The 1.8 A crystal structure of alpha1 acid glycoprotein Orosomucoid solved by UV RIP reveals the broad drug binding activity of this human plasma lipocalin

alpha1 Acid glycoprotein AGP is an important drug binding protein in human plasma and, as an acute phase protein, it has a strong influence on pharmacokinetics and pharmacodynamics of many pharmaceuticals. We report the crystal structure of the recombinant unglycosylated human AGP at 1.8 resolution, which was solved using the new method of UV radiation damage induced phasing UV RIP . AGP reveals a typical lipocalin fold comprising an eight stranded beta; barrel. Of the four loops that form the entrance to the ligand binding site, loop 1, which connects beta strands A and B, is among the longest observed so far and exhibits two full turns of an alpha helix. Furthermore, it carries one of the five N linked glycosylation sites, while a second one occurs underneath the tip of loop 2. The branched, partly hydrophobic, and partly acidic cavity, together with the presumably flexible loop 1 and the two sugar side chains at its entrance, explains the diverse ligand spectrum of AGP, which is known to vary with changes in glycosylation patter

Microtubule-Destabilizing Agents: Structural and Mechanistic Insights from the Interaction of Colchicine and Vinblastine with Tubulin

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Characterization of photocycle intermediates in crystalline photoactive yellow protein.

The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice

Glomerular endothelial surface layer acts as a barrier against albumin filtration

Glomerular endothelium is highly fenestrated, and its contribution to glomerular barrier function is the subject of debate. In recent years, a polysaccharide-rich endothelial surface layer (ESL) has been postulated to act as a filtration barrier for large molecules, such as albumin. To test this hypothesis, we disturbed the ESL in C57Bl/6 mice using long-term hyaluronidase infusion for 4 weeks and monitored albumin passage using immunolabeling and correlative light-electron microscopy that allows for complete and integral assessment of glomerular albumin passage. ESL ultrastructure was visualized by transmission electron microscopy using cupromeronic blue and by localization of ESL binding lectins using confocal microscopy. We demonstrate that glomerular fenestrae are filled with dense negatively charged polysaccharide structures that are largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact. Both retention of cationic ferritin in the glomerular basement membrane and systemic blood pressure were unaltered. Enzyme treatment, however, induced albumin passage across the endothelium in 90% of glomeruli, whereas this could not be observed in controls. Yet, there was no net albuminuria due to binding and uptake of filtered albumin by the podocytes and parietal epithelium. ESL structure and function completely recovered within 4 weeks on cessation of hyaluronidase infusion. Thus, the polyanionic ESL component, hyaluronan, is a key component of the glomerular endothelial protein permeability barrier

Conversion of mature human beta-cells into glucagon-producing alpha-cells

Conversion of one terminally differentiated cell type into another (or transdifferentiation) usually requires the forced expression of key transcription factors. We examined the plasticity of human insulin-producing beta-cells in a model of islet cell aggregate formation. Here, we show that primary human beta-cells can undergo a conversion into glucagon-producing alpha-cells without introduction of any genetic modification. The process occurs within days as revealed by lentivirus-mediated beta-cell lineage tracing. Converted cells are indistinguishable from native alpha-cells based on ultrastructural morphology and maintain their alpha-cell phenotype after transplantation in vivo. Transition of beta-cells into alpha-cells occurs after beta-cell degranulation and is characterized by the presence of beta-cell-specific transcription factors Pdx1 and Nkx6.1 in glucagon(+) cells. Finally, we show that lentivirus-mediated knockdown of Arx, a determinant of the alpha-cell lineage, inhibits the conversion. Our findings reveal an unknown plasticity of human adult endocrine cells that can be modulated. This endocrine cell plasticity could have implications for islet development, (patho)physiology, and regeneration