Enhanced proteolytic clearance of plasma Aß by peripherally administered neprilysin does not result in reduced levels of brain Aß in mice.

Accumulation of ß-amyloid (Aß) in the brain is believed to contribute to the pathology of Alzheimer's Disease (AD). Aß levels are controlled by the production of Aß from amyloid precursor protein, degradation by proteases, and peripheral clearance. In this study we sought to determine whether enhancing clearance of plasma Aß with a peripherally administered Aß-degrading protease would reduce brain Aß levels through a peripheral sink. Neprilysin (NEP) is a zinc-dependent metalloprotease that is one of the key Aß-degrading enzymes in the brain. We developed a NEP fusion protein with in vitro degradation of Aß and a 10 day plasma half-life in mouse. Intravenous administration of NEP to wild-type and APP23 transgenic mice resulted in dose-dependent clearance of plasma Aß. However, this did not correspond to reduced levels of soluble brain Aß with treatment up to 5 weeks in WT mice or formic acid-extractable brain Aß with 3 month treatment in aged APP23. In contrast, intracranial injection of NEP resulted in an acute decrease in soluble brain Aß. We found no change in amyloid precursor protein gene expression in mice treated with intravenous NEP, suggesting that the lack of effects in the brain following this route of administration was not caused by compensatory upregulation of Aß production. Taken together, these results suggest a lack of a robust peripheral Aß efflux sink through which brain amyloid burdens can be therapeutically reduced.

Bicyclic carbamates as inhibitors of papain-like cathepsin proteases.

A 6-oxa-1-aza-bicyclo[3.2.1]octan-7-one system inhibits the proteolytic activity of several cysteine proteases belonging to the papain family. In vitro mechanistic studies and in silico calculations suggest that the minimal pi-overlap between the bridgehead nitrogen and the carbonyl leads to a considerable weakening of the urethane system, making it susceptible to nucleophilic attack from the active site thiol group. The resulting covalent adduct is slowly hydrolyzed, releasing the hydroxypiperidine product of the inhibitor. Synthesis and testing of a set of analogs led to variable protease subtype selectivities ranging from micromolar to nanomolar potencies.

Self-assembled small-molecule microarrays for protease screening and profiling.

Small-molecule microarrays are attractive for chemical biology as they permit the analysis of hundreds to thousands of interactions in a highly miniaturized format. Methods to prepare small-molecule microarrays from combinatorial libraries by a self-assembly process based on the sequence-specific hybridization of peptide nucleic acid (PNA) encoded libraries to oligonucleotide arrays are presented. A systematic study of the dynamic range for multiple detection agents, including direct fluorescence of attached fluorescein and cyanine-3 dyes, antibody-mediated fluorescence amplification, and biotin-gold nanoparticle detection, demonstrated that individual PNA-encoded probes can be detected to concentrations of 10 pM on the oligonucleotide microarrays. Furthermore, a new method for parallel processing of biological samples by using gel-based separation of probes is presented. The methods presented in this report are exemplified through profiling two closely related cysteine proteases, cathepsin K and cathepsin F, across a 625-member PNA-encoded tetrapeptide acrylate library. A series of the specific cathepsin K and F inhibitors identified from the library were kinetically characterized and shown to correlate with the observed microarray profile, thus validating the described methods. Importantly, it was shown that this method could be used to obtain orthogonal inhibitors that displayed greater than tenfold selectivity for these closely related cathepsins.

Preliminary crystallographic analysis of the cysteine desulfurase IscS from Escherichia coli.

IscS is a widely distributed cysteine desulfurase that catalyzes the pyridoxal phosphate dependent beta-elimination of sulfur from L-cysteine and plays a central role in the delivery of sulfur to a variety of metabolic pathways. Crystals of Escherichia coli IscS have been obtained by the hanging-drop vapor-diffusion method using polyethylene glycol (PEG) as a precipitant. Initial seed crystals were obtained using PEG 6000 and sodium acetate, and diffraction-quality crystals were grown using a mixture of PEG 2000 and PEG 10 000 in the presence of sodium citrate. A complete native X-ray diffraction data set was collected from a single crystal at 103 K to a resolution of 2.1 A. The crystals belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 73.7086, b = 101.9741, c = 108.617 A (alpha = beta = gamma = 90 degrees ). Analysis of the Matthews equation and self-rotation function suggest two molecules per asymmetric unit, consistent with the presence of a single dimeric molecule.

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo.

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.