ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 8, pp. 1449-1459 © Pleiades Publishing, Ltd., 2026.
1449
Dynamics of Aspartate Isomerization
and Its Cellular Implications: Evidence
for Isoaspartate Formation in ER-Folded Proteins
and Stress-Responsive Regulation of PCMT1
Burcu Biterge
Department of Molecular Biology and Genetics, Faculty of Science and Letters,
Istanbul Technical University, 34467 Istanbul, Turkey
e-mail: bitergesut@itu.edu.tr
Received March 25, 2026
Revised July 8, 2026
Accepted July 20, 2026
Abstract— Spontaneous isomerization of aspartate and deamidation of asparagine residues into isoaspartate
(isoAsp) constitute major non-enzymatic post-translational modifications that alter protein structure, stability,
and turnover. The repair enzyme protein L-isoaspartate O-methyltransferase (PCMT1) catalyzes methylation of
isoAsp residues, thereby preventing their accumulation and preserving proteome integrity. Although PCMT1
has been studied extensively in cytoplasm and nucleus, its relationship to endoplasmic reticulum (ER) pro-
teostasis remains poorly understood. Here, we investigated dynamics of aspartate isomerization within the
cell, focusing on isoAsp accumulation and the regulation of PCMT1 localization under physiological and
stress conditions. Using immunofluorescence, subcellular fractionation, and in vitro methylation assays, we
detected isoAsp-modified proteins within the ER-enriched fractions of HeLa cells. We found that ER stress
induction enhanced formation of isoAsp-containing proteins, with MG132 treatment producing the highest
accumulation. PCMT1 expression increased under both stress conditions, accompanied by distinct subcellular
redistribution between the cytoplasmic and nuclear compartments. These observations indicate that ER-fold-
ed proteins are susceptible to spontaneous aspartate isomerization, and that PCMT1 activity dynamically
responds to proteostatic stress. Our findings provide the first experimental evidence linking isoAsp forma-
tion within the ER to PCMT1-mediated protein repair, thereby integrating chemical instability with cellular
quality-control pathways. This study establishes a structural and cellular framework for understanding the
dynamics of aspartate isomerization in the cell and underscores significance of PCMT1 in maintaining
proteostasis under stress conditions.
DOI: 10.1134/S0006297926600894
Keywords: aspartate isomerization, isoAspartate, protein damage, PCMT1, ER stress, proteostasis
INTRODUCTION
Proteins accumulate several spontaneous non-en-
zymatic chemical modifications over their lifetime
due to the action of damaging agents. Spontaneous
isomerization of aspartate and deamidation of aspar-
agine amino acids into isoaspartate (isoAsp) represent
the most abundant posttranslational modification in
proteins [1]. Deamidation begins immediately after
the protein synthesis with conversion of L-aspar-
tyl residues into unstable L-succinimidyl interme-
diates (Fig. 1). In most cases, succinimide is rapidly
converted into L-isoaspartyl residues, which are rec-
ognized as “damage” signals by the cell and must
be removed either by repair or degradation [2].
Methylation of the “damaged” isoAsp residues and
their restoration to “normal” aspartate is catalyzed
by the protein L-isoaspartate O-methyltransferase
(PCMT1) [3]. PCMT1 is a ubiquitously expressed and
evolutionarily conserved enzyme, which is involved
in several cellular processes [4]. PCMT1 has two
isoforms, and the long isoform has an endoplas-
mic reticulum (ER)-retention signal at its C-terminal
end [5].
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Fig. 1. PCMT1-mediated repair mechanism of spontaneously arisen isoaspartate residues. L-aspartyl and L-asparaginyl res-
idues could undergo spontaneous conversion into L-succinimidyl intermediates, which subsequently generate L-isoaspartyl
residues. PCMT1 catalyzes methylation of L-isoaspartyl residues to form an unstable L-isoaspartyl-O-methyl ester, which
undergoes spontaneous demethylation and re-formation of the succinimidyl intermediate. Repeated cycles of methylation
and hydrolysis facilitate conversion of damaged L-isoaspartyl residues into the normal L-aspartyl form, thereby contributing
to the repair of spontaneously damaged proteins.
PCMT1 mediated methylation of isoAsp residues
could also act as a signal that targets isoAsp-contain-
ing proteins for proteasomal degradation [6]. In line
with this, we have previously shown that histone H4
is methylated by PCMT1 at aspartate 24 (H4D24me),
which accumulates within the cell upon proteasomal
inhibition [7]. Through this degradation mechanism,
PCMT1 plays an important role in protein homeostasis
by maintaining the turnover of chemically damaged
or aged proteins. In the cell, ER is the major site of
protein homeostasis regulation, which provides a bal-
ance between protein synthesis and degradation. Itis
also responsible for quality control, where defective or
misfolded proteins are eliminated through proteasomal
degradation via the endoplasmic reticulum (ER)-asso-
ciated protein degradation pathway (ERAD) [8, 9]. Dis-
ruptions in the ER homeostasis result in ER stress and
activation of the unfolded protein response (UPR) [10].
IRE1 (inositol-requiring protein 1), PERK (protein ki-
nase R-like endoplasmic reticulum kinase), and ATF6
(activating transcription factor-6) are the major play-
ers of the UPR pathway that either restore ER homeo-
stasis or induce apoptotic cell death [11].
There is growing evidence in the literature, which
collectively indicates that the concepts of aspartate
deamidation, protein accumulation, proteasomal deg-
radation, and ER stress are interconnected. Further-
more, a recent proteomics study showed significant
enrichment of isoAsp-containing proteins in the mem-
brane-related subcellular components (such as ER)
across various tissues in PCMT1 knock-out mice [12].
Despite these studies, we still lack a complete picture
of the dynamics of aspartate isomerization in the cell.
Therefore, in this study we aimed to better charac-
terize the intracellular site of isoAsp accumulation
under physiological and ER stress conditions, which
could help us gain a deeper understanding of the dy-
namics of aspartate isomerization in the cell and its
role in disease pathogenesis.
MATERIALS AND METHODS
Mammalian cell culture and UPR induction.
HeLa cervical cancer cell line was cultured at 37°C
under 5% CO
2
and 95% humidity in Dulbecco’s mod-
ified Eagle’s medium (DMEM), high glucose (4.5 g/L)
supplemented with 10% Fetal Bovine Serum (FBS),
1% L-glutamine (200 mM), and 1× penicillin/strep-
tomycin (100×) solution. The cells were washed in
phosphate-buffered saline (PBS) and removed from
the dish by incubation with trypsin-EDTA solution.
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To induce direct and indirect ER stress, the cells
were treated either with Tunicamycin (1-5 μg/ml
for 6-24 h), an inhibitor of N-linked glycosylation,
or with MG132 (carbobenzoxy-Leu-Leu-leucinal), a
proteasomal inhibitor (20 mM for 2-6-24 h), respec-
tively. ER stress induction and activation of the UPR
pathway were evaluated by monitoring the levels of
UPR markers: phosphorylated IRE1 (pS724) and BiP
(GRP78, Glucose-Regulated Protein 78 kDa).
Generation of stable cell lines. HeLa cells were
transfected with 10 μg of a pcDNA3 plasmid overex-
pressing wild type PCMT1-FLAG-2HA fusion protein
or an empty vector and 100 μL of ExGen500 transfec-
tion reagent (Fermentas). Transfected cells were in-
cubated for 48 hours before the selection agent G418
(Calbiochem) was added to the medium. The cells sta-
bly expressing wild type PCMT1 were selected by cul-
turing for 2-3 weeks with occasional replenishment of
fresh medium every 2-3 days. After the selection, cells
were maintained in complete medium supplemented
with 1 mg/ml G418.
Immunofluorescence staining. Cells were grown
on 8-mm coverslips in 10 cm plates and incubated with
5 μM tunicamycin or 20 μM MG132 for 24 h. Thecov-
erslips were washed in PBS and fixed in 4% parafor-
maldehyde for 10 min. After washing them in 1× PBS,
the cells were permeabilized in 0.6% Triton X-100 in
PBS for 20 min at room temperature. The cells were
blocked with 4% BSA-0.2% Tween 20-PBS for 1 h at
room temperature and incubated with anti-α-H4D24me
(in house-made, see [7]) and anti-α-PCMT1 (Abcam,
ab97446) primary antibodies overnight at 4°C. After
washing in 0.2% Tween 20-PBS, the cells were incu-
bated with a FITC-labelled secondary antibody (Pro-
teintech #SA00003-2). The cells were counterstained
with DAPI (4′,6-diamidino-2-phenylindole), mounted
in Fluoroshield mounting medium (Abcam #ab104139),
sealed, and stored at 4°C in the dark. The cells were
imaged using an Olympus BX53 system and analyzed
with the help of Cellsense software (version1.17).
Fluorescence intensity quantification was per-
formed on fluorescence images acquired using iden-
tical microscope settings for all experimental groups.
Individual nuclei and cytoplasmic regions were man-
ually outlined as regions of interest (ROIs) based on
DAPI staining, and mean fluorescence intensity (MFI)
was measured using ImageJ 1.54 (National Institutes
of Health, USA). Background fluorescence was de-
termined from a cell-free region of each image and
subtracted from the measured fluorescence intensity.
Statistical significance was determined using the un-
paired two-tailed Student’s t-test.
Subcellular fractionation. 5×10
7
cells for each
experimental group (control, MG132, and TUN) were
resuspended in 10 mM Tris-HCl pH 7.4 buffer supple-
mented with 250 mM sucrose and sonicated for lysis.
Cellular debris were removed via centrifugation at
700g. The supernatant containing total protein frac-
tion was taken into a new tube and centrifuged at
15.000g for 20 min to remove mitochondria. The re-
maining supernatant was labelled as “crude-ER”. This
ER-enriched fraction was layered slowly onto a su-
crose gradient (2 ml of 2 M – 3 ml of 1.5 M – 3 ml of
1.3 M) and centrifuged in Beckman polyallomer tubes
(Cat. no: 342412) using a Beckman L-100 Ultracentri-
fuge at 100.000g for 70 min at 4°C. The remaining
pellet was resuspended in 100 μl of 1× PBS (pH 7.4)
and labelled as ER fraction.
Immunoblotting. Total cell lysates were prepared
in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0,
1% NP-40, 0.1% SDS and 0.5% Na-DOC), followed by
sonication; protein concentration was determined
using BCA protein assay. Equal amounts of proteins
were denatured in 1× Laemmli buffer at 95°C for
5 min, briefly centrifuged and separated in a 10%
SDS-polyacrylamide gel. Proteins were transferred to
PVDF membranes with 0.45 μm pore size and stained
with a Ponceau S dye (CST#59803). Membranes were
blocked in 5% BSA and 0.2% Tween 20 containing
1× TBS for 1 h at room temperature. Next, the mem-
branes were incubated with primary antibodies (Re-
combinant α-IRE1 (phospho S724) Abcam #ab243665;
α-BiP CST #3183; α-Calreticulin CST #2891) diluted
in blocking buffer at 4°C and incubated overnight
on a horizontal shaking platform. After incubation,
membranes were washed 3×5 min in 0.2% Tween 20
containing 1× TBS (TBS-T). Next, incubation with an
anti-rabbit IgG, HRP-linked secondary antibody (CST,
Cat. no. 7074) was carried out, followed by washing
and incubation with an ECL reagent for signal gener-
ation (Biorad Clarity Max Western ECL). Imaging was
done using a LiCor Odyssey FC Imaging System.
Quantification of isoAspartate formation.
In vitro methylation assays were carried out as de-
scribed in[7]. Briefly, subcellular fractions containing
equal amounts of protein (10 μg) were incubated with
1 μg of recombinant PCMT1 and 55 mM SAM (S-ade-
nosyl methionine) in 1× HMT buffer (50 mM Tris-HCl
pH 8.0 and 50 mM CH
3
CO
2
K) at30°C for1 h. Thereac-
tion was stopped by adding 10 μl of 0.3 M phosphoric
acid to the tubes. The samples were centrifuged at
14.000g for 10 min. The supernatants were taken into
new tubes and analyzed by HPLC for quantification
of S-adenosyl-L-homocysteine (SAH).
RESULTS
In order to have a better understanding of the
intracellular distribution of isoAsp accumulation
under physiological conditions, we first set out to
perform immunofluorescence staining of HeLa cells
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Fig. 2. PCMT1 overexpression alters subcellular distribution of H4D24me. a) Representative immunofluorescence imag-
es of HeLa cells transfected with an empty vector (EV) or vector encoding wild-type PCMT1. Cells were stained with
the anti-H4D24me antibody (green) and imaged at 100× magnification. DNA stained with DAPI (blue) marks the nucleus.
Scale bars: 20 μm. b) Quantification of cytoplasmic H4D24me fluorescence intensity demonstrated significant reduction
following PCMT1 overexpression. Fluorescence intensity was quantified using Fiji/ImageJ software. Data are presented as
a mean ± SD. c) Quantification of nuclear H4D24me fluorescence intensity revealed no significant difference between the
EV- and PCMT1-transfected cells. ns, not significant; *** p < 0.001.
using in-house made histone H4D24me antibod-
ies that were previously characterized [7] (Fig. 2a).
Although histones are almost exclusively nuclear
proteins, immunofluorescence analysis revealed that
the H4D24me signal was predominantly detected in
the cytoplasmic/perinuclear compartment, displaying
a subcellular distribution similar to that of ER-asso-
ciated proteins. Quantitative analysis showed that the
cytoplasmic staining was significantly reduced follow-
ing PCMT1 overexpression (Fig. 2b), while no signifi-
cant difference in the nuclear H4D24me fluorescence
between the control and the PCMT1-overexpressing
cells was detected (Fig. 2c).
Next, we wanted to investigate whether ER is
the site of accumulation for isoAsp-containing dam-
aged proteins before degradation by the proteosome.
For this purpose, we first induced ER stress in HeLa
cells via tunicamycin or MG132 treatment to en-
rich for isoAsp-containing damaged proteins. Tu-
nicamycin is a chemical agent that inhibits the
GPT (UDP-N-acetylglucosamine-dolichol phosphate
N-acetylglucosamine-1-phosphatetransferase) enzyme.
Inhibition of GPT enzyme blocks glycoprotein syn-
thesis and causes accumulation of unfolded proteins
in the ER, which eventually results in ER-stress [13].
MG132 is a reversible proteasome inhibitor that im-
pairs the proteasome-dependent protein degradation,
resulting in accumulation of misfolded proteins and
indirect activation of ER-stress [14]. Next, we pre-
pared subcellular fractions from these cells by ul-
tracentrifugation, which we used for the PCMT1-me-
diated in vitro methylation assays for quantification
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Fig. 3. Experimental workflow for investigating the PCMT1-mediated isoaspartate methylation during proteotoxic stress.
HeLa cells were treated with either tunicamycin or MG132 for ER-stress induction. Cells were fractionated by differential
centrifugation to obtain sequential subcellular fractions. In vitro methylation assays were performed using recombinant
PCMT1 in the presence of S-adenosyl-L-methionine (SAM) as the methyl donor to catalyze methylation of isoaspartyl (isoAsp)
residues, generating methylated isoAsp (isoAsp-me) and S-adenosyl-L-homocysteine (SAH). Methylation products were sub-
sequently analyzed with high-performance liquid chromatography (HPLC) to quantify the PCMT1-mediated methylation
activity.
of isoAsp accumulation. PCMT1 enzyme utilizes SAM
(S-adenosyl methionine) as a substrate and releases
SAH (S-adenosyl homocysteine), which can be quan-
tified by HPLC analysis to determine the overall level
of isoAspartate formation (summarized in Fig. 3).
Prior to the fractionation experiments, we op-
timized conditions for induction of ER-stress and
showed UPR activation in the cells treated with tu-
nicamycin and MG132 as evident by the increase
in the BiP and pIRE1 signals, respectively (Fig. 4a).
We selected the treatment conditions, which resulted
in the highest level of UPR activation (5 μM tunicamy-
cin, 24 h and 20 μM MG132, 24 h) for the preparation
of subcellular fractions. Here, we first obtained an
ER-enriched (crude-ER) fraction by removing the cel-
lular debris and mitochondria. Then, the ER-enriched
fraction was ultracentrifuged to precipitate the ER
into a pellet. Efficiency of fractionation was moni-
tored by detecting the levels of the ER-resident pro-
tein calreticulin, which served as an ER-marker. We
detected similar levels of calreticulin in the crude-
ER and ultracentrifuged ER-pellet fractions (Fig. 4b).
On the other hand, the highest level of PCMT1 was
detected in the crude-ER fraction, which could sug-
gest increased PCMT1 activity and isoAspartate ac-
cumulation in this fraction. Therefore, the crude-ER
fraction was selected for the in vitro methylation as-
says as the representative ER fraction.
One commonly used method of quantifying
isoAspartate levels in a given sample relies on de-
tection of the products of in vitro methylation assay
reactions using recombinant PCMT1 enzyme [2]. This
reaction catalyzes methylation of isoAsp residues into
isoAsp-O-methyl esters, which utilizes SAM (S-adeno-
syl methionine) as a methyl-group donor and releas-
es SAH (S-adenosyl homocysteine) as a byproduct.
Quantification of SAH levels by HPLC is indicative
of the levels of isoAsp accumulation in the sample
(Fig. 4c). Using this technique, we determined the lev-
els of isoAspartate both in the total lysate and the in
the ER fractions in the control and UPR-induced cells.
In line with our hypothesis, we were able to detect
isoAsp accumulation within the ER. Interestingly, the
highest amount of isoAsp accumulation in the ER was
obtained after MG132 treatment, in comparison with
the control cells or tunicamycin treatment (Fig. 4d,
p < 0.05).
Finally, we assessed the potential effect of ER-
stress induction on the expression level and subcel-
lular localization of PCMT1 by immunofluorescence
staining. We had previously reported that PCMT1
is both a cytoplasmic and a nuclear protein [7].
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Fig. 4. Proteotoxic stress increases isoaspartate accumulation in endoplasmic reticulum. a) Optimization for the ER stress
activation in the cells treated with tunicamycin and MG132. α-BiP and α-pIRE1 signals indicate UPR induction. b) Preparation
of subcellular fractions from HeLa cells. α-Calreticulin signal serves as an ER-marker. c) a representative chromatograph
of the SAH produced after in vitro methylation assays. Quantification of isoAsp levels within the ER fractions of the un-
treated control, MG132- and Tunicamycin treated cells are quantified and presented in (d). Both MG132 and tunicamycin
treatment significantly increased isoAsp accumulation in the ER compared with the untreated controls. Data are presented
as a mean ± SD. *** p < 0.001.
Intriguingly, we observed increased localization of
PCMT1 in the nucleus after the MG132-treatment,
while in the Tunicamycin-treated cells, the PCMT1
localization was more cytoplasmic (Fig. 5). Further-
more, an increased PCMT1 signal in the ER-stress in-
duced cells in comparison with the untreated cells
was detected.
DISCUSSION
Maintenance of protein homeostasis is essential
for cellular function, particularly in post-mitotic tis-
sues where proteins are retained for prolonged peri-
ods. While protein folding, degradation, and chaper-
one-mediated quality control have been extensively
investigated, spontaneous chemical modifications that
accumulate during protein aging represent an equally
important but often overlooked issue of proteostasis.
Among these modifications, isomerization of aspar-
tate and deamidation of asparagine residues result-
ing in isoaspartate (isoAsp) formation are ubiquitous
age-associated reactions that compromise protein
integrity. In the present study, we demonstrate that
isoAsp- containing proteins accumulate within the
ER-enriched fractions during proteotoxic stress and
that the level of PCMT1 expression and its intracel-
lular localization are dynamically altered following
activation of the UPR pathway. These findings estab-
lish a previously unrecognized connection between
spontaneous protein damage, isoAsp repair, and ER
protein quality control.
Methylation of aspartic acid residues was first
described in the literature in the 1980s as a possi-
ble step of repairing aged membrane proteins in
erythrocytes [3]. Since then, accumulation of isoAsp
and deamidated residues in proteins has been
shown to have significant biological consequences.
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Fig. 5. Proteotoxic stress induces nuclear and cytoplasmic accumulation of PCMT1. a) Representative immunofluorescence
images of HeLa cells under control conditions or following treatment with MG132/tunicamycin. Cells were stained with
anti-H4D24me antibody (green) and DNA stained with DAPI (blue) marks the nucleus. Images are taken at 20× magnifi-
cation. Scale bar: 100 μm. b) Quantification of nuclear PCMT1 fluorescence intensity demonstrated a significant increase
following MG132 and tunicamycin treatment compared with the untreated controls. c) Quantification of cytoplasmic PCMT1
fluorescence intensity also revealed significantly increased PCMT1 levels in both MG132- and tunicamycin-treated cells, with
the greatest increase observed following tunicamycin treatment. Fluorescence intensity was quantified using ImageJ after
background subtraction by measuring mean fluorescence intensity within the manually defined nuclear and cytoplasmic
regions of interest. Data are presented as a mean ± SD. * p < 0.05, ** p < 0.01.
For instance, these modifications have been observed
particularly in the long-lived proteins such as crys-
tallins in the lens, extracellular matrix proteins, and
histones [7, 15, 16]. Conversion of Asp into isoAsp
residues has been linked with loss of function and
misfolding as it disrupts protein structure, affects its
solubility, and makes the protein prone to aggrega-
tion [17]. Furthermore, isoAsp containing peptides/
proteins are harder to degrade as the isoaspartyl
bonds are resistant to most proteolytic enzymes [18].
Biological relevance of these modifications is par-
ticularly evident in neurodegenerative disorders.
IsoAsp residues have been identified in several ag-
gregation-prone proteins, including amyloid-β, tau,
α-synuclein, β-synuclein, and SOD1, all of which play
central roles in Alzheimer’s disease, Parkinson’s dis-
ease, and amyotrophic lateral sclerosis [19-22]. Many
of these proteins are themselves substrates of the
cellular quality-control pathways, suggesting that the
spontaneous isoAsp formation could further increase
the burden placed on the already compromised pro-
teostasis systems. isoAsp formation within Aβ pro-
motes β-sheet aggregation and enhances neurotoxic-
ity [23, 24]. Similarly, the Tau protein that is a key
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component of neurofibrillary tangles contains Asn
and Asp residues prone to isomerization, and forma-
tion of isoAsp impairs microtubule binding and accel-
erates aggregation [25].
Formation of isoAsp residues represents a form
of “molecular aging”, and the primary cellular de-
fense against the deleterious effects of such sponta-
neous damage are counteracted by the PCMT1-medi-
ated methylation and repair. Physiological importance
of this pathway has been convincingly demonstrat-
ed in the PCMT1-knockout mice, which accumulate
high levels of isoAsp in the brain facilitating devel-
opment of progressive seizures, neurodegeneration,
and premature death [26, 27]. These findings estab-
lished PCMT1 as an essential component of neuro-
nal protein quality control. Notably, PCMT1 activity
is especially high in the tissues containing long-lived
proteins, such as brain, lens, and erythrocytes, where
spontaneous protein damage accumulates over time.
Previous studies have further suggested that PCMT1
efficiency declines with aging [28]. Together with the
high metabolic demand of the brain for SAM and its
relatively low regenerative protein turnover, these
modifications have higher probability to occur in the
brain [1, 29].
A major finding of the present study is the
demonstration of the presence of isoAsp-containing
proteins within the ER-enriched fractions and their
accumulation following proteotoxic stress. Although
previous proteomic studies have identified isoAsp-con-
taining proteins in the membrane-associated cellular
fractions [12], direct evidence for their presence with-
in the ER-associated compartments has been limited.
Using an in vitro methylation assay coupled with
HPLC analysis, we observed significantly increased
isoAsp levels in the ER fractions following both prote-
asome inhibition and tunicamycin-induced ER stress,
with MG132 producing the most pronounced increase.
Toour knowledge, this represents the first experimen-
tal evidence supporting presence of isoAsp-containing
proteins within the ER-associated fractions during
proteotoxic stress.
ER provides a favorable environment for sponta-
neous protein damage. As the principal site of fold-
ing and maturation of the secretory and membrane
proteins, ER exposes proteins to prolonged residence
times while they undergo glycosylation, oxidative
folding, and disulfide bond formation. Mildly alka-
line luminal conditions together with oxidative stress
favor succinimide formation from susceptible aspar-
agine and aspartate residues [30, 31]. Furthermore,
accumulation of unfolded proteins during ER stress
exposes normally buried amino acids, potentially ac-
celerating spontaneous deamidation and isomeriza-
tion. The resulting chemically damaged proteins are
expected to further compromise folding efficiency,
thereby amplifying ER stress and promoting activation
of the UPR pathway[32]. Although our study does not
directly demonstrate that isoAsp formation initiates
ER stress, the observed accumulation of isoAsp-con-
taining proteins within the ER fractions suggests that
spontaneous protein aging constitutes an additional
source of proteotoxic burden under stress conditions.
These findings could also be viewed within the
broader framework of the proteostasis network. Cel-
lular protein homeostasis is generally maintained
through coordinated actions of molecular chaperones,
ubiquitin-proteasome system, autophagy, and ER-asso-
ciated degradation (ERAD). Protein repair mechanisms
have received comparatively little attention within
this network. During ERAD, terminally misfolded pro-
teins are retrotranslocated from the ER lumen into the
cytosol, ubiquitinated, and subsequently degraded by
the proteasome [33]. Considering that PCMT1 is pre-
dominantly a cytosolic enzyme rather than an ER-res-
ident protein, retrotranslocation provides a plausible
opportunity for the repair of isoAsp-containing sub-
strates before their complete degradation. Consistent
with this hypothesis, we showed that both MG132 and
tunicamycin treatment increased PCMT1 expression.
The elevated PCMT1 levels likely reflect an adaptive
response to the increased accumulation of chemical-
ly damaged proteins generated during proteotoxic
stress. Interestingly, different stress stimuli produced
distinct patterns of intracellular localization. MG132
induced a more pronounced increase in the nucle-
ar PCMT1, whereas tunicamycin resulted in stronger
cytoplasmic accumulation. These observations suggest
that PCMT1 localization is dynamically regulated ac-
cording to the nature of the cellular stress response.
One possible explanation is stress-dependent nucle-
ocytoplasmic redistribution of PCMT1. Alternatively,
different PCMT1 isoforms could be differentially ex-
pressed under specific stress conditions. The longer
PCMT1 isoform contains a C-terminal ER-retention se-
quence [5], raising the intriguing possibility that the
individual isoforms participate in distinct aspects of
cellular protein quality control. Although our experi-
ments cannot distinguish between these possibilities,
they provide a rationale for future studies aiming at
examining the isoform-specific regulation of PCMT1
during proteotoxic stress.
Our immunofluorescence analysis further demon-
strated that PCMT1 overexpression markedly reduced
the cytoplasmic H4D24 methylation without signifi-
cantly affecting nuclear staining. Histones are among
the longest-lived proteins in mammalian cells and,
therefore, represent substrates that are particularly
susceptible to spontaneous deamidation and isom-
erization. Preferential reduction of the cytoplasmic
H4D24 methylation may reflect enhanced repair or
turnover of the methylated substrates rather than
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simple accumulation of the repair intermediates.
Although the molecular basis of this observation re-
mains to be established, it supports the notion that
PCMT1 activity influences intracellular processing of
isoAsp-containing proteins.
Several limitations of this study should be ac-
knowledged. First, the study was performed exclu-
sively in HeLa cells, and validation in neuronal mod-
els will be necessary to determine the physiological
relevance of these observations in the tissues most
affected by protein aging. Second, the HPLC-based as-
say quantifies total isoAsp content but does not iden-
tify individual proteins carrying these modifications.
Future proteomic analyses using mass spectrometry
will, therefore, be required to define the ER-associat-
ed isoAsp proteome and determine which substrates
are preferentially repaired by PCMT1. Finally, the
mechanisms responsible for stress-dependent chang-
es in PCMT1 localization remain unresolved and war-
rant further investigation.
CONCLUSION
Our findings highlight dynamic interactions
between protein damage, cellular repair capacity,
and stress adaptation mechanisms. Detection of the
isoAsp-modified proteins within the ER raises the
possibility that spontaneous chemical modifications
contribute to the proteostatic burden. The stress-
responsive modulation of PCMT1 suggests that this
enzyme functions as part of a broader adaptive net-
work coordinating cytosolic and nuclear quality-con-
trol systems with ER-associated degradation. While
the present work establishes an important founda-
tion linking isoAsp chemistry to ER homeostasis,
future research integrating biochemical, structural,
and cellular approaches would be essential to eluci-
date dynamics of aspartate isomerization and precise
mechanistic role of PCMT1 in maintaining proteosta-
sis under stress conditions.
Funding
This work was supported by The Scientific and Tech-
nological Research Council of Türkiye (TUBITAK),
project no. 219Z278.
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest
The author of this work declares that she has no con-
flicts of interest.
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