Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

Much evidence indicates that, during activation of seven-transmembrane (7TM) receptors, the intracellular segments of the transmembrane helices (TMs) move apart with large amplitude, rigid body movements of especially TM-VI and TM-VII. In this study, AspIII:08 (Asp113), the anchor point for monoamine binding in TM-III, was used as the starting point to engineer activating metal ion sites between the extracellular segments of the beta2-adrenergic receptor. Cu(II) and Zn(II) alone and in complex with aromatic chelators acted as potent (EC50 decreased to 0.5 microm) and efficacious agonists in sites constructed between positions III:08 (Asp or His), VI:16 (preferentially Cys), and/or VII:06 (preferentially Cys). In molecular models built over the backbone conformation of the inactive rhodopsin structure, the heavy atoms that coordinate the metal ion were located too far away from each other to form high affinity metal ion sites in both the bidentate and potential tridentate settings. This indicates that the residues involved in the main ligand-binding pocket will have to move closer to each other during receptor activation. On the basis of the distance constraints from these activating metal ion sites, we propose a global toggle switch mechanism for 7TM receptor activation in which inward movement of the extracellular segments of especially TM-VI and, to some extent, TM-VII is coupled to the well established outward movement of the intracellular segments of these helices. We suggest that the pivots for these vertical seesaw movements are the highly conserved proline bends of the involved helices.     

Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies

The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2'-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.     

Structure-guided engineering of xylitol dehydrogenase cosubstrate specificity

Xylitol dehydrogenase (XDH) is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol. For efficient xylose utilization at high flux rates, cosubstrates should be recycled between the NAD+-specific XDH and the NADPH-preferring xylose reductase, another enzyme in the pathway. To understand and alter the cosubstrate specificity of XDH, we determined the crystal structure of the Gluconobacter oxydans holoenzyme to 1.9 angstroms resolution. The structure reveals that NAD+ specificity is largely conferred by Asp38, which interacts with the hydroxyls of the adenosine ribose. Met39 stacked under the purine ring and was also located near the 2' hydroxyl. Based on the location of these residues and on sequence alignments with related enzymes of various cosubstrate specificities, we constructed a double mutant (D38S/M39R) that was able to exclusively use NADP+, with no loss of activity.     

Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation—the Phase II conjugation reaction—of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3′-phosphoadenosine-5′-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.     

Inflammatory and Immunological parameters in adults with Down syndrome

Background  

The increase in life expectancy within the general population has resulted in an increasing number of elderly adults, including patients with Down syndrome (DS), with a current life expectancy of about 50 years. We evaluate the parameters of humoral and cellular immune response, the quantitative expression of the regulator of calcineurin1 gene (RCAN1) and the production of cytokines. The study group consisted of adults DS (n = 24) and a control group with intellectual disability without Down syndrome (ID) (n = 21) and living in a similar environmental background. It was evaluated serology, immunophenotyping, the quantitative gene expression of RCAN1 and the production of cytokines.     

In Vitro-Stimulated IL-6 Monocyte Secretion and In Vivo Peripheral Blood T Lymphocyte Activation Uniquely Predicted 15-Year Survival in Patients with Head and Neck Squamous Cell Carcinoma

The study was performed in order to determine whether peripheral blood monocyte in vitro function, and lymphocyte in vivo activation at diagnosis, was associated with HPV tumor infection status and 15-year survival in head and neck squamous cell carcinoma (HNSCC) patients. Sixty-five patients from a consecutive cohort of newly diagnosed HNSCCs, together with 18 control patients, were included in the study. Monocyte responsiveness was assessed by measuring monocyte in vitro interleukin (IL)-6 secretions after 24 hours of LPS stimulation in cultures with a serum-free medium. T lymphocyte activation was determined as the fraction of CD71-positive cells on CD3-positive cells by flow cytometry, whereas HPV infection was determined by PCR on formalin-fixed paraffin-embedded (FFPE) tumor tissue. Disease-specific survivals and overall survivals were determined 15 years following inclusion. HPV-positive HNSCC patients had a lower monocyte LPS-stimulated IL-6 response. A high LPS-stimulated monocyte IL-6 response predicted a decreased survival rate (P=0.019). A high percentage of CD71-positive T lymphocytes also predicted an impaired prognosis (P=0.021). The predictive power of IL-6 monocyte LPS-stimulated responses was retained when adjusted for age, gender and TNM stage of the patients. The monocyte and T lymphocyte survival predictions were independent of each other. The survival was particularly low with a combined high activated monocyte and T lymphocyte status. In a multivariate analysis, IL-6 secretion and the percentage of CD71-positive T lymphocytes both uniquely predicted survival independent of HPV infection status. It is postulated that the natural and adaptive immune systems are separately and additionally linked to the clinical aggressiveness of HNSCCs.