Structural basis of cooperativity in human UDP-glucose dehydrogenase

Background

UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.

Methodology

Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.

Conclusion

In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD⁺ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD⁺ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.     

BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

Background

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells.

Methodology/Principal Findings

We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation.

Conclusions/Significance

Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance.     

Diet and ranging behavior of the endangered Javan gibbon (Hylobates moloch) in a submontane tropical rainforest

Altitude influences forest structure and food abundance and distribution, which in turn affect primate feeding and ranging patterns. Javan gibbons (Hylobates moloch) are endemic to forests spanning a broad range of altitudes on Java, Indonesia. Most information about Javan gibbon behavior comes from studies in lowland forests, while the vast majority of wild gibbons remaining inhabit hill and lower montane forests. We studied the diets, activity patterns, and ranging behavior of three gibbon groups in hill/lower montane (950-1,100?m asl) forest in the Gunung Halimun-Salak National Park (GHSNP) from April 2008 to March 2009. The mean home range size was 37?ha and the mean daily path length was 1,180?m. The study groups spent 36% of time feeding, 41% resting, 15% traveling, 6% engaging in social behavior, and 2% in aggressive interactions. Fruit was the most important food (63% of feeding time) followed by leaves (24%), and flowers (12%). Our results suggest that Javan gibbons in higher elevation habitats have substantially larger home ranges than lowland populations, despite broad similarity in their activity budgets and diets. Conservation managers should consider the effects of altitude and habitat quality on gibbon ranging behavior when developing habitat corridors, selecting sites for translocation or reintroduction projects, and designating and managing protected areas.     

Beraprost enhances the APC function of B cells by upregulating CD86 expression levels

Lipid mediators are emerging as important regulators of the immune system. Based on our previous result that shows strong expression of prostacyclin synthase in the germinal center, we investigated whether prostacyclin would regulate the APC function of B cells. Owing to the very short half-life of prostacyclin in experimental conditions, we used a more stable analog, beraprost. Beraprost increased the amounts of the costimulatory molecule CD86 but not CD80 on the surface of activated B cells in time- and dose-dependent manners. However, the enhancing effect of beraprost was not observed on memory B cells, centroblasts, and centrocytes. Beraprost required BCR and CD40 signals to upregulate CD86 expression levels. Other prostanoids such as PGE(2), 6-keto-PGF(1α), and PGF(2α) failed to alter CD86 expression levels, whereas other prostacyclin analogs were as potent as beraprost. Results carried out with receptor antagonists revealed that beraprost enhanced CD86 levels by binding to prostacyclin receptor IP and by increasing intracellular cAMP concentrations. Beraprost-treated B cells potently stimulated allogeneic T cells, which was significantly abolished by CD86 neutralization. Our data imply an unrecognized cellular and molecular mechanism about the germinal center reactions.     

A tyrosine-sulfated CCR5-mimetic peptide promotes conformational transitions in the HIV-1 envelope glycoprotein

The HIV-1 envelope glycoprotein is a trimeric complex of heterodimers composed of a surface glycoprotein, gp120, and a transmembrane component, gp41. The association of this complex with CD4 stabilizes the coreceptor-binding site of gp120 and promotes the exposure of the gp41 helical region 1 (HR1). Here, we show that a 15-amino-acid peptide mimetic of the HIV-1 coreceptor CCR5 fused to a dimeric antibody Fc domain (CCR5mim-Ig) bound two gp120 molecules per envelope glycoprotein complex and by itself promoted HR1 exposure. CCR5mim-Ig also stabilized the association of a CD4-mimetic peptide with the envelope glycoprotein. A fusion of the CD4- and CCR5-mimetic peptides, DM1, bound gp120 and neutralized R5, R5X4, and X4 HIV-1 isolates comparably to CD4, and they did so markedly more efficiently than either peptide alone. Our data indicate that the potency of DM1-Ig derives from its avidity for the HIV-1 envelope glycoprotein trimer and from the bidirectional induction of its receptor-mimetic components. DM1 has significant advantages over other inhibitors that target both coreceptor and CD4-binding sites, and it may serve as a lead for a new class of HIV-1 inhibitor peptides.     

The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.     

Development of a reference material using methamphetamine abusers' hair samples for the determination of methamphetamine and amphetamine in hair

In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers' hair samples were collected, homogenized and finally bottled. The concentration of each bottle was determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC-MS) after derivatization with trifluoroacetic anhydride (TFAA). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64+/-1.24 and 0.54+/-0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.     

Polyethyleneimine-mediated chemical extraction of cytoplasmic His-tagged inclusion body proteins from Escherichia coli

The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His-tagged model proteins: gloshedobin, a thrombin-like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine-induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost-prohibitive spermine and EDTA. The residual PEI in the post-extraction mixture was efficiently counteracted by addition of Mg2+, allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI-mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.