Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves b-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in b-casein turnover when the assay temperature is raised from 37 to 45 8C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identi®cation of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insu cient to assign these`orphan' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Brie¯y, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identi®ed they can be used as pharmacological tools to explore receptor function and relationship to disease.

This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd 15 nM). The screen was performed at uHTS rates (i.e., >100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality.

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.

Pseudomonic acid A (PS-A)1 is a potent antibiotic that acts through inhibition of bacterial isoleucyl-tRNA synthetase (IleRS, E) (1, 2). As part of an effort to understand the precise mechanism of the interaction of PS-A and its analogues with IleRS we have undertaken a detailed characterization of the kinetics and thermodynamics of substrate and inhibitor binding by this enzyme (3, 4). These experiments showed that PS-A analogues are reversible, slow tight-binding inhibitors that form an highly stabilized inhibitor complex with overall dissociation constants in the picomolar range. Comparison of PS-A analogues with inhibitors that were non-hydrolyzable analogues of the normal activated enzyme intermdiate, Ile-AMP, showed that both classes of inhibitor bound to IleRS in a manner that was mutually exclusive with each other and with the substrates, L-isoleucine (Ile) and Mg⅐ATP, but that very different enzyme conformations were induced upon binding (as shown by differences in IleRS tryptophan fluorescence) (4). In this report, we describe experiments in which we attempted to gain additional information on the binding of substrates and inhibitors to IleRS using different approaches to the more classical techniques which we had previously employed (e.g. state-state and transient kinetics (3, 4)).One technique for investigating changes in the conformation of proteins as a result of ligand binding is to test their effect upon proteolysis (e.g. Ref. 5), and this has previously been shown to yield useful information on several aminoacyl-tRNA synthetases (e.g. Refs. 6 -9). Here, we used this approach in two distinct ways. First, the effect of substrate and inhibitor binding on IleRS proteolysis patterns was investigated in an attempt to identify protein determinants for binding. Second, a homogeneous real-time assay capable of monitoring ligand protection from IleRS proteolysis using fluorescence polarization was developed which could be potentially used to screen for new inhibitors.