Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[3H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated 3H-methanol. All α-synuclein proteins accumulated isoaspartate at ~1% of molecules/day, ~20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of 3H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ~57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein

D-amino acids in aging erythrocytes.

Mature human erythrocytes are highly differentiated cells which have lost the ability to biosynthesize proteins de novo. During cell aging in circulation, erythrocyte proteins undergo spontaneous postbiosynthetic modifications, regarded as "protein fatigue" damage, which include formation of isomerized and/or racemized aspartyl residues. These damaged proteins cannot be replaced by new molecules; nevertheless, data support the notion that they can be repaired to a significant extent, through an enzymatic transmethylation reaction. This repair reaction has therefore been used as a means to monitor the increase of altered aspartyl residues in erythrocyte membrane proteins during cell aging. The relationship between protein repair and aspartyl racemization in red blood cell stress and disease is discussed

Isoaspartyl Formation in Creatine Kinase B Is Associated with Loss of Enzymatic Activity; Implications for the Linkage of Isoaspartate Accumulation and Neurological Dysfunction in the PIMT Knockout Mouse

Isoaspartate (isoAsp) formation is a common type of spontaneous protein damage that is normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). PIMT-KO (knockout) mice exhibit a pronounced neuropathology highlighted by death from an epileptic seizure at 30 to 60 days after birth. The mechanisms by which isoaspartyl damage disrupts normal brain function are incompletely understood. Proteomic analysis of the PIMT-KO mouse brain has shown that a number of key neuronal proteins accumulate high levels of isoAsp, but the extent to which their cellular functions is altered has yet to be determined. One of the major neuronal targets of PIMT is creatine kinase B (CKB), a well-characterized enzyme whose activity is relatively easy to assay. We show here that (1) the specific activity of CKB is significantly reduced in the brains of PIMT-deficient mice, (2) that in vitro aging of recombinant CKB results in significant accumulation of isoAsp sites with concomitant loss of enzymatic activity, and (3) that incubation of in vitro aged CKB with PIMT and its methyl donor S-adenosyl-L-methionine substantially repairs the aged CKB with regard to both its isoAsp content and its enzymatic activity. These results, combined with similarity in phenotypes of PIMT-KO and CKB-KO mice, suggests that loss of normal CKB structure and function contributes to the mechanisms by which isoAsp accumulation leads to CNS dysfunction in the PIMT-KO mouse