Crystal structure of human phosphoglucose isomerase and analysis of the initial catalytic steps

The second enzyme in the glycolytic pathway, phosphoglucose isomerase (PGI), catalyses an intracellular aldose-ketose isomerization. Here we describe the human recombinant PGI structure (hPGI) solved in the absence of active site ligands. Crystals isomorphous to those previously reported were used to collect a 94% complete data set to a limiting resolution of 2.1 A. From the comparison between the free active site hPGI structure and the available human and rabbit PGI (rPGI) structures, a mechanism for protein initial catalytic steps is proposed. Binding of the phosphate moiety of the substrate to two distinct elements of the active site is responsible for driving a series of structural changes resulting in the polarisation of the active site histidine, priming it for the initial ring-opening step of catalysis.     

Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage

We showed previously that the intracellular transport of sphingolipids (SLs) is altered in SL storage disease fibroblasts, due in part to the secondary accumulation of free cholesterol. In the present study we examined the mechanism of cholesterol elevation in normal human skin fibroblasts induced by treatment with SLs. When cells were incubated with various natural SLs for 44 h, cholesterol levels increased 25-35%, and cholesterol esterification was reduced. Catabolism of the exogenous SLs was not required for elevation of cholesterol because (i) a non-hydrolyzable and a degradable SL analog elevated cellular cholesterol to similar extents, and (ii) incubation of cells with various SL catabolites, including ceramide, had no effect on cholesterol levels. Elevated cholesterol was derived primarily from low density lipoproteins (LDL) and resulted from up-regulation of LDL receptors induced by cleavage of the sterol regulatory element-binding protein-1. Upon SL treatment, cholesterol accumulated with exogenous SLs in late endosomes and lysosomes. These results suggest a model in which excess SLs present in endocytic compartments serve as a "molecular trap" for cholesterol, leading to a reduction in cholesterol at the endoplasmic reticulum, induction of sterol regulatory element-binding protein-1 cleavage, and up-regulation of LDL receptors.     

Integration of hypoxic dilation signaling pathways for skeletal muscle resistance arteries

Mediator contributions to hypoxic dilation of rat gracilis muscle resistance arteries were determined by measuring dilation, vascular smooth muscle hyperpolarization, and metabolite production after incremental hypoxia. Nitric oxide (NO) synthase inhibition abolished responses to mild hypoxia, whereas COX inhibition impaired responses to more severe hypoxia by 77%. Blocking 20-hydroxyeicosatetraenoic acid (20-HETE) impaired responses to moderate hypoxia. With only NO systems intact, responses were maintained with mild hypoxia (88% normal) mediated via K(Ca) channels. When only COX pathways were intact, responses to moderate-severe hypoxia were largely retained (79% of normal) mediated via K(ATP) channels. Vessel responses to moderate hypoxia were retained with only 20-HETE systems intact mediated via K(Ca) channels. NO production increased 5.6-fold with mild hypoxia; greater hypoxia was without further effect. With increased hypoxia, 20-HETE levels fell to 40% of control values. 6-keto-PGF(1alpha) levels were not altered with mild hypoxia, but increased 4.6-fold with severe hypoxia. These results suggest vascular reactivity to progressive hypoxia represents an integration of NO production (mild hypoxia), PGI(2) production (severe hypoxia), and reduced 20-HETE levels (moderate hypoxia).     

Formation of a copper specific binding site in non-native states of beta-2-microglobulin

A debilitating complication of long-term hemodialysis is the deposition of beta-2-microglobulin (beta2m) as amyloid plaques in the joint space. We have recently shown that Cu(2+) can be a contributing, if not causal, factor at concentrations encountered during dialysis therapy. The basis for this effect is destabilization and incorporation of beta2m into amyloid fibers upon binding of Cu(2+). In this work, we demonstrate that while beta2m binds Cu(2+) specifically in the native state, it is binding of Cu(2+) by non-native states of beta2m which is responsible for destabilization. Mutagenesis of potential coordinating groups for Cu(2+) shows that native state binding of Cu(2+) is mediated by residues and structures that are different than those which bind in non-native states. An increased affinity for copper by non-native states compared to that of the native state gives rise to overall destabilization. Using mass spectrometry, NMR, and fluorescence techniques, we show that native state binding is localized to H31 and W60 and is highly specific for Cu(2+) over Zn(2+) and Ni(2+). Binding of Cu(2+) in non-native states of beta2m is mediated by residues H13, H51, and H84, but not H31. Although denatured beta2m has characteristics of a globally unfolded state, it nevertheless demonstrates the following strong specificity of binding: Cu(2+) > Zn(2+) > Ni(2+). This requires the existence of a well-defined structure in the unfolded state of this protein. As Cu(2+) effects are reported in many other amyloidoses, e.g., PrP, alpha-synuclein, and Abeta, our results may be extended to the emerging field of divalent ion-associated amyloidosis.     

"Superhumanized" antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28

Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.     

Melanophore appearance in wild and red tilapia embryos

Red tilapia has aroused interest in many countries for its commercial potential. This tilapia strain combines a desirable coloration and appearance with other advantageous farming characteristics. To study the early appearance of melanophore pigmentation in tilapia, a red tilapia strain originating from Thailand and a wild type coloration of Oreochromis niloticus were used as broodstock to produce artificially wild x wild and red x red progenies. The larvae were assessed periodically up to the first feeding and were recorded. Wild type fish showed a regular appearance of stellate melanophores. In the red strain, the pattern of chromatophores varies from total absence of black spotting to different degrees of macromelanophore distribution. Comparison between red and wild types showed that these two tilapia can be easily scored at day 7. Further, we present indications that the pigmentation over the body develops independently of the initial degree of pigmentation.