Post-translational Transformation of Methionine to Aspartate Is Catalyzed by Heme Iron and Driven by Peroxide: A NOVEL SUBUNIT-SPECIFIC MECHANISM IN HEMOGLOBIN*

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H₂O₂). Using H₂¹⁸O₂, we verified incorporation of ¹⁸O into the Asp carboxyl side chain confirming the role of H₂O₂ in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.     

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln²⁶³ serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.     

Crystal Structure of α-1,4-Glucan Lyase,a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-d-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (β/α)₈-barrel catalytic domain with B and B′ subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp⁵⁵³ and Asp⁶⁶⁵ are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite −1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.     

Role of 4-1BB Receptor in the Control Played by CD8+ T Cells on IFN-γ Production by Mycobacterium tuberculosis Antigen-Specific CD4+ T Cells

Background

Antigen-specific IFN-γ producing CD4⁺ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-γ production without affecting protective IFN-γ is a challenge in tuberculosis research.

Methods and Findings

Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4⁺ T cell-mediated IFN-γ response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-γ response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8⁺ T cells which suppressed IFN-γ-secreting CD4⁺ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-γ responses by CD4⁺ T cells in protein-boosted mice without affecting the low protective IFN-γ-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8⁺ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-γ inhibition did not require soluble IL-10, TGF-β, XCL-1 and MIP-1β. In vivo Ag85B stimulation induced 4-1BB expression on CD8⁺ T cells and in vivo 4-1BB ligation reduced the activation, IFN-γ production and expansion of Ag85B-specific CD4⁺ T cells of DNA-primed and protein-boosted mice.

Conclusion/Significance

Antigen-specific suppressor CD8⁺ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-γ-secreting CD4⁺ T cells. The selective expression of 4-1BB only on CD8⁺ T cells in mice developing a massive, non-protective IFN-γ response opens novel strategies for intervention in tuberculosis pathology and vaccination through T-cell co-stimulatory-based molecular targeting.     

Combined Antigen-Specific Interferon-γ and Interleukin-2 Release Assay (FluoroSpot) for the Diagnosis of Mycobacterium tuberculosis Infection

Background

To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.

Methods

Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.

Results

Correlation of IFN- γ⁺ spot-forming cells in EliSpot-IGRA and FluoroSpot were R² = 0.67 for ESAT-6 and R² = 0.73 for CFP-10. The number of IL-2^- IFN- γ⁺ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ⁺ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.

Conclusions

Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice.     

Conformational Preference of ‘CαNN’ Short Peptide Motif towards Recognition of Anions

Among several ‘anion binding motifs’, the recently described ‘C^αNN’ motif occurring in the loop regions preceding a helix, is conserved through evolution both in sequence and its conformation. To establish the significance of the conserved sequence and their intrinsic affinity for anions, a series of peptides containing the naturally occurring ‘C^αNN’ motif at the N-terminus of a designed helix, have been modeled and studied in a context free system using computational techniques. Appearance of a single interacting site with negative binding free-energy for both the sulfate and phosphate ions, as evidenced in docking experiments, establishes that the ‘C^αNN’ segment has an intrinsic affinity for anions. Molecular Dynamics (MD) simulation studies reveal that interaction with anion triggers a conformational switch from non-helical to helical state at the ‘C^αNN’ segment, which extends the length of the anchoring-helix by one turn at the N-terminus. Computational experiments substantiate the significance of sequence/structural context and justify the conserved nature of the ‘C^αNN’ sequence for anion recognition through “local” interaction.     

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates

We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.     

Diosgenin from Dioscorea bulbifera: Novel Hit for Treatment of Type II Diabetes Mellitus with Inhibitory Activity against α-Amylase and α-Glucosidase

Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, ¹H NMR and ¹³C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus.     

Kinetics of Isomerization and Inversion of Aspartate 58 of αA-Crystallin Peptide Mimics under Physiological Conditions

Although proteins consist exclusively of L-amino acids, we have reported that aspartyl (Asp) 58 and Asp 151 residues of αA-crystallin of eye lenses from elderly cataract donors are highly inverted and isomerized to D-β, D-α and L-β-Asp residues through succinimide intermediates. Of these Asp isomers, large amounts of D-β- and L-β-isomers are present but the amount of D-α-isomer is not significant. The difference in abundance of the Asp isomers in the protein may be due to the rate constants for the formation of the isomers. However, the kinetics have not been well defined. Therefore, in this study, we synthesized a peptide corresponding to human αA-crystallin residues 55 to 65 (T⁵⁵VLD⁵⁸SGISEVR⁶⁵) and its isomers in which L-α-Asp at position 58 was replaced with L-β-, D-β- and D-α-Asp and determined the rate of isomerization and inversion of Asp residues under physiological conditions (37°C, pH7.4). The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 3 times higher than the rate constant for dehydration from L-β-Asp peptide to L-succinimidyl peptide. The rate constant for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide was about 5 times higher than the rate constant for hydrolysis from L-succinimidyl peptide to L-α-Asp peptide. The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 2 times higher than the rate constant for dehydration from D-α-Asp peptide to D-succinimidyl peptide. The rate constants for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide and for hydrolysis from D-succinimidyl peptide to D-β-Asp peptide were almost equal. Using these rate constants, we calculated the change in the abundance ratios of the 4 Asp isomers during a human lifespan. This result is consistent with the fact that isomerized Asp residues accumulate in proteins during the ageing process.     

CD4-CD8-αβ and γδ T Cells Display Inflammatory and Regulatory Potentials during Human Tuberculosis

T-cells play an important role controlling immunity against pathogens and therefore influence the outcome of human diseases. Although most T-lymphocytes co-express either CD4 or CD8, a smaller T-cell subset found the in the human peripheral blood that expresses the αβ or γδ T-cell-receptor (TCR) lacks the CD4 and CD8 co-receptors. These double negative (DN) T-cells have been shown to display important immunological functions in human diseases. To better understand the role of DN T-cells in human Mycobacterium tuberculosis, we have characterized their frequency, activation and cytokine profile in a well-defined group of tuberculosis patients, categorized as severe and non-severe based on their clinical status. Our data showed that whereas high frequency of αβ DN T-cells observed in M. tuberculosis-infected patients are associated with disease severity, decreased proportion of γδ DN T-cells are found in patients with severe tuberculosis. Together with activation of CD4⁺ and CD8⁺ T-cells, higher frequencies of both αβ and γδ DN T-cells from tuberculosis patients also express the chronic activation marker HLA-DR. However, the expression of CD69, an early activation marker, is selectively observed in DN T-cells. Interestingly, while αβ and γδ DN T-cells from patients with non-severe tuberculosis display a pro-inflammatory cytokine profile, characterized by enhanced IFN-γ, the γδ DN T-cells from patients with severe disease express a modulatory profile exemplified by enhanced interleukin-10 production. Overall, our findings suggest that αβ and γδ DN T-cell present disparate immunoregulatory potentials and seems to contribute to the development/maintenance of distinct clinical aspects of TB, as part of the complex immunological network triggered by the TB infection.     

Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K _i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K _i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn²⁺ ions, in particular the αS583C mutant showing a K _i for Zn²⁺of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K _i, the later mutation generating a Ca²⁺blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na⁺ions at an external Na⁺binding site preventing ion permeation through the channel pore.     

Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in Lactobacillus johnsonii 100-100

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [¹⁴C]taurocholic acid that were increased as much as threefold over control levels. [¹⁴C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.     

Combinatorial Pharmacogenetic Interactions of Bucindolol and β1, α2C Adrenergic Receptor Polymorphisms

Background

Pharmacogenetics involves complex interactions of gene products affecting pharmacodynamics and pharmacokinetics, but there is little information on the interaction of multiple genetic modifiers of drug response. Bucindolol is a β-blocker/sympatholytic agent whose efficacy is modulated by polymorphisms in the primary target (β₁ adrenergic receptor [AR] Arg389 Gly on cardiac myocytes) and a secondary target modifier (α_2C AR Ins [wild-type (Wt)] 322–325 deletion [Del] on cardiac adrenergic neurons). The major allele homozygotes and minor allele carriers of each polymorphism are respectively associated with efficacy enhancement and loss, creating the possibility for genotype combination interactions that can be measured by clinical trial methodology.

Methodology

In a 1,040 patient substudy of a bucindolol vs. placebo heart failure clinical trial, we tested the hypothesis that combinations of β₁389 and α_2C322–325 polymorphisms are additive for both efficacy enhancement and loss. Additionally, norepinephrine (NE) affinity for β₁389 AR variants was measured in human explanted left ventricles.

Principal Findings

The combination of β₁389 Arg+α_2C322–325 Wt major allele homozygotes (47% of the trial population) was non-additive for efficacy enhancement across six clinical endpoints, with an average efficacy increase of 1.70-fold vs. 2.32-fold in β₁389 Arg homozygotes+α_2C322–325 Del minor allele carriers. In contrast, the minor allele carrier combination (13% subset) exhibited additive efficacy loss. These disparate effects are likely due to the higher proportion (42% vs. 8.7%, P = 0.009) of high-affinity NE binding sites in β₁389 Arg vs. Gly ARs, which converts α_2CDel minor allele-associated NE lowering from a therapeutic liability to a benefit.

Conclusions

On combination, the two sets of AR polymorphisms 1) influenced bucindolol efficacy seemingly unpredictably but consistent with their pharmacologic interactions, and 2) identified subpopulations with enhanced (β₁389 Arg homozygotes), intermediate (β₁389 Gly carriers+α_2C322–325 Wt homozygotes), and no (β₁389 Gly carriers+α_2C322–325 Del carriers) efficacy.     

Complete integrin headpiece opening in eight steps

Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the α_IIbβ₃ integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca²⁺, α1 helix, α1’ helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward α_IIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.     

Structural and Dynamic Properties of the Human Prion Protein

Prion diseases involve the conformational conversion of the cellular prion protein (PrP^C) to its misfolded pathogenic form (PrP^Sc). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrP^C to PrP^Sc.     

The celA Gene, Encoding a Glycosyl Hydrolase Family 3 β-Glucosidase in Azospirillum irakense, Is Required for Optimal Growth on Cellobiosides

The CelA β-glucosidase of Azospirillum irakense, belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C₃, C₄, and C₅ oligosaccharides. The growth of a ΔcelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 β-glucosidases appear to be functional alternatives for the GHF1 β-glucosidases in the assimilation of β-glucosides by other bacteria.     

Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via β₂-adrenoceptor (β₂-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α_2A-and α_2C-AR (α_2A/2C-AR^-/-). These findings suggest that β₂-AR is not the single adrenoceptor involved in bone turnover regulation and show that α₂-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α_2A/2C-AR^-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α₂-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α₂-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α_2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α_2CAR^-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (μCT) analysis showed that α_2C-AR^-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α_2C-AR^-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α_2C-AR^-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α_2C-AR^-/- mice. Altogether, these findings suggest that α_2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α_2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.     

NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO₄ to NAD⁺ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD⁺. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD⁺-bound states. ITC measurements of RslTpt1 binding to NAD⁺ (K_D ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD⁺-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP⁺, which contains an adenylate 2′-PO₄ (mimicking the substrate RNA 2′-PO₄), binds with lower affinity (K_D ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD⁺ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD⁺ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.     

Effect of Short- and Long-Range Interactions on Trp Rotamer Populations Determined by Site-Directed Tryptophan Fluorescence of Tear Lipocalin

In the lipocalin family, the conserved interaction between the main α-helix and the β-strand H is an ideal model to study protein side chain dynamics. Site-directed tryptophan fluorescence (SDTF) has successfully elucidated tryptophan rotamers at positions along the main alpha helical segment of tear lipocalin (TL). The rotamers assigned by fluorescent lifetimes of Trp residues corroborate the restriction expected based on secondary structure. Steric conflict constrains Trp residues to two (t, g ⁻) of three possible χ₁ (t, g ⁻, g ⁺) canonical rotamers. In this study, investigation focused on the interplay between rotamers for a single amino acid position, Trp 130 on the α-helix and amino acids Val 113 and Leu 115 on the H strand, i.e. long range interactions. Trp130 was substituted for Phe by point mutation (F130W). Mutations at positions 113 and 115 with combinations of Gly, Ala, Phe residues alter the rotamer distribution of Trp130. Mutations, which do not distort local structure, retain two rotamers (two lifetimes) populated in varying proportions. Replacement of either long range partner with a small amino acid, V113A or L115A, eliminates the dominance of the t rotamer. However, a mutation that distorts local structure around Trp130 adds a third fluorescence lifetime component. The results indicate that the energetics of long-range interactions with Trp 130 further tune rotamer populations. Diminished interactions, evident in W130G113A115, result in about a 22% increase of α-helix content. The data support a hierarchic model of protein folding. Initially the secondary structure is formed by short-range interactions. TL has non-native α-helix intermediates at this stage. Then, the long-range interactions produce the native fold, in which TL shows α-helix to β-sheet transitions. The SDTF method is a valuable tool to assess long-range interaction energies through rotamer distribution as well as the characterization of low-populated rotameric states of functionally important excited protein states.