Cleavage of alpha-synuclein by calpain: potential role in degradation of fibrillized and nitrated species of alpha-synuclein

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.     

Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.     

Amyloid fibril formation and chaperone-like activity of peptides from alphaA-crystallin

AlphaA-crystallin (alphaAC), a major component of eye lens, exhibits chaperone-like activity and is responsible for maintaining eye lens transparency. Synthetic peptides which corresponded to the putative substrate-binding site of alphaAC have been reported to prevent aggregation of proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we found that these peptides, alphaAC(70-88), the peptide corresponding to amino acids 70-88 of alphaAC (KFVIFLDVKHFSPEDLTVK), and alphaAC(71-88), suppressed the amyloid fibril formation of amyloid beta protein (Abeta). On the other hand, while alphaAC(71-88) exhibited chaperone-like activity toward insulin, alphaAC(70-88) and alphaAC(70-88)K70D promoted rapid growth of aggregates consisting of insulin and these peptides in their solution mixtures. Interestingly, we found that alphaAC(71-88) itself can also form amyloid fibrils. It is possible that the chaperone-like activity of the alphaAC peptides is potentially related to their propensity for amyloid fibril formation. Analysis of variants of the alphaAC peptides suggested that F71 is important for amyloid formation, and interestingly, this same residue has previously been found to be essential for chaperone-like activity. Amyloid fibril formation was also observed with the shorter peptide, alphaAC(70-76)K70D, showing that the ability to form amyloid fibrils is maintained even with significant deletion of the C-terminal sequence. The formation of amyloid fibril was suppressed in alphaAC(70-88), suggesting that the K70 in the substrate binding site may play a role in suppressing the amyloid fibril formation of alphaAC, which agreed with recent proposals about the presence of an aggregation suppressor in the region flanking aggregation-prone hydrophobic sequences.     

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.     

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation

The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high M_r disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils.     

Inhibition of α-synuclein fibril assembly by small molecules: Analysis using epitope-specific antibodies

The conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases. Existing treatments are at best symptomatic. Accordingly, small molecule inhibitors of amyloid fibril formation and their mechanisms are of great interest. Here we report that the conformational changes undergone by α -synuclein as it assembles into amyloid fibrils can be detected by epitope-specific antibodies. We show that the conformations of polyphenol-bound α-synuclein monomers and dimers differ from those of unbound monomers and resemble amyloid fibrils. This strongly suggests that small molecule inhibitors bind and stabilize intermediates of amyloid fibril formation, consistent with the view that inhibitor-bound molecular species are on-pathway intermediates.     

Formation of a high affinity lipid-binding intermediate during the early aggregation phase of alpha-synuclein

The alpha-synuclein (alpha-syn) protein is clearly implicated in Parkinson's disease (PD). Mutations or triplication of the alpha-syn gene leads to early onset PD, possibly by accelerating alpha-syn oligomerization. alpha-syn interacts with lipids, and this membrane binding activity may relate to its toxic activity. To understand how the alpha-syn aggregation state affects its lipid binding activity we used surface plasmon resonance to study the interaction of wild-type and mutant alpha-syn with a charged phospholipid membrane, as a function of its aggregation state. Apparent dissociation constants for alpha-syn indicated that an intermediate species, present during the lag phase of amyloid formation, binds with an increased affinity to the membrane surface. Formation of this species was dependent upon the rate of fibril formation. Fluorescence anisotropy studies indicate that only upon the formation of amyloid material can alpha-syn perturb the acyl-chain region of the lipid bilayer. Circular dichroism spectroscopy showed that upon aging, both wild-type and mutant alpha-syn lose their ability to form lipid-bound alpha-helical species once they become fibrillar. These results indicate that alpha-syn forms a high affinity lipid binding intermediate species during fibril formation. Oligomeric alpha-syn is known to be toxic, and it is feasible that the high affinity binding species described here may correspond to a toxic species involved in PD.     

Biochemical properties of cytochrome c nitrated by peroxynitrite

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.     

Protein Polymerization into Fibrils from the Viewpoint of Nucleation Theory

The assembly of various proteins into fibrillar aggregates is an important phenomenon with wide implications ranging from human disease to nanoscience. Using general kinetic results of nucleation theory, we analyze the polymerization of protein into linear or helical fibrils in the framework of the Oosawa-Kasai (OK) model. We show that while within the original OK model of linear polymerization the process does not involve nucleation, within a modified OK model it is nucleation-mediated. Expressions are derived for the size of the fibril nucleus, the work for fibril formation, the nucleation barrier, the equilibrium and stationary fibril size distributions, and the stationary fibril nucleation rate. Under otherwise equal conditions, this rate decreases considerably when the short (subnucleus) fibrils lose monomers much more frequently than the long (supernucleus) fibrils, a feature that should be born in mind when designing a strategy for stymying or stimulating fibril nucleation. The obtained dependence of the nucleation rate on the concentration of monomeric protein is convenient for experimental verification and for use in rate equations accounting for nucleation-mediated fibril formation. The analysis and the results obtained for linear fibrils are fully applicable to helical fibrils whose formation is describable by a simplified OK model.     

Apolipoprotein C-II amyloid fibrils assemble via a reversible pathway that includes fibril breaking and rejoining

Alzheimer's and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.     

Monomeric 14-3-3ζ Has a Chaperone-Like Activity and Is Stabilized by Phosphorylated HspB6

Members of the 14-3-3 eukaryotic protein family predominantly function as dimers. The dimeric form can be converted into monomers upon phosphorylation of Ser(58) located at the subunit interface. Monomers are less stable than dimers and have been considered to be either less active or even inactive during binding and regulation of phosphorylated client proteins. However, like dimers, monomers contain the phosphoserine-binding site and therefore can retain some functions of the dimeric 14-3-3. Furthermore, 14-3-3 monomers may possess additional functional roles owing to their exposed intersubunit surfaces. Previously we have found that the monomeric mutant of 14-3-3ζ (14-3-3ζ(m)), like the wild type protein, is able to bind phosphorylated small heat shock protein HspB6 (pHspB6), which is involved in the regulation of smooth muscle contraction and cardioprotection. Here we report characterization of the 14-3-3ζ(m)/pHspB6 complex by biophysical and biochemical techniques. We find that formation of the complex retards proteolytic degradation and increases thermal stability of the monomeric 14-3-3, indicating that interaction with phosphorylated targets could be a general mechanism of 14-3-3 monomers stabilization. Furthermore, by using myosin subfragment 1 (S1) as a model substrate we find that the monomer has significantly higher chaperone-like activity than either the dimeric 14-3-3ζ protein or even HspB6 itself. These observations indicate that 14-3-3ζ and possibly other 14-3-3 isoforms may have additional functional roles conducted by the monomeric state.     

Pre-Steady-State Kinetic Analysis of the Elongation of Amyloid Fibrils of β2-Microglobulin with Tryptophan Mutagenesis

Amyloid fibrils elongate seed dependently, with preformed fibrils providing a template for propagation of amyloidogenic conformation. Most seeding experiments use relatively few seed fibrils in comparison with monomers, resembling steady-state enzyme kinetics. Pre-steady-state kinetics should also be useful for characterizing the elongation process. With β₂-microglobulin (β₂-m), a protein responsible for dialysis-related amyloidosis, we measured the pre-steady-state kinetics of fibril elongation at pH 2.5, conditions under which the monomer is largely unfolded. β₂-m has Trp residues at positions 60 and 95. We used three single Trp mutants and fluorescence spectroscopy to study structural change upon fibril elongation. To focus on conformational change in monomers, we prepared seeds with a mutant without a Trp residue. At a fixed concentration of monomeric β₂-m, the apparent rate of fibril elongation increased with an increase in the concentration of seeds and then saturated, suggesting the accumulation of a rate-limiting intermediate. Importantly, saturation occurred at a seed/monomer ratio of around 10, as expressed by the concentration of the monomer. Because the number of monomers constituting the seed fibrils is much larger than 10, the results suggest that the elongation process is limited by “non-active-site binding.” Spectral analysis indicated that, upon this non-active-site binding, both Trp60 and Trp95 are exposed to the solvent, and then only Trp60 is buried upon transition to the fibrils. We propose a new model of fibril elongation in which non-active-site binding plays a major role.     

Nucleation of Polymorphic Amyloid Fibrils

One and the same protein can self-assemble into amyloid fibrils with different morphologies. The phenomenon of fibril polymorphism is relevant biologically because different fibril polymorphs can have different toxicity, but there is no tool for predicting which polymorph forms and under what conditions. Here, we consider the nucleation of polymorphic amyloid fibrils occurring by direct polymerization of monomeric proteins into fibrils. We treat this process within the framework of our newly developed nonstandard nucleation theory, which allows prediction of the concentration dependence of the nucleation rate for different fibril polymorphs. The results highlight that the concentration dependence of the nucleation rate is closely linked with the protein solubility and a threshold monomer concentration below which fibril formation becomes biologically irrelevant. The relation between the nucleation rate, the fibril solubility, the threshold concentration, and the binding energies of the fibril building blocks within fibrils might prove a valuable tool for designing new experiments to control the formation of particular fibril polymorphs.     

Evidence for stepwise formation of amyloid fibrils by the mouse prion protein

The full-length mouse prion protein, moPrP, is shown to form worm-like amyloid fibrils at pH 2 in the presence of 0.15 M NaCl, in a slow process that is accelerated at higher temperatures. Upon reduction in pH to 2, native moPrP transforms into a mixture of soluble β-rich oligomers and α-rich monomers, which exist in a slow, concentration-dependent equilibrium with each other. It is shown that only the β-rich oligomers and not the α-rich monomers, can form worm-like amyloid fibrils. The mechanism of formation of the worm-like amyloid fibrils from the β-rich oligomers has been studied with four different physical probes over a range of temperatures and over a range of protein concentrations. The observed rate of fibrillation is the same, whether measured by changes in ellipticity at 216 nm, in thioflavin fluorescence upon binding, or in the mean hydrodynamic radius. The observed rate is significantly slower when monitored by total scattering intensity, suggesting that lateral association of the worm-like fibrils occurs after they form. The activation energy for worm-like fibril formation was determined to be 129 kJ/mol. The observed rate of fibrillation increases with an increase in protein concentration, but saturates at protein concentrations above 50 μM. The dependence of the observed rate of fibrillation on protein concentration suggests that aggregate growth is rate-limiting at low protein concentration and that conformational change, which is independent of protein concentration, becomes rate-limiting at higher protein concentrations. Hence, fibril formation by moPrP occurs in at least two separate steps. Longer but fewer worm-like fibrils are seen to form at low protein concentration, and shorter but more worm-like fibrils are seen to form at higher protein concentrations. This observation suggests that the β-rich oligomers grow progressively in size to form critical higher order-oligomers from which the worm-like amyloid fibrils then form.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.     

Collagen fibrils in vitro grow from pointed tips in the C- to N-terminal direction.

Growth of collagen fibrils was examined in a system in which collagen monomers are generated by specific enzymic cleavage of type IpCcollagen with procollagen C-proteinase. Fibrils formed at 37 degrees C had highly tapered and symmetrical pointed tips. The pattern of cross-striations in the pointed tips indicated that all the molecules were oriented so that the N-termini were directed towards the tip. At 29 degrees C and 32 degrees C, the fibrils formed were thicker. One end of fibrils formed at 29 degrees C was blunt, and the other was pointed. Growth of the fibrils was exclusively from pointed tips. Occasionally a spear-like projection appeared at a blunted end. The spear-like projection then became a new pointed tip for growth in the opposite direction. The results suggested a model for fibril growth with at least three distinct binding sites for monomers. In the model, the pointed tip is the site with the highest affinity for the binding of monomers and most probably defines the critical concentration for fibril assembly. The main shaft of the fibril is a site with very low affinity for binding. The blunted end defines a low-affinity binding site where monomers can bind in opposite orientation to produce growth from a new pointed end.     

Spatial extent of charge repulsion regulates assembly pathways for lysozyme amyloid fibrils

Formation of large protein fibrils with a characteristic cross β-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path.     

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.     

Tyrosine phosphorylation/dephosphorylation regulates peroxynitrite-mediated peptide nitration

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tyrosine and tryptophan residues are especially susceptible to attack by reactive nitrogen species. On the other hand, protein tyrosine phosphorylation has gained much attention in respect to cellular regulatory events and signal transduction. Peroxynitrite-mediated nitration of peptide YPPPPPW and phosphopeptide pYPPPPPW were studied at pH 7.4. The predominant nitrated products were separated and identified by reverse phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS). The nitration sites were established by tandem electrospray ionization-mass spectrometry (LC-MS/MS). A regulatory effect of tyrosine phosphorylation/dephosphorylation on peptide nitration was observed. YPPPPPW was predominantly nitrated at tyrosine residue while pYPPPPPW was nitrated at tryptophan one. Our results can help in understanding the biochemical significance of the relationship of tyrosine phosphorylation and nitration in proteins.     

Proteolytic degradation of tyrosine nitrated proteins

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.