The truncated protein is more toxic to mitochondria than full-length protein and diminishes the effect of PDI on α-syn fibrillation. Here, we use nuclear magnetic resonance spectroscopy to show that the truncation alters α-syn conformation, resulting in an attractive interaction of the N-terminus with membranes and molecular chaperone, protein disulfide isomerase (PDI). ![]() Nevertheless, little is known about the mechanisms underlying the role of C-terminal truncation on the cytotoxicity and aggregation of α-syn. C-terminally truncated α-syn is found in the brain of PD patients, reduces cell viability and tends to form fibrils. Α-Synuclein (α-syn) is the main protein component of Lewy bodies, the major pathological hallmarks of Parkinson’s disease (PD). The mobility of molecular mass markers are indicated on the left. The black region defines amino acids 61–95, also known as NAC (see " Discussion " ). E, Schematic summarizing the approximate location of the three major fragments (F1–F3) resistant to proteolytic digestion as determined by antibody recognition and apparent molecular mass on SDS-PAGE. The epitope of each antibody is also indicated in parentheses above each panel. Western blot analysis of assembled -syn undigested () or digested () with proteinase K was performed using the antibodies indicated above each panel. D, the protected peptides were identified by using antibodies mapped to specific epitopes (26). In A–C, proteins were resolved on 15% SDS-polyacrylamide gels and visualized by staining with Coomassie Blue R-250. ![]() Note that proteolytic resistant fragments are only observed with -syn. C, -syn, -syn, and 71– 82 were incubated under assembly conditions (100 mM sodium acetate, pH 7.0, at 37 ☌ for 2 days with shaking) and undigested () or digested () with proteinase K for 30 min. ![]() Unassembled (A) or polymerized (B) human -syn was untreated (U) or digested with proteinase K for the time indicated above each lane. Within -syn filaments, the central hydrophobic region is protected against proteolytic digestion. ,, and /, -syn, -syn, and /-syn, respectively A76E and A76R, respective mutant proteins. A total of 5 g or 50 ng of protein was loaded for each set of assembly experiments analyzed by Coomassie Blue staining or Western blot analysis, respectively. Proteins resolved by SDS-PAGE were visualized with Coomassie Blue R-250 (CB) or by Western blot analysis with antibodies specific to -syn (LB 509) or -syn (Syn 207). F, A76E and A76R mutants of -syn were incubated alone or with -syn for 48 h at 37 ☌ with shaking. D and E, -syn incubated alone or with either -syn or 71– 82 at 37 ☌ for 48 h with shaking. B and C, syn proteins (2.5 or 5 mg/ml) were incubated at 37 ☌ for 48 h with continuous shaking. A, unassembled proteins at 5 mg/ml were predominantly in the superntant fractions. " Superntant (S) and pellet (P) fractions were loaded on 12% polyacrylamide gels. ![]() Assembly of protein was monitored by sedimentation at 100,000 g for 20 min as described under " Materials and Methods. Thus, we have identified key sequence elements necessary for the assembly of human α-synuclein into filaments, and theseĮlements may be exploited as targets for the design of drugs that inhibit α-synuclein fibrillization and might arrest diseaseĬentrifugal sedimentation analysis of the assembly of recombinant synuclein proteins and derivatives thereof. To form filaments, and these peptides promote fibrillization of full-length human α-synuclein in vitro. To proteolytic digestion in α-synuclein filaments and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize Not assemble into filaments in vitro 2) the rate of α-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this regionĪbrogates assembly 3) this stretch of 12 amino acids appears to form the core of α-synuclein filaments, because it is resistant The following observations: 1) human β-synuclein is highly homologous to α-synuclein but lacks these 12 residues, and it does Here, we report that aġ2-amino acid stretch (71VTGVTAVAQKTV82) in the middle of the hydrophobic domain of human α-synuclein is necessary and sufficient for its fibrillization based on That these inclusions impair vital metabolic processes and compromise vialibity of affected cells. Neuronal and oligodendrocytic aggregates of fibrillar α-synuclein define several diseases of the nervous system.
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