Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus

The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo‐form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA‐bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant‐derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin‐3‐carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of monoterpene biotransformation in two pseudomonads

Aims: To study the metabolic profile of Pseudomonas rhodesiae and Pseudomonas fluorescens in water–organic solvent systems using terpene substrates for both growth and biotransformation processes and to determine the aerobic or anaerobic status of these degradation pathways. Materials and Methods: Substrates from pinene (α‐pinene, α‐pinene oxide, β‐pinene, β‐pinene oxide, turpentine) and limonene (limonene, limonene‐1,2‐oxide, orange peel oil) families were tested. For the bioconversion, the terpene‐grown biomass was concentrated and used either as whole cells or as a crude enzymatic extract. Conclusion: Pseudomonas rhodesiae was the most suitable biocatalyst for the production of isonovalal from α‐pinene oxide and did not metabolize limonene. Pseudomonas fluorescens was a more versatile micro‐organism and metabolized limonene in two different ways. The first (anaerobic, cofactor‐independent, noninducible) allowed limonene elimination by synthesizing α‐terpineol. The second (aerobic, cofactor‐dependent) involved limonene‐1,2‐oxide as an intermediate for energy production through a β‐oxidation process. Significance and Impact of the Study: Enzymatic isomerization of β‐ to α‐pinene was described for the first time for both strains. Alpha‐terpineol production by P. fluorescens was very efficient and appeared as a promising alternative for the commercial production of this bioflavour.     

Characterization of mice deficient in interleukin-1β converting enzyme

Interleukin-1β converting enzyme (ICE) processes the inactive proIL-1β to the proinflammatory mature IL-1β. ICE belongs to a family of cysteine proteases that have been implicated in apoptosis. To address the biological functions of ICE, we generated ICE-deficient mice through gene targeting technology. ICE-deficient mice developed normally, appeared healthy, and were fertile. Peritoneal macrophages from ICE-deficient mice underwent apoptosis normally upon ATP treatment. Thymocytes from young ICE-deficient mice also underwent apoptosis when triggered by dexamethasone, gamma irradiation, or aging. ICE-deficient mice had a major defect in the production of mature IL-1β and had impaired IL-1α production on LPS stimulation in vitro and in vivo. ICE-deficient mice were resistant to LPS-induced endotoxic shock. J. Cell. Biochem. 64:27–32. © 1997 Wiley-Liss, Inc.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Improved Detection of Human Antibodies to a Plasmodium Antigen Using a Peptide Modified with Aib Residues

A 17-mer sequence was selected as a model to study the influence of modifications of terminal ends both on the conformation of a peptide and on its antigenicity towards naturally developing antibodies. This sequence corresponded to a tandemly repeated motif, found in a long repetitive region, with high helical propensity, of a Plasmodium falciparum liver-stage antigen (LSA-1), immunogenic in man. Our model peptide was synthesized with ionizable or non-ionizable ends, or modified in both extremities by introduction of the helix-promoting residue α-aminoisobutyric acid (Aib). Helical contribution, absent in the 17 amino-acid sequence possessing ionizable ends, was detectable when non-ionizable ends were introduced, and dramatically increased in the Aib-modified analogue. The presence of ionizable ends totally abolished reactivity towards human sera, otherwise detectable with the peptide possessing non-ionizable ends. While modification by Aib residues was neither detrimental nor beneficial to antigenicity in solution, it clearly resulted in an improved sensitivity of the specific antibody detection when used as solid-phase antigen in ELISA. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational flexibility and loss of structural rigidity for a model hexapeptide,GRGDTP: 1H‐NMR and molecular dynamics studies

The NMR and molecular dynamics methods are used to study the conformations of a hexapeptide, GRGDTP, which has been shown to be accessible to various types of cell‐adhesion based cellular behaviors such as cell‐to‐matrix interactions, cell differentiation, immunogenicity development, gene expression, angiogenesis, metastasis, sex determination and gamete fusion. ¹H‐NMR results indicate the existence of weak 5→2 hydrogen bonded β‐turn type‐III. Molecular simulation studies using a mixed protocol of distance geometry, constrained minimization, restrained molecular dynamics followed by energy minimization resulted additional conformations that include about 64% of population of inverse γ‐turn (HB, 3→1) and about 35% population of γ‐turn (HB, 4→2). The inter‐proton distances observed in γ‐and inverse γ‐turns are also consistent with the NMR constraints. The variable internal hydrogen bonding due to γ‐turns initiated at Gly 1 and Arg 2 , and its tendency to inter‐convert between γ‐and inverse γ‐turn conformations imply that the peptide is flexible in nature. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 460–471, 2013.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

Structural and functional study of FK domain of Fstl1

Fstl1 is a TGF‐β superfamily binding protein which involved in many pathological processes. The function of Fstl1 has been widely elucidated, but its structural characterization has not been explored. Here we solved the high‐resolution crystal structure of FK domain of murine Fstl1, analyzed its unique characteristics, and investigated its contribution to the function of full‐length Fstl1. We found that Fstl1‐FK forms a stable dimer in both solution and crystal, which suggest that this protein may function as a dimer during its interaction with TGF‐β, a molecule known to form dimer during activation process. We also found this FK domain is indispensable for the proper function of Fstl1 during the transduction of TGF‐β signaling. These observations provide important insights into the understanding of Fstl1 and may facilitate the exploration of this molecule in clinical study.     

Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

Crystal structure of a GH1 β‐glucosidase from Hamamotoa singularis

A GH1 β‐glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4′‐galactosyllactose, a tri‐galactooligosaccharide, at 3.0 and 2.1 Å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction.