In recent decades, evidence has been accumulating showing the important role of urokinase-type plasminogen activator (uPA) in growth, invasion, and metastasis of malignant tumours. The evidence comes from results with animal tumour models and from the observation that a high level of uPA in human tumours is associated with a poor patient prognosis. It therefore initially came as a surprise that a high tumour level of the uPA inhibitor plasminogen activator inhibitor-I (PAI-I) is also associated with a poor prognosis, the PAI-I level in fact being one of the most informative biochemical prognostic markers. We review here recent investigations into the possible tumour biological role of PAI-I, performed by animal tumour models, histological examination of human tumours, and new knowledge about the molecular interactions of PAI-I possibly underlying its tumour biological functions. The exact tumour biological functions of PAI-I remain uncertain but PAI-I seems to be multifunctional as PAI-I is expressed by multiple cell types and has multiple molecular interactions. The potential utilisation of PAI-I as a target for anti-cancer therapy depends on further mapping of these functions.

To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridgeconstrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent K i , which at 25°C and pH 7.4 was ϳ500 nM. At the same conditions, the equilibrium dissociation constant K D , monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ϳ400 nM. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg 4 of upain-1 as the P 1 residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P 1 , P 2 , P 3 , P 4 , and the P 5 residues of upain-1. Substitution with alanine of the P 2 residue, Trp 3 , converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P 2 residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.Serine proteases of the trypsin family (clan SA) have many physiological and pathophysiological functions. There is therefore extensive interest in generating specific inhibitors to be used for pharmacological interference with their enzymatic activity. Moreover, serine proteases are classical subjects for studies of catalytic and inhibitory mechanisms.Serine protease-catalyzed peptide bond hydrolysis proceeds through a tetrahedral transition state formed by a nucleophilic attack on the carbonyl group of the substrate P 1 amino acid by the hydroxyl group of Ser 195 (using the chymotrypsin template numbering (1)), with His 57 and Asp 102 acting as a charge relay system. The protonated His 57 functions as a general acid to facilitate collapse of the tetrahedral intermediate that is stabilized through interactions at the oxyanion hole and main chain ␤-strand-type hydrogen bonds between the P 1 -P 3 and P 2 Ј amino acids of the substrate and residues within the polypeptide binding cleft, as well as specific contacts within the S 1 , S 2 , S 3 , S 1 Ј, and S 2 Ј pockets, which bind respective side chains of the P 1 , P 2 , P 3 , P 1 Ј, and P 2 Ј residues (for reviews, see Refs. 2 and 3). Substrate specificity is governed by the fit of the P residues into their corresponding S-pockets as ...

Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in ␤-sheet A and detachment of ␤-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg 273 and Asp 224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn 267 . Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.The serpins constitute a family of proteins of which the majority are inhibitors of serine proteases (1). Of decisive importance for the inhibitory mechanism is the surface-exposed reactive center loop (RCL) 5 between ␤-strands s5A and s1C (Fig. 1). The scissile P 1 -P 1 Ј bond in the RCL is attacked by the serine protease, but at the enzyme-acyl intermediate stage, where the active site serine of the protease and the P 1 residue of the serpin are linked by an ester bond, the N-terminal part of the RCL inserts as s4A thereby pulling the protease to the opposite pole of the serpin and distorting its active site. The required energy stems from stabilization of the serpin in the "relaxed" conformation with an inserted RCL, as compared with the "stressed," active conformation with a surface-exposed RCL. The conformational changes associated with the insertion of s4A in serpins are well described by x-ray crystal structure analyses and include rearrangements in a region around s1A, s2A, hD, and hE referred to as the flexible joint region (reviewed in Ref.2).Plasminogen activator inhibitor-1 (PAI-1) and antithrombin III are unique among serpins as they can spontaneously adopt the relaxed conformation without prior cleavage of the RCL. This is referred to as latency transition and involves insertion of the N-terminal part of the intact RCL as s4A while the C-terminal part and the connec...

BackgroundHomeobox genes are essential for embryonic patterning and cell fate determination. They are regulated mostly at the transcriptional level. In particular, Prep1 regulates Hox transcription in association with Pbx proteins. Despite its nuclear role as a transcription factor, Prep1 is located in the cytosol of mouse oocytes from primary to antral follicles. The homeodomain factor Bicoid (Bcd) has been shown to interact with 4EHP (eukaryotic translation initiation factor 4E homolog protein) to repress translation of Caudal mRNA and to drive Drosophila embryo development. Interestingly, Prep1 contains a putative binding motif for 4EHP, which may reflect a novel unknown function.Methodology/Principal FindingsIn this paper we show by confocal microscopy and deconvolution analysis that Prep1 and 4EHP co-localize in the cytosol of growing mouse oocytes, demonstrating their interaction by co-immunoprecipitation and pull-down experiments. A functional 4EHP-binding motif present in Prep1 has been also identified by mutagenesis analysis. Moreover, Prep1 inhibits (>95%) the in vitro translation of a luciferase reporter mRNA fused to the Hoxb4 3′UTR, in the presence of 4EHP. RNA electrophoretic mobility shift assay was used to demonstrate that Prep1 binds the Hoxb4 3′UTR. Furthermore, conventional histology and immunohistochemistry has shown a dramatic oocyte growth failure in hypomorphic mouse Prep1i/i females, accompanied by an increased production of Hoxb4. Finally, Hoxb4 overexpression in mouse zygotes showed a slow in vitro development effect.ConclusionsPrep1 has a novel cytoplasmic, 4EHP-dependent, function in the regulation of translation. Mechanistically, the Prep1-4EHP interaction might bridge the 3′UTR of Hoxb4 mRNA to the 5′ cap structure. This is the first demonstration that a mammalian homeodomain transcription factor regulates translation, and that this function can be possibly essential for the development of female germ cells and involved in mammalian zygote development.

Meis1 overexpression induces tumorigenicity but its activity is inhibited by Prep1 tumor suppressor. Why does overexpression of Meis1 cause cancer and how does Prep1 inhibit? Tumor profiling and ChIP-sequencing data in a genetically-defined set of cell lines show that: 1) The number of Meis1 and Prep1 DNA binding sites increases linearly with their concentration resulting in a strong increase of “extra” target genes. 2) At high concentration, Meis1 DNA target specificity changes such that the most enriched consensus becomes that of the AP-1 regulatory element, whereas the specific OCTA consensus is not enriched because diluted within the many extra binding sites. 3) Prep1 inhibits Meis1 tumorigenesis preventing the binding to many of the “extra” genes containing AP-1 sites. 4) The overexpression of Prep1, but not of Meis1, changes the functional genomic distribution of the binding sites, increasing seven fold the number of its “enhancer” and decreasing its “promoter” targets. 5) A specific Meis1 “oncogenic” and Prep1 “tumor suppressing” signature has been identified selecting from the pool of genes bound by each protein those whose expression was modified uniquely by the “tumor-inducing” Meis1 or tumor-inhibiting Prep1 overexpression. In both signatures, the enriched gene categories are the same and are involved in signal transduction. However, Meis1 targets stimulatory genes while Prep1 targets genes that inhibit the tumorigenic signaling pathways.