40
Aggregation, Protein
All these enzymes, which catalyze the
pairing of cysteine residues in disulFde-
bridged proteins, have functional domains
pertaining to the thioredoxin superstruc-
ture.
Another type of enzyme, peptidyl–prolyl
cis–trans isomerases, facilitates the fold-
ing of some proteins by catalyzing the
cis–trans isomerization of X-Pro peptide
bonds.
Two
classes
of
unrelated
pro-
teins demonstrate this activity, those that
bind cyclosporin, which are known as
cy-
clophilins
, and those that bind ±K506. The
cellular function of these enzymes is im-
portant, since cyclosporin and ±K506 are
potent immunosuppressors that regulate
T-cells activation. Both classes of pep-
tidyl–prolyl isomerases are ubiquitous,
and abundant in prokaryotes and eukary-
otes. The sequences of several members
of each family are known, and the three-
dimensional structures of at least one
member of each family have been eluci-
dated by X-ray crystallography and multidi-
mensional NMR. Their role is to accelerate
the cis-trans isomerization of X-pro pep-
tide bonds when this process is the rate-
limiting step in protein folding. Although
they do not present structural similarity,
both exhibit a hydrophobic binding cleft
favoring the rotamase activity by excluding
water molecules.
4
Protein Aggregation in the Cellular
Environment
4.1
The Formation of Inclusion Bodies
The overexpression of genes introduced in
foreign hosts frequently results in aggre-
gated nonnative proteins called
inclusion
bodies
. In cells, inclusion bodies appear as
unordered amorphous aggregates clearly
separated from the rest of the cytoplasm;
they form a highly refractive area when
observed microscopically. A great variety
of experimental studies indicates that the
formation of inclusion bodies results from
partially folded intermediates in the in-
tracellular folding pathway and not from
either totally unfolded or native proteins.
4.1.1
Occurrence of Inclusion Bodies
Inclusion bodies were Frst identiFed in
the blood cells of patients with abnormal
hemoglobins, the resulting pathology be-
ing anemia. Pathological point mutants of
hemoglobin aggregate into inclusion bod-
ies; this is the case for hemoglobin K¨oln
(Val98Met on the
β
chain) and hemoglobin
Sabine (Leu91Pro on the
β
chain). Similar
deposits have been described in studies on
the metabolism of abnormal proteins sub-
jected to covalent modiFcation in
E. coli
.
The formation of aggregates also occurs
when cells are subjected to heat shock.
The
in vivo
folding pathway of tailspike
endorhamnosidase of Salmonella phage
22 is a well-documented system studied
by J.King’s group. ±urthermore, it is one
of the few systems in which the
in vivo
folding pathway has been compared with
the
in vitro
refolding pathway. The protein
is a trimer of 666 amino acids. The sec-
ondary structure is predominantly
β
-sheet.
Newly synthesized polypeptide chains re-
leased from the ribosome generate an
early partially folded intermediate. This in-
termediate further evolves into a species
sufFciently
structured
for
chain–chain
recognition. In the following step, an in-
completely folded trimer is formed upon
close association with the latter species.
The protrimer is then transformed into
thena
t
iveta
i
lsp
ike
.Ac
leard
if
ferencebe
-
tween the physicochemical properties of
the intermediates and the native state has
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