enhanced accessibility of hydrophobic substrates to the active site can be achieved, while preserving the functional structure of the enzyme. Our results provide a molecular picture on the nature of the stability of proteins in nonaqueous media.

Evolution of xylan substitution patterns in gymnosperms and angiosperms: implications for xylan interaction with cellulose

The xylan decoration pattern in all lineages of gymnosperms permits hydrogen bonding of xylan to cellulose. The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls.

Cellulose Aggregation under Hydrothermal Pretreatment Conditions

Cellulose, the most abundant biopolymer on Earth, represents a resource for sustainable production of biofuels. Thermochemical treatments make lignocellulosic biomaterials more amenable to depolymerization by exposing cellulose microfibrils to enzymatic or chemical attacks. In such treatments, the solvent plays fundamental roles in biomass modification, but the molecular events underlying these changes are still poorly understood. Here, the 3D-RISM-KH molecular theory of solvation has been employed to analyze the role of water in cellulose aggregation under different thermodynamic conditions. The results show that, under ambient conditions, highly structured hydration shells around cellulose create repulsive forces that protect cellulose microfibrils from aggregating. Under hydrothermal pretreatment conditions, however, the hydration shells lose structure, and cellulose aggregation is favored. These effects are largely due to a decrease in cellulose-water interactions relative to those at ambient conditions, so that cellulose-cellulose attractive interactions become prevalent. Our results provide an explanation to the observed increase in the lateral size of cellulose crystallites when biomass is subject to pretreatments and deepen the current understanding of the mechanisms of biomass modification.

Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 Å resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting β-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

Supramolecular Interactions in Secondary Plant Cell Walls: Effect of Lignin Chemical Composition Revealed with the Molecular Theory of Solvation

Plant biomass recalcitrance, a major obstacle to achieving sustainable production of second generation biofuels, arises mainly from the amorphous cell-wall matrix containing lignin and hemicellulose assembled into a complex supramolecular network that coats the cellulose fibrils. We employed the statistical-mechanical, 3D reference interaction site model with the Kovalenko-Hirata closure approximation (or 3D-RISM-KH molecular theory of solvation) to reveal the supramolecular interactions in this network and provide molecular-level insight into the effective lignin-lignin and lignin-hemicellulose thermodynamic interactions. We found that such interactions are hydrophobic and entropy-driven, and arise from the expelling of water from the mutual interaction surfaces. The molecular origin of these interactions is carbohydrate-π and π-π stacking forces, whose strengths are dependent on the lignin chemical composition. Methoxy substituents in the phenyl groups of lignin promote substantial entropic stabilization of the ligno-hemicellulosic matrix. Our results provide a detailed molecular view of the fundamental interactions within the secondary plant cell walls that lead to recalcitrance.