A critical role of the amyloid precursor protein (APP) in Alzheimer's disease (AD) pathogenesis has been well established. However, the physiological function of APP remains elusive and much debated. We reported previously that the APP family of proteins is essential in mediating the developing neuromuscular synapse. In the current study, we created a conditional allele of APP and deleted APP in presynaptic motor neuron or postsynaptic muscle. Crossing these alleles onto the APP-like protein 2-null background reveals that, unexpectedly, inactivating APP in either compartment results in neuromuscular synapse defects similar to the germline deletion and that postsynaptic APP is obligatory for presynaptic targeting of the high-affinity choline transporter and synaptic transmission. Using a HEK293 and primary hippocampus mixed-culture assay, we report that expression of APP in HEK293 cells potently promotes synaptogenesis in contacting axons. This activity is dependent on neuronal APP and requires both the extracellular and intracellular domains; the latter forms a complex with Mint1 and Cask and is replaceable by the corresponding SynCAM (synaptic cell adhesion molecule) sequences. These in vitro and in vivo studies identify APP as a novel synaptic adhesion molecule. We postulate that transsynaptic APP interaction modulates its synaptic function and that perturbed APP synaptic adhesion activity may contribute to synaptic dysfunction and AD pathogenesis.

Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemü ller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579 -582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5-to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-␤-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.The ubiquitin-proteasome system is the major pathway for degrading proteins in the cytosol and nucleus in eukaryotic cells (1, 2). Proteins marked for degradation by an attachment of a polyubiquitin chain are hydrolyzed by the 26 S proteasome in an ATP-dependent manner (3-5). This complex consists of the 20 S core proteasome, in which proteolysis occurs, and two 19 S regulatory complexes (6, 7). 20 S proteasomes also exist in archaea and many eubacteria, which lack both 26 S proteasomes and ubiquitin (8). The 20 S proteasome from Thermoplasma acidophilum has proven to be especially useful for studies of the proteasome's structure and catalytic mechanism (9, 10). Like the eukaryotic core particle, the T. acidophilum proteasome is a cylindrical complex consisting of four superimposed 7-membered rings (11). However, these archaeal particles are simpler and more symmetric in organization. The two outer rings are composed of identical ␣-subunits, and its inner rings are composed of identical ␤-subunits, each of which contains an active site. These 14 active sites are located on the inner surface of this particle.The narrow openings in the ␣-rings serve as sites of substrate entrance into the inner chamber of the cylinder where proteolysis occurs. Globular proteins are too large to traverse these openings and need to be unfolded to be translocated into the 20 S particle for degradation (11). Presumably, t...

The development of inhibitors of protein tyrosine kinases (PTKs) is a promising approach to obtaining new therapeutic agents for a variety of diseases, particularly cancer. However, the discovery of peptide-based inhibitors has been hindered by the lack of small peptide substrate sequences (i.e. five residues or less) with which a variety of inhibitor designs could be readily evaluated by replacing the Tyr with natural and unnatural amino acids. These prototypical small peptide inhibitors could then form the basis for designing analogous conformationally constrained, peptide-mimetic or non-peptide inhibitors with improved therapeutic potential. In this study we have identified the best known small peptide substrate for the PTK pp60c-src, which is the parent of the src family of nonreceptor PTKs. This pentapeptide substrate, Ac-Ile-Tyr-Gly-Glu-Phe-NH2, has a Km of 368 microM and Vmax of 1.02 mumol/min/mg when tested utilizing the assay methodology of Budde et al. (Anal. Biochem. 1992, 200, 347-351) after a series of modifications were made to more closely simulate the conditions inside a typical mammalian cell. This substrate was designed from information obtained by Songyang et al. (Nature 1995, 373, 536-539) with a 2.5 billion member combinatorial library of peptide substrates for pp60c-src. A second pentapeptide substrate, Ac-Glu-Asp-Ala-Ile-Tyr-NH2, with a weaker binding affinity (Km = 880 microM) but improved Vmax (1.86 mumol/min/mg), was also identified. This peptide was designed from the pp60c-src autophosphorylation sequence and information obtained by Songyang et al. (Ibid.) and Till et al. (J. Biol. Chem. 1994, 269, 7423-7428) with combinatorial libraries of peptide substrates. These new substrates provide sufficient binding affinities and rates of phosphorylation to be utilized for evaluating the relative effectiveness of various reversible and mechanism-based irreversible inhibitor designs for pp60c-src while appended to easily prepared small peptides.

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About 70% of zebrafish (Danio rerio) genes are orthologues of the human's, which are of great interests, but still largely unknown for their functions. Recently, a report on human histone PARylation factor 1 (HPF1/C4orf27) showed that it is involved in DNA damage response along with poly (ADP-ribose) polymerase 1 (PARP1). However, its function in living organism remains unclear. Given that zebrafish has showed its values in modeling human diseases and physiology, we characterized a zebrafish homolog of human HPF1 by sequence alignment. We also analyzed its expression pattern during early development and among adult tissues. Furthermore, knocking down hpf1 by morpholinos affected zebrafish early development. Our work provides a novel clue for the mechanism of genome stability and early embryogenesis.