Reversible pegylation prolongs the hypotensive effect of atrial natriuretic peptide

Natriuretic peptides (NP), including atrial natriuretic peptide (ANP), induce potent natriuresis and vasodilation and thereby generate hypotension in vivo. Despite intensive efforts, clinical application of NP as an antihypertensive agent is limited because of their short biological half-life and poor bioavailability. Recently, we have developed a strategy that facilitates slow release of peptides from PEG-peptide inactive conjugates, based on reversible pegylation. Peptides prepared by this approach undergo slow, spontaneous chemical hydrolysis at physiological conditions, releasing the native active peptide/protein drug from the inactive conjugates over prolonged periods. A PEG chain of 30 kDa was linked covalently to the alpha-amino side chain of the hormone via a MAL-Fmoc-NHS spacer, yielding PEG 30-Fmoc-ANP, a prodrug that releases the native hormone upon incubation at physiological conditions. Bolus administration of native ANP to Wistar rats receiving adrenaline yields a short, transitory effect in lowering blood pressure (BP), reaching a maximum at 2 min, and then returning to control values after 12 to 25 min. In contrast, administration of PEG 30-Fmoc-ANP lowered BP following a lag period of 50 min, and maintained low BP for a period exceeding 60 min. Saline or PEG 30-Fmoc-Alanine were not effective in lowering BP in Wistar rats. These results show that the novel compound, PEG 30-Fmoc-ANP, is a reversible pegylated prodrug derivative that facilitates a prolonged BP lowering effect in rats and may be considered as a candidate for development into an antihypertensive drug.     

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment

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The ferrous-oxy complex of human aromatase

In this communication, we document the self-assembly of heterologously expressed truncated human aromatase (CYP19) into nanometer scale phospholipids bilayers (Nanodiscs). The resulting P450 CYP19 preparation is stable and can tightly associate with the substrate androstenedione to form a nearly complete high-spin ferric protein. Ferrous CYP19 in Nanodiscs was mixed anaerobically in a rapid-scan stopped-flow with atmospheric dioxygen and the formation of the ferrous-oxy complex observed. First order decay of the oxy-complex to release superoxide and regenerate the ferric enzyme was monitored kinetically. Surprisingly, the ferrous-oxy complex of aromatase is more stable than that of hepatic CYP3A4, opening the path to precisely determine the biochemical and biophysical properties of the reaction cycle intermediates in this important human drug target.     

FtsZ Dynamics during the Division Cycle of Live Escherichia coli Cells

The dynamics and assembly of bacterial cell division protein FtsZ were monitored in individual, growing and dividing Escherichia coli cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ. Cells expressing FtsZ-GFP at levels less than or equivalent to that of wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence. During the late stages of cytokinesis, which constituted the last one-fourth of the cell cycle, the lumen of the FtsZ ring disappeared as the whole structure condensed. At this time, loops of FtsZ-GFP polymers emanated outward from the condensing ring structure and other unstable fluorescent structures elsewhere in the cell were also observed. Assembly of FtsZ rings at new division sites occurred within 1 min, from what appeared to be single points. Interestingly, this nucleation often took place in the predivisional cell at the same time the central FtsZ ring was in its final contraction phase. This demonstrates directly that, at least when FtsZ-GFP is being expressed, new division sites have the capacity to become fully functional for FtsZ targeting and assembly before cell division of the mother cell is completed. The results suggest that the timing of FtsZ assembly may be normally controlled in part by cellular FtsZ concentration. The use of wide-field optical sectioning microscopy to obtain sharp fluorescence images of FtsZ structures is also discussed.     

Optimized small‐molecule pull‐downs define MLBP1 as an acyl‐lipid‐binding protein

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC‐MS to identify candidate binding ligands. We optimized this method using ABA–PYL interactions and show that ABA co‐purifies with wild‐type PYL5 but not a binding site mutant. The K_d of PYL5 for ABA is 1.1 μm , which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37 START domain‐related proteins, which resulted in the identification of ligands that co‐purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co‐purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.     

A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface

In Escherichia coli, initial assembly of the Z ring for cell division requires FtsZ plus the essential Z ring‐associated proteins FtsA and ZipA. Thermosensitive mutations in ftsA, such as ftsA27, map in or near its ATP binding pocket and result in cell division arrest at non‐permissive temperatures. We found that purified wild‐type FtsA bound and hydrolysed ATP, whereas FtsA27 was defective in both activities. FtsA27 was also less able to localize to the Z ring in vivo. To investigate the role of ATP transactions in FtsA function in vivo, we isolated intragenic suppressors of ftsA27. Suppressor lesions in the ATP site restored the ability of FtsA27 to compete with ZipA at the Z ring, and enhanced ATP binding and hydrolysis in vitro. Notably, suppressors outside of the ATP binding site, including some mapping to the FtsA‐FtsA subunit interface, also enhanced ATP transactions and exhibited gain of function phenotypes in vivo. These results suggest that allosteric effects, including changes in oligomeric state, may influence the ability of FtsA to bind and/or hydrolyse ATP.