N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 91

trafficking, and increased N-homocysteinylation of key proteins important for
synapse formation, function, and plasticity.
Kinesin and dynein are motor proteins involved in vesicular transport along the
microtubules from one part of neuronal cell to another. In rat hippocampal neuronal
cells cultured in folate-deficient medium that induces hyperhomocysteinemia,
kinesin and dynein become N-homocysteinylated, which leads to protein aggrega-
tion and reduced interaction with tubulin [299]. The colocalization of Hcy with
kinesin and dynein and the occurrence of cellular protein aggregates are
demonstrated by immunohistochemistry using antibodies against kinesin, tubulin,
and Hcy-glutaraldehyde-BSA conjugates. These interactions are also confirmed by
immunoprecipitation and Western blotting and quantified by the proximity ligation
assay in which a pair of oligonucleotide-labeled secondary antibodies generates a
signal only when the two probes are bound in close proximity. The signal from each
interacting pair is visualized as an individual fluorescent dot; the number of dots is
quantified by counting and assigned to a specific subcellular location on micros-
copy images. The proximity ligation assay shows that N-homocysteinylation of
kinesin and dynein increases 29- and 37-fold, respectively, in the folate-deficient
cells, compared with folate-replete cells. At the same time the interactions of
kinesin and dynein with tubulin decrease 3.2- and 3.6-fold, respectively, in the
folate-deficient cells. Similar changes occur when the rat neuronal cells are treated
with Hcy-thiolactone, supporting a conclusion that N-homocysteinylation of
kinesin and dynein causes aggregation and prevents their interactions with tubulin.
LC-MS/MS analyses identify Lys1218 residue in dynein as being N-homo-
cysteinylated [299].

5.5 Quantification of N -Hcy-Protein

The discovery that N-Hcy-protein is formed biologically in living cells relied on the
use of radiolabeled [ 35 S]methionine and [ 35 S]homocysteine as precursors [73, 74].
In those experiments confluent cultures of human cells in 3.5-cm dishes are labeled
for 24-48 h with 0.1 mCi/mL [ 35 S]methionine or [ 35 S]homocysteine (5-100 M) in

methionine-free medium supplemented with fetal bovine serum and other
requirements. Streptomycin is not used in these experiments because it reacts
with Hcy-thiolactone (Table 3.4 ) [74]. Extracellular and cellular proteins are
treated with DTT to remove free and disulfide-linked Hcy. Covalently incorporated
[ 35 S]Hcy and [ 35 S]Met are released from the radiolabeled protein by hydrolysis
with 6 N HCl, 25 mM DTT (1 h, 120 C); under these conditions protein N-linked
Hcy is converted to [ 35 S]Hcy-thiolactone [139]. [ 35 S]Hcy-thiolactone is separated
from [ 35 S]Met by two-dimensional TLC on 5 cm

μ

4 cm cellulose or silica gel

plates using as little as 5 L acid-hydrolyzed sample (Fig. 3.7e, f ). The first-

dimension solvent is butanol/acetic acid/water (4/1/1, v/v), and the second-dimen-
sion solvent is isopropanol/ethyl acetate/water/ammonia (12/12/1/0.12, v/v). [ 35 S]
Hcy-thiolactone is visualized by autoradiography using Kodak BioMax X-ray film

μ