Cerebral deposition of amyloid β peptide (Aβ) is an early and critical feature of Alzheimer's disease. Aβ generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: β-secretase and γ-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of β-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of β-secretase cleavage products, and these were cleaved exactly and only at known β-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of β-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as β-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for β-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.
Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in ␣-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes -and ␥-synuclein. ␣-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for -or ␥-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between ␣-and -synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of ␣-, -, and ␥-synuclein show the same natively unfolded structure. While over time ␣-synuclein forms the previously described fibrils, no fibrils could be detected for -and ␥-synuclein under the same conditions. Most importantly, -and ␥-synuclein could not be cross-seeded with ␣-synuclein fibrils. However, under conditions that drastically accelerate aggregation, ␥-synuclein can form fibrils with a lag phase roughly three times longer than ␣-synuclein. These results indicate that -and ␥-synuclein are intrinsically less fibrillogenic than ␣-synuclein and cannot form mixed fibrils with ␣-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to ␣-synuclein and are also abundant in brain. Parkinson's disease (PD)1 is a neurodegenerative disorder that predominantly affects dopaminergic neurons in the nigrostriatal system but also affects several other regions of the brain. Pathological hallmarks of PD are Lewy bodies and Lewy neurites (1-3), which also accumulate in dementia with Lewy bodies (4) but not in a variety of other neurodegenerative disorders. Recently, two dominant mutations in ␣-synuclein have been linked to familial early onset PD (5, 6). This has put ␣-synuclein at the center of investigations into the pathogenesis of PD.␣-Synuclein is closely related to two other proteins, -and ␥-synuclein (Fig. 1A). With 78% similarity -synuclein has been called an "almost carbon copy" of ␣-synuclein (7), and it was not trivial to generate antibodies that clearly distinguish both forms (8); ␥-synuclein shares 60% similarity at the amino acid level with ␣-synuclein (Fig. 1A). All three synucleins are highly expressed in the human brain and show a strikingly similar regional distribution. They are all expressed in the thalamus, substantia nigra, caudate nucleus, amygdala, and the hippocampus (9). Moreover, ␣-and -synuclein even share the same subcellular distribution; they colocalize to presynaptic terminals in primary hippocampal neurons (10), and they show a virtually complete overlap in human and mouse brain sections as demonstrated by double-stained confocal microscopy (11). No ␣-or -synuclein-specific synapses were identified (11). The high expression of -and ␥-synuclein in the substantia nigra and their similarity to ␣-synuclein ...
The HIV-1 protein Vpr is critical for a number of viral functions including a unique ability to arrest T-cells at a G2/M checkpoint and induce subsequent apoptosis. It has been shown to interact specifically with the second UBA (ubiquitin associated) domain found in the DNA repair protein HHR23A, a highly evolutionarily conserved protein. This domain is a commonly occurring sequence motif in some members of the ubiquitination pathway, UV excision repair proteins, and certain protein kinases. The three dimensional structure of the UBA domain, determined by NMR spectroscopy, is presented. The protein domain forms a compact three-helix bundle. One side of the protein has a hydrophobic surface that is the most likely Vpr target site.
Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement of the cystine in the cyclic heptapeptide cyclo(CYVNVPC) by D-alpha-acetylthialysine or D-alpha-lysine gave cyclo(YVNVP(D-alpha-acetyl-thiaK)) (22) and cyclo(YVNVP(D-alpha-acetyl-K)) (30), which showed improved binding 10-fold relative to that of the control peptide KPFYVNVEF (1). NMR spectroscopy and molecular modeling experiments indicate that a beta-turn conformation centered around YVNV is essential for high-affinity binding. X-ray structure analyses show that the linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a beta-turn conformation. Our data confirm the unique structural requirements of the ligand binding site of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by the stabilization of the beta-turn conformation by three intramolecular hydrogen bonds (one mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.
Immature myeloid cells have been shown to transduce signals through a carboxyl-terminally truncated isoform of Stat5. This functionally distinct signal transducer and activator of transcription isoform is generated through a unique protein-processing event. Evaluation of numerous cell lines has determined that there is a direct correlation between the expression of truncated Stat5 and protease activity. Moreover, protease activity is found only in the myeloid and not in lymphoid progenitors. To further characterize the protease small quantities have been purified to near homogeneity. Studies on this purified material indicate that the protease has an apparent molecular mass of ϳ25 kDa and is active over a wide range of pH values. The protease will also cleave both activated (i.e. tyrosine-phosphorylated) and inactivate Stat5. Although this activity is sensitive to phenylmethylsulfonyl fluoride, it is notably not sensitive to several other serine protease inhibitors. Additional studies have led to the identification of the unique site where the protease cleaves Stat5. Mutagenesis of this site renders Stat5 resistant to cleavage. Consistent with the model that Stat5 cleavage is important for early myeloid development, introduction of a "non-cleavable" isoform of Stat5 into FDC-P1 cells (a myeloid progenitor line) leads to significant phenotypic changes.
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