N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 89

5.4.5.5 N -Hcy-Tau

Tau, an essential component of neuronal cytoskeleton, binds and stabilizes micro-
tubule proteins (MTP), regulates nucleation and assembly of tubuli, is important for
neuronal integrity, and plays a role in Alzheimer's disease. The MTP network is
highly dynamic and regulates cell division and intracellular transport [349]. Tau
exists in six alternatively spliced isoforms. Highly conserved 31 amino acid repeats
in the C-terminal domain of the tau protein constitute the MTP binding region of tau
[350]. The C-terminal domain of tau contains three or four (3R and 4R) contiguous
microtubule-binding repeats. Tau isoform 4R stabilizes MTP more efficiently than
3R and is overexpressed in Alzheimer's disease [351]. The positively charged
lysine residues in the MTB binding regions of tau interact with the negative C
terminus of tubulin in a sequence-specific fashion [352].
Interactions of N-Hcy-tau containing 1.6, 7.2, and 25.2 mol N-Hcy/mol protein
with MTP and tubulin were studied by ultracentrifugation and SDS-PAGE analyses
[353]. After ultracentrifugation of mixtures containing MTP and N-Hcy-tau or
control unmodified tau, N-Hcy-tau is found predominantly in the supernatant,
while control tau is found in the pellet. The extent of tau binding to MTP is
inversely related to the extent of tau N-homocysteinylation. These results suggest
that N-homocysteinylation reduces tau's affinity for the MTP.
In addition to binding to MTP, tau regulates nucleation and assembly of tubulin.
Tau increases the rate of MTP elongation by decreasing tubulin dissociation during
assembly. This lowers the critical concentration for elongation. In the presence of
N-Hcy-tau, critical concentration of tubulin increases from 1.95

0.18 μ Mto

2.42 M. Thus, N-Hcy-tau fails to decrease tubulin dissociation, and the

extent of tubulin assembly is reduced as the extent of tau N-homocysteinylation
increases [353].

0.31 μ

5.4.5.6 N -Hcy-Caseins

Bovine -caseins are intrinsically unstructured proteins with differ-

ent aggregation and micelle formation propensities. Collectively, the caseins make
up about 80 % of the total milk protein (30-35 g/L). The caseins can coagulate into
curds and separate from other proteins, mostly lactoglobulins, which remain soluble
at 3 g/L in the whey. The milk protein carries small amounts of N-linked Hcy:
1.09

α (S1)-, β -, and κ

0.01 μ M in total milk (which also contains 3.45 0.25 μ M tHcy) and

1.51 M tHcy) [297].

The modification of individual caseins with Hcy-thiolactone generates proteins
containing 1.5, 2.1, and 1.3 N-linked Hcy residues per one

0.34 μ M in whey (which also contains 5.4 2.5 μ

-casein
molecule, respectively [354]. Studies of the fluorescence of Trp residues, thioflavin
T, and 1-anilino-8-naphthalene sulfonate, as well as CD spectroscopy studies, show
that N-homocysteinylation causes increase in

β -, α (S1)-, and κ

-sheet structures. Furthermore,
N-Hcy-caseins acquire increased propensity to aggregate. The sizes of aggregates

β