alpha-chymotrypsin and 3-nitrotyrosine

Alpha-synuclein (alpha-syn) is a major protein component of the neuropathological hallmarks of Parkinson's disease and related neurodegenerative disorders termed synucleinopathies. Neither the mechanism of alpha-syn fibrillization nor the degradative process for alpha-syn has been elucidated. Previously, we showed that wild-type, mutated, and fibrillar alpha-syn proteins are substrates of calpain I in vitro. In this study, we demonstrate that calpain-mediated cleavage near and within the middle region of soluble alpha-syn with/without tyrosine nitration and oxidation generates fragments that are unable to self-fibrillize. More importantly, these fragments prevent full-length alpha-syn from fibrillizing. Calpain-mediated cleavage of alpha-syn fibrils composed of wild-type or nitrated alpha-syn generate C-terminally truncated fragments that retain their fibrillar structure and induce soluble full-length alpha-syn to co-assemble. Therefore, calpain-cleaved soluble alpha-syn inhibits fibrillization, whereas calpain-cleaved fibrillar alpha-syn promotes further co-assembly. These results provide insight into possible disease mechanisms underlying synucleinopathies since the formation of alpha-syn fibrils could be causally linked to the onset/progression of these disorders.

Topics: alpha-Synuclein; Calpain; Chymotrypsin; Humans; Hydrolysis; Microscopy, Immunoelectron; Nerve Degeneration; Nerve Tissue Proteins; Nitrates; Parkinson Disease; Peptide Fragments; Peroxynitrous Acid; Recombinant Proteins; Solubility; Synucleins; Tyrosine

Doppel (Dpl) is a glycosylphosphatidylinositol-anchored protein expressed in the testis. It exhibits 26% sequence identity with the prion protein (PrP) but lacks the octarepeat region implicated as the major copper-binding domain. Contrary to expectations, Cu(II) induced a 26% reduction in the intrinsic fluorescence of Dpl(27-154) and a calculated K(d) for a single-site model of 0.16 +/- 0.08 microm. Other metals had minimal effects on fluorescence quenching. Matrix-assisted laser desorption ionization mass spectrometry of a Dpl peptide revealed binding of copper (but not other metals) to the helical alphaB/B'-loop-alphaC subregion of Dpl. Fluorescence quenching and equilibrium dialysis analyses of this Dpl(101-145) peptide were compatible with a binding site of K(d) = 0.4 microm. Diethylpyrocarbonate footprinting (Qin, K., Yang, Y., Mastrangelo, P., and Westaway, D. (2002) J. Biol. Chem. 277, 1981-1990) of Dpl(27-154) defined one residue/molecule was protected by copper from diethylpyrocarbonate adduct formation, and reiteration of this analysis with Dpl(101-145) suggested that His(131) may contribute to Cu(II) binding. Taken together, our data indicate that the alpha-helical region of mouse Dpl possesses a selective copper-binding site with a submicromolar K(d) and perhaps one or more lower affinity sites. Although metallated forms of Dpl might exist in vivo, analyses of Tg(Dpl)10329 mice were inconsistent with reports that Dpl expression is associated with increased carbonylation and nitrosylation of brain proteins. Thus, rather than comprising an important source of free radical damage, copper binding may serve to modulate the activity, stability, or localization of the Dpl protein.

Topics: Animals; Binding Sites; Binding, Competitive; Blotting, Western; Carbon; Chymotrypsin; Circular Dichroism; Copper; Diethyl Pyrocarbonate; Dose-Response Relationship, Drug; Free Radicals; GPI-Linked Proteins; Histidine; Kinetics; Mice; Nitrogen; Peptides; Prions; Protein Binding; Proteins; PrPC Proteins; Recombinant Proteins; Spectrometry, Fluorescence; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tyrosine

The proteasomal pathway is responsible for processes essential for cell viability, including the selective degradation of oxidized proteins. An age-dependent loss in proteasome function has been reported in many tissues, but has not been examined in the retina. In this study, we evaluated proteasome function and protein oxidation in retinal homogenates from young adult and old F344BN rats. For retinal proteasome from old rats, we observed an 80% decrease in the rate of casein degradation and a 75% loss in chymotrypsin-like activity. This loss in activity could be partially accounted for by a 50% reduction in expression of the 20S proteasome. The regulatory complex PA700 and the inducible beta-subunit, LMP7, which is associated with the chymotrypsin-like activity, were expressed in equivalent concentrations relative to the 20S catalytic core in both young and old rats. Immunochemical analysis using antibodies that recognize the protein oxidative modifications, nitrotyrosine and 4-hydroxy-2-nonenal, showed that retinal proteins from old rats exhibited the greatest immunoreactivity. These results suggest that the age-related loss in proteasome function contributes to the accumulation of oxidized retinal proteins. Thus, the combined effect of an increase in oxidized proteins and inactivation of the protease responsible for ridding the cell of oxidized proteins places the aged retina at greater risk for irreversible damage caused by oxidative stress.

Topics: Aging; Aldehydes; Animals; Blotting, Western; Caseins; Chymotrypsin; Cysteine Endopeptidases; Eye Proteins; Male; Multienzyme Complexes; Oxidation-Reduction; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Inbred BN; Rats, Inbred F344; Retina; Tyrosine

Nitration of tyrosine-26 at pH 9.0 in bacteriorhodopsin does not change its absorption spectrum but lowers the apparent pK of the alkaline transition to a blue-shifted chromophore from about pH 12.0 to 10.6. This effect is reversed by reducing the nitrotyrosine-26 to aminotyrosine which demonstrates that the protonation state of tyrosine-26 and the alkaline chromophore transition are correlated. Nitration of tyrosine-64 resulted in a shift of the purple complex from 570 to 535 nm at neutral pH. The alkaline transition pK of such a nitrated membrane was below 10 but was clearly independent of the protonation state of tyrosine-64 because it is not reversed by reduction of the nitrotyrosine. Nitrotyrosine-26 showed spectral properties similar to L-nitrotyrosine in aqueous environment while nitrotyrosine-64 showed only a 360-nm absorbance in the apomembrane but not in the retinal-containing membrane. Both tyrosines are accessible to water-soluble reagents.

Topics: Bacteriorhodopsins; Carotenoids; Chemical Phenomena; Chemistry; Chymotrypsin; Halobacterium; Hydrogen-Ion Concentration; Methane; Photochemistry; Spectrophotometry; Structure-Activity Relationship; Tetranitromethane; Tyrosine