cells
ArticleA Novel Approach: Investigating the Intracellular Clearance
Mechanism of Glyceraldehyde-Derived Advanced Glycation
End-Products Using the Artificial Checkpoint Kinase 1 d270KD
Mutant as a Substrate Model
Kenji Takeda 1,2, * , Akiko Sakai-Sakasai 1 , Kouji Kajinami 2
1

2*

Citation: Takeda, K.; Sakai-Sakasai,A.; Kajinami, K.; Takeuchi, M. A
Novel Approach: Investigating theIntracellular Clearance Mechanism of
Glyceraldehyde-Derived Advanced
Glycation End-Products Using theArtificial Checkpoint Kinase 1

and Masayoshi Takeuchi 1

Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku,
Uchinada-Machi, Ishikawa 920-0293, Japan; asakasai@kanazawa-med.ac.jp (A.S.-S.);takeuchi@kanazawa-med.ac.jp (M.T.)
Department of Cardiology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-Machi,
Ishikawa 920-0293, Japan; kajinami@kanazawa-med.ac.jp
Correspondence: ktakeda@kanazawa-med.ac.jp; Tel.: +81-76-218-8456

Abstract: Advanced glycation end-products (AGEs), formed through glyceraldehyde (GA) as anintermediate in non-enzymatic reactions with intracellular proteins, are cytotoxic and have been
implicated in the pathogenesis of various diseases. Despite their significance, the mechanisms underlying the degradation of GA-derived AGEs (GA-AGEs) remain unclear. In the present study, we
found that N-terminal checkpoint kinase 1 cleavage products (CHK1-CPs) and their mimic protein,d270WT, were degraded intracellularly post-GA exposure. Notably, a kinase-dead d270WT variant(d270KD) underwent rapid GA-induced degradation, primarily via the ubiquitin–proteasome pathway. The high-molecular-weight complexes formed by the GA stimulation of d270KD were abundantin the RIPA-insoluble fraction, which also contained high levels of GA-AGEs. Immunoprecipitation
experiments indicated that the high-molecular-weight complexes of d270KD were modified by GAAGEs and that p62/SQSTM1 was one of its components. The knockdown of p62 or treatment withchloroquine reduced the amount of high-molecular-weight complexes in the RIPA-insoluble fraction,
indicating its involvement in the formation of GA-AGE aggregates. The present results suggestthat the ubiquitin–proteasome pathway and p62 play a role in the degradation and aggregation of
intracellular GA-AGEs. This study provides novel insights into the mechanisms underlying GA-AGE
metabolism and may lead to the development of novel therapeutic strategies for diseases associatedwith the accumulation of GA-AGEs.

d270KD Mutant as a Substrate Model.
Cells 2023, 12, 2838. https://doi.org/
10.3390/cells12242838

Keywords: advanced glycation end-products (AGEs); glyceraldehyde (GA); glyceraldehyde-derived
AGEs; toxic AGEs (TAGEs); p62/SQSTM1; CHK1-CPs

Academic Editor: Huabo Su
Received: 14 November 2023
Revised: 4 December 2023

1. Introduction

Accepted: 12 December 2023

Sugars are a vital source of energy and essential nutrients for living organisms. However, modifications to the lysine and arginine residues of proteins in vivo, which resultin the formation of crosslinks, may significantly change the steric structures of proteins,thereby affecting their activities and physical properties. This process is known as glycation or the Maillard reaction and is divided into two stages: an early-stage reaction that
produces Amadori transfer products and a late-stage reaction that results in advanced
glycation end-products (AGEs) through oxidation, dehydration, and condensation [1].
AGEs have been implicated in the pathogenesis of various diseases [2,3], such as diabetes,
liver disease [4], atherosclerosis [5], Alzheimer’s disease, and aging [6].
AGEs represent a diverse cluster of compounds. For instance, Nε-(carboxymethyl)lysine(CML) and Nε-(carboxyethyl)lysine (CEL) are AGEs formed through reactions with glyoxal(GO), methylglyoxal (MGO), and lysine residues in proteins. AGEs can be broadly classified

Published: 14 December 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Cells 2023, 12, 2838. https://doi.org/10.3390/cells12242838

https://www.mdpi.com/journal/cells

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into exogenously derived dietary AGEs, formed through the heat processing of foods likemilk and dairy products [7,8], and endogenously generated AGEs that accumulate within
the body. The development of diseases due to endogenous AGE accumulation is postulated
to involve two pathways: (1) the activation of downstream cellular signaling through the
RAGE receptor, inducing oxidative stress and inflammation [9] and (2) cross-linking withintracellular proteins, disrupting normal physiological functions. Additionally, reports
emphasize the AGE interactions with the gut microbiota and their consequential effects [7].
AGEs are generated in vivo not only from glucose, but also from, for example,
metabolic intermediates of glucose, degradation products, and Maillard reaction intermediates [10,11]. In the classification spectrum of more than 40 AGEs identified to date [9],glyceraldehyde (GA) plays a crucial role in the formation of GA-AGEs (GA-derived AGEs)
through intricate metabolic pathways [3]. In fructolysis, fructokinase (FK) phosphorylates
fructose (Fru), forming Fru-1-phosphate (F-1-P). The subsequent cleavage by aldolase B
yields dihydroxyacetone-phosphate and GA. Concurrently, glycolysis metabolizes GA-3phosphate (GA-3-P) through GA-3-P dehydrogenase (GAPDH), ultimately leading to pyruvate. Reduced GAPDH activity results in intracellular accumulation of GA-3-P, promoting
non-enzymatic reactions that contribute to GA formation. Moreover, under hyperglycemic
conditions, the polyol pathway is activated and glucose is reduced by aldose reductase
to form sorbitol, which is subsequently oxidized by sorbitol dehydrogenase to form Fru.
These excess GAs, arising from impaired glucose metabolism, non-enzymatically react withproteins, ultimately leading to the formation of the GA-AGEs implicated in diseases associated with diabetic complications, insulin resistance, heart disease, Alzheimer’s disease,
hypertension, nonalcoholic steatohepatitis, and cancer [3]. The toxic effects of GA-AGEs
and their specific structures that are responsible for toxicity remain unclear. However, anantibody targeting GA-AGEs was shown to effectively mitigate neurotoxicity caused byserum AGEs in patients with diabetic nephropathy undergoing hemodialysis [12]. Other
antibodies targeting different types of AGEs or CML did not exert similar protective effects.
These findings indicate that only AGE structures containing epitopes recognized by theanti-GA-AGE antibody are toxic. They are referred to as toxic AGEs (TAGEs) and are
distinct from other known GA-AGEs, such as 3-hydroxy-5-hydroxymethyl-pyridinium(GLAP), triosidines, and MG-H1. None of these structures showed AGE-specific fluorescence or protein cross-linking. Two compounds with a 1,4-dihydropyrazine ring thatshowed fluorescence and had cross-links were identified as TAGE candidate structures(PCT/JP2019/34195).
The late-stage Maillard reaction is irreversible and once GA-AGEs are formed, there
are no known enzymes that specifically remove the sites modified by glycation. Glycation
modifications have been suggested to compromise cellular homeostasis by inducing theloss of function of various biomolecules or by forming toxic aggregates, which inactivate
other normal essential proteins [3]. The ubiquitin–proteasome pathway and autophagy
pathway are intracellular systems that remove defective proteins and aggregates. These
two pathways are not independent of each other, and they function cooperatively [13–16].
Recent studies suggested that AGEs are closely associated with autophagy [17]. Their effects
on autophagy are complex, with some studies indicating that they inhibit autophagy [18,19]
and others suggesting that they induce autophagy [20–24]. Therefore, the coordinated roleof the two intracellular degradation mechanisms in the AGE clearance process remains
unclear. Although AGEs are formed and accumulate in proteins with a long turnover
due to a decrease in the activities of intracellular protein quality control mechanisms withaging, the mechanisms underlying intracellular GA-AGE degradation and removal have
yet to be clarified. A more detailed understanding of these mechanisms is critical for thedevelopment of effective therapeutic strategies that mitigate the detrimental effects of
GA-AGE accumulation.

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In the present study, we found that N-terminal checkpoint kinase 1 cleaved products(CHK1-CPs) and their mimetic protein (d270WT) were susceptible to intracellular degradation upon the administration of GA, and also that a kinase-dead mutant (d270KD) of
d270WT exhibited more rapid GA-responsive degradation with the formation of highmolecular-weight complexes (typical features indicative of GA-AGE conversion). d270KD,
which is rapidly degraded with the generation of GA-AGEs, may serve as a useful modelfor GA-AGE formation in future research on the molecular mechanisms that act on theclearance of GA-AGEs that accumulate in cells.
2. Materials and Methods
2.1. Antibodies
The following primary antibodies were used: anti-mouse IgG2a magnetic beads(M076-11), anti-DYKDDDDK (FLAG)-tag magnetic beads (M185-11), anti-V5-tag magnetic
beads (M16711), anti-normal rabbit IgG (PM035), and an anti-DYKDDDDK (FLAG)-tag(PM020) for immunoprecipitation, and anti-HA-tag (M132-3), anti-α-tubulin-HRP, andanti-microtubule-associated protein 1 light chain 3 (LC3; M186-3) for Western blottingfrom Medical & Biological Laboratories (MBL, Nagoya, Japan); anti-phospho-SQSTM1/p62(Ser403) (#39786), anti-SQSTM1/p62 (#5114), and anti-HUWE1 (#5695) from Cell Signaling Technologies (CST, Beverley, MA, USA); anti-V5-HRP (R961-25) from Thermo Fisher
Scientific (Waltham, MA, USA); anti-FLAG-HRP (015-22391) from Wako Pure Chemicals(Osaka, Japan); anti-ubiquitin (#AUB01) from Cytoskeleton (Denver, CO, USA); and antiSPRTN (HPA025073) from Sigma-Aldrich (St Louis, MO, USA). An anti-GA-AGE (TAGE)
antibody was prepared as previously reported [25]. The anti-TAGE antibody specifically
identified unique epitopes other than the known structures derived from GA, such as
GLAP and triosidines. However, it did not recognize well-known AGEs containing CML
and Nε-(carboxyethyl)lysine (CEL) or bind to other AGEs derived from reducing sugars
and carbonyl molecules, including pyrraline, pentosidine, crossline, argpyrimidine, GO- or
MGO-lysine dimers, and GO- or MGO-derived hydroimidazolone.
2.2. Cell Culture
COS-7 cells were obtained from the American Type Culture Collection and cultured
in Dulbecco’s modified Eagle medium (DMEM + GlutaMax medium; Gibco BRL, GlandIsland, NY, USA) supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa,
KS, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (both from Gibco BRL) at
37 ◦ C in a humidified atmosphere of 5% CO2 . HeLa cells (a gift from Dr. Sumiyo Akazawa,
Kanazawa Medical University, Japan) were cultured in minimum essential medium (MEMalpha + GlutaMax medium; Gibco BRL, Waltham, MA, USA) supplemented with 10% FBS,
100 U/mL penicillin, and 100 µg/mL streptomycin at 37 ◦ C in a humidified atmosphere of
5% CO2 . Cells were subcultured when they reached 70–80% confluence. In experiments
where GA (purity 98% or higher; catalog number: 17014-94, Nacalai Tesque, Tokyo, Japan)
was applied to cells, GA or phosphate buffer was added at the concentrations indicated.
In the aminoguanidine (AG; purity 97% or higher; catalog number: 328-26432, FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan)-induced suppression experiment, AG
at the indicated concentrations was added to cells 2 h prior to the treatment with GA.
Regarding the proteasome inhibitor treatment, cells were treated with 10 µM MG-132(Sigma-Aldrich, St. Louis, MO, USA) or DMSO (Nacalai Tesque, Kyoto, Japan) for 6 h. In
the autophagy inhibition experiments, 50 µM chloroquine (Sigma-Aldrich) or phosphatebuffer was added to HeLa cells 20 h prior to the GA treatment.
2.3. Lactate Dehydrogenase (LDH) Cytotoxicity Assay
LDH released into the culture supernatant was measured using a LDH cytotoxicity
assay kit (Nacalai Tesque, Kyoto, Japan) following the manufacturer’s instructions. Briefly,
HeLa cells were plated on 24-well plates (5 × 104 cells/well). The cells were stimulatedwith 4 mM GA or phosphate buffer for the indicated time. The culture medium (100 µL)

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was collected and mixed with 100 µL of the substrate solution. After an incubation at room
temperature for 20 min, 50 µL of the stop solution was added and the absorbance wasmeasured at 490 nm using an iMark™ Microplate Reader (Bio-Rad, Hercules, CA, USA).
LDH levels in the cell culture supernatant collected from the GA-treated cell group werenormalized relative to the cultured medium collected from the control group (phosphatebuffer-treated group).
2.4. Generation of Expression Plasmid Constructs
Flag-tagged CHK1 expression constructs, including the wild-type (WT) form andits kinase-dead mutant form (KDmut), were previously described [26]. Briefly, cDNA
encoding full-length rat Chk1 was amplified from rat cardiac myocyte cDNA using aforward primer containing the Flag-tag sequence at the 50 end and then subcloned into
the pcDNA4/HisMax vector. Xpress- and 6 × His-tag sequences at the N terminus were
removed from the vector by PCR using the KOD-plus-mutagenesis kit (Toyobo, Osaka,
Japan) according to the manufacturer’s instructions. Deletion at the C terminus and point
mutations in Flag-Chk1 expression vectors were created by PCR using the KOD-plusmutagenesis kit. To generate the d270KD-EGFP-V5 expression construct, full-length EGFP
cDNA was amplified by PCR using the pEGFP-C1 vector (BD Biosciences, San Jose, CA,
USA) and subcloned into the pcDNA3.1 vector (Thermo Fisher Scientific). The open reading
frame of EGFP with the V5-tag and 6 × His-tag sequences at the C terminus was thenamplified using the vector as a template by PCR using PrimeSTAR Max DNA polymerase,
subcloned into the pENTR/D-TOPO vector (Thermo Fisher Scientific) using the TOPO®
cloning procedure, and then transformed into the mammalian expression vector pDEST30(Thermo Fisher Scientific) using LR clonase II enzyme. Fragments containing the N-terminal
region of Chk1 (amino acids 1-270) in pFlag-Chk1Wt and its KDmut (d270KD) vectors
were cloned in the vector in-frame with the gene that encodes the EGFP-V5/6 × His-tag at
the 30 end using the In-Fusion® HD PCR cloning kit (Takara, Shiga, Japan). To generate
the d270KD-ZsGreen expression construct, full-length ZsGreen1 cDNA was amplified
by PCR using the ZsGreen1-1 vector (Clontech, Palo Alto, CA, USA) and the fragments
were cloned into the vector in-frame with the gene encoding Flag-d270KD at the 30 end
using the In-Fusion® HD PCR cloning kit (Takara, Shiga, Japan). HA-ubiquitin was a giftfrom Edward Yeh (Addgene plasmid #18712). All constructs were sequenced to ensure
proper ligation in the correct frame and Taq polymerase fidelity using the ABI PRISM TM
310 genetic analyzer (Applied Biosystems, Foster City, CA, USA).
2.5. Transfection
Regarding transient plasmid DNA transfection, cells were seeded on 35, 60, 100 mm,
or 6-well tissue culture plates, cultured in complete growth medium, and then transfected using FuGENE® 4K (Promega, Madison, WI, USA) according to the manufacturer’s
protocol. The transfection of siRNA was performed using the RNAiMax Transfection
reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations.
SignalSilence® siRNA (#6241, Cell Signaling Technologies) targeting Chk1, Silencer SelectsiRNA (Ambion, Valencia, CA, USA) targeting Huwe1 (siRNA ID: s19596 and s19597),
SQSTM1 (siRNA ID: s16962), SPRTN (siRNA ID: s38329), or scrambled siRNA (Silencer
Select Negative Control #1, catalogue #4390843) was used. The final siRNA concentration
was 50 nM for SignalSilence® siRNAs and 5 nM for Silencer Select siRNAs.
2.6. Western Blot
Transfected COS-7 and HeLa cells were lysed in lysis buffer (CelLytic™M cell lysis
reagent; Sigma-Aldrich) containing proteinase inhibitors and phosphatase inhibitors (bothfrom Nacalai Tesque). After the cellular debris was removed by centrifugation, protein concentrations in the supernatants were measured using the Qubit protein assay kit (Thermo
Fisher Scientific). To detect GA-AGEs (TAGE) or high-molecular-weight complexes, cells
were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na

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deoxycholate, and 0.1% SDS), centrifuged, and separated into supernatant (soluble fraction)and pellet (insoluble fraction). After the addition of SDS sample buffer to each fraction,
the RIPA-insoluble fraction was sonicated. All samples were boiled at 95 ◦ C for 5 min,separated on 5–20 or 12.5% SDS-PAGE gels, and analyzed by Western blotting. Target proteins were visualized by Chemi-Lumi One L, Super (Nacalai Tesque), or Immobilon Forte
Western HRP Substrate (Millipore, Bedford, MA, USA). A densitometric quantification of
the resultant blots was performed using NIH ImageJ software (version 1.54d).
2.7. In Vitro GA-AGE Modification Assay
The in vitro GA-AGE modification of the d270KD protein was performed according
to previous methods with some changes. Briefly, HeLa cells transfected with Flag-tagfused d270KD (Flag-d270KD) were lysed in CelLytic™M cell lysis reagent containing
proteinase inhibitors and phosphatase inhibitors. After the cellular debris was removed bycentrifugation, total cellular proteins were incubated with anti-Flag antibody-immobilized
magnetic beads at 4 ◦ C overnight, and the resulting immunoprecipitates were washedthree times with lysis reagent. After the elution of Flag-d270KD with 40 µL of Flag peptide(2 mg/mL), 200 µL of PBS was added. The eluted samples, including Flag-d270KD, werethen ultrafiltered and concentrated with Amicon Ultra 0.5 (10 K) (Millipore) to remove the
FLAG peptide. This purified Flag-d270KD recombinant protein (2.5 µg) was incubated in
50 µL of PBS for 20 h with or without the addition of 4 mM GA. After the addition of SDS
sample buffer to these reaction mixtures, they were boiled at 95 ◦ C for 5 min.
2.8. In Vivo Ubiquitination Assay
For the in vivo ubiquitylation assays, the d270KD-EGFP expression vector d270KDEGFP was used to transfect COS-7 cells (100 mm plates) for 48 h. The cells were treatedwith 10 µM MG132 and then harvested after 8 h. The cell pellets were resuspended
in denaturing buffer (1.5% SDS, 50 mM Tris–HCl pH 7.5, and 5 mM DTT) followed byboiling for 10 min, and were then diluted 10-fold with CelLytic™M cell lysis reagent
containing proteinase inhibitors and phosphatase inhibitors. After the cellular debris
was removed by centrifugation, the extracted proteins were immunoprecipitated with
anti-V5 antibody-immobilized magnetic beads and subjected to the assay described above.
Immunoprecipitates were analyzed by Western blotting with anti-ubiquitin antibodies oranti-V5 antibodies.
2.9. Luciferase Assay
The gene encoding luciferase was amplified by PCR using the pGL4.54 vector (Promega)
as a template with the forward primer 50 -ACAAACACACTTAACATGGAAGATGCCAAAA
AC-30 and reverse primer 50 -GTAACAGGCCTTCTACACGGGCGATCTTGCCGCCC-30 .
The pFlag-d270KD plasmid vector was amplified by PCR using the forward primer 50 -TA
GAAGGGCCTGTACCTAGGATCCAGT-30 and reverse primer 50 -GTTAAGTGGTTTGTTATACCATCTA-30 to form a linear strand. The luciferase gene was then inserted downstreamof the d270KD gene using the InFusion HD cloning kit (Clontech, Palo Alto, CA, USA).
The accuracy of the gene insertion site was confirmed by a sequence analysis. Twenty-fourhours after the transfection of the d270KD fusion luciferase expression vector (d270KD-Luc)
into cells cultured in 6-well plates, the firefly luciferase reporter assay was performed using
the Luciferase Reporter Assay System (Promega Madison, WI, USA). Three independent
assays were performed under each condition.
2.10. Fluorescence Imaging Analysis
COS-7 cells were grown in six-well plates containing collagen-coated glass coverslips(diameter of 12 mm, Iwaki, Shizuoka, Japan) and transfected with the d270KD-EGFP
expression vector at 60% confluence using FuGENE® 4K as described above. After an
incubation for 24 h in complete medium, the cells were treated with or without GA (2 mM)
for the indicated time and fixed with ice-cold 4% paraformaldehyde in PBS for 10 min.

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The fixed cells were washed three times for 5 min each with PBS and then mounted with
Prolong gold antifade reagent/DAPI (Thermo Fisher Scientific). All fluorescence images
were obtained using a digital high-definition microscope system (BZ-9000, Keyence, Osaka,
Japan) with the following filter sets: OP-66834, Ex360/40 Em460/50; OP-66836, Ex470/40
Em535/50; OP-66838, Ex560/40 Em630/60.
2.11. Immunoprecipitation Assay of High-Molecular-Weight Complexes Formed by a
GA Stimulation
The d270KD-ZsGreen expression vector was transfected into HeLa cells (100 mm
plates) for 48 h. The cells were treated with 2 mM GA and then harvested after 3 h. The
cells were lysed in RIPA buffer containing proteinase inhibitors and phosphatase inhibitors,
centrifuged, and separated into supernatant (soluble fraction) and pellet (insoluble fraction).
After the addition of solubilization buffer containing a high concentration of SDS (2.0%
SDS, 50 mM Tris-HCl pH 7.5) to the pellet, the RIPA-insoluble fraction was sonicated. Afterremoving the high concentration of SDS from the fraction using the Pierce™ SDS-PAGE
Sample Prep Kit (Thermo Fisher Scientific), ultrafiltration was performed using Amicon
Ultra 0.5 (30 K) (Millipore), while the solvent of the fraction was replaced with CelLytic™M
cell lysis reagent containing proteinase inhibitors and phosphatase inhibitors. The antiFLAG antibody (MBL) or normal rabbit IgG (MBL) was added to the soluble and insoluble
fractions, which were then incubated at 4 ◦ C overnight. The coupling of the antibodies to
Dynabeads Protein A (Thermo Fisher Scientific) was performed by adding CelLytic™Mequilibrated Dynabeads directly to both fractions, followed by an incubation at room
temperature for 30 min. Immunoprecipitated Dynabead complexes were washed once with
CelLytic™M reagent and three times with TBS-T. Proteins bound to the Dynabead–antibody
complexes were eluted by adding 50 µL of the FLAG peptide (2 mg/mL). After the additionof SDS sample buffer to each fraction, all samples were boiled at 95 ◦ C for 5 min, separated
on 5–20% SDS-PAGE gels, and analyzed by Western blotting with the indicated antibodies.
2.12. Statistical Analysis
All numerical results are reported as the mean ± SEM of at least three measurements.
Statistical analyses were performed using the paired Student’s t-test, a one-way ANOVA
followed by Dunnett’s post hoc test, or a two-way ANOVA followed by the Bonferroni post
hoc test with GraphPad Prism software (Version 5.0, GraphPad Prism Software, San Diego,
CA, USA). Differences were considered to be significant at p < 0.05 and were denoted by anasterisk in the graphs.
3. Results
3.1. Identification of a Constitutive KD Mutant of CHK1 That Is Rapidly Modified with GA-AGEs
and Degraded in Cells after GA Stimulation
We previously reported that the prolonged stimulation of cells with GA, an intermediate metabolite of GA-AGEs, induced DNA damage stress and necrosis [3]. CHK1 is aserine–threonine kinase that plays a central role in cell cycle checkpoints and DNA stress responses [27]. However, the effects of the GA stimulation on CHK1 (GA-AGE modifications)in cells remain unclear. Therefore, we initially stimulated HeLa cells with 2 mM GA andobserved the formation of high-molecular-weight complexes of endogenous CHK1 protein
after 4 h (Figure 1a). Furthermore, when a FLAG-tag-fused CHK1 (Flag-CHK1) expression
vector was introduced into HeLa cells and stimulated with GA in the same manner, theformation of high-molecular-weight complexes of exogenous CHK1 protein was strongly
promoted (Figure 1b).

Cells 2023, 12, 2838

tions) in cells remain unclear. Therefore, we initially stimulated HeLa cells with 2 mM GA
and observed the formation of high-molecular-weight complexes of endogenous CHK1
protein after 4 h (Figure 1a). Furthermore, when a FLAG-tag-fused CHK1 (Flag-CHK1)
expression vector was introduced into HeLa cells and stimulated with GA in the same
7 of 26
manner, the formation of high-molecular-weight complexes of exogenous CHK1 protein
was strongly promoted (Figure 1b).

Figure 1. Effects of the GA treatment on GA-AGE formation with wild-type CHK1 and its N-terminal
Figure 1. Eﬀects of the GA treatment on GA-AGE formation with wild-type CHK1 and its N-termiandcelllysateswerecollected
atat
thenalcleavage
cleavageproducts.
products.(a)(a)HeLa
HeLacells
cellswere
weretreatedtreatedwith
with2 2mM
mMGA
GA
and
cell
lysateswerecollected
indicated times for a Western blot analysis. The densitometric quantification of endogenous CHK1
high-molecular-weight complex and monomer values relative to the non-treatment group (0 h) is
shown. The data were analyzed using a one-way ANOVA followed by Dunnett’s post hoc test andare shown as the mean ± SEM of three independent experiments. * p < 0.01, ** p < 0.001. (b) The
expression vector of wild-type CHK1 (Flag-CHK1) was transfected into HeLa cells and stimulatedwith 2 mM GA 24 h later. Cell lysates were collected at the indicated times and subjected to a WesternCells 2023, 12, 2838

8 of 26

blot analysis. The densitometric quantification of Flag-CHK1 high-molecular-weight complex,
monomer, and CP values relative to the non-treatment group is shown. The data were analyzed using
a one-way ANOVA followed by Dunnett’s post hoc test and are shown as the mean ± SEM of three independent experiments. * p < 0.01, ** p < 0.001. (c) HeLa cells were transfected with scrambled control
siRNA or SPRTN siRNA and transfected 24 h later with a Flag-CHK1 expression vector. Cell lysates
were collected after 48 h and subjected to a Western blot analysis using the indicated antibodies. The
densitometric quantification of Flag-CHK1 values relative to the control group (Control) is shown.
The data were analyzed using the paired Student’s t-test and are shown as the mean ± SEM of three
independent experiments. * p < 0.001. The positions of the bands corresponding to CHK1-CPs areindicated by arrows.

The C terminus of CHK1 was previously reported to be cleaved by SPRTN metalloprotease, resulting in N-terminal CHK1 fragments that were more active than the full-length
protein [28]. In our experimental model using HeLa cells, a few N-terminal CHK1 fragmentsof endogenous CHK1 were detected in the basal state; however, a number of N-terminal
CHK1 cleavage products (CPs) were clearly present in HeLa cells transiently expressing
Flag-CHK1 (Figure 1b). As expected, the protein levels of N-terminal CHK1-CPs detected
at approximately 30 kDa were suppressed in cells transfected with siRNA against SPRTN(Figure 1c). These results are consistent with previous findings showing that SPRTN cleaved
the C terminus of CHK1 at multiple sites to form these N-terminal CHK1 cleaved forms.
Upon a stimulation with 2 mM GA, a decrease in N-terminal CHK1-CPs was observed after
1 h. On the other hand, non-cleaved (i.e., full-length) Flag-CHK1 showed no GA-stimulated
reductions in monomeric bands within 4 h. Therefore, N-terminal CHK1-CPs were more
susceptible to the effects of GA-stimulated glycation modifications earlier.
To investigate the GA-stimulated degradation of N-terminal CHK1-CPs in more detail,we generated a mutant expression vector (Flag-d270WT) lacking the C-terminal regulatory
domain, as in N-terminal CHK1-CPs. As expected, a Western blot analysis showed a timedependent decrease in the protein levels of Flag-d270WT transiently expressed in HeLa
cells upon a stimulation with 2 mM GA (Figure 2a). In addition, a band of high-molecularweight complexes of Flag-d270WT, which are characteristic of glycation modifications(GA-AGE conversion), was weakly detected from 2 h after the GA stimulation. The KD
mutant (Flag-d270KD), in which the kinase was constitutively inactivated by a singleamino acid substitution (a mutation in which the 36th alanine is replaced by phenylalanine:
A36F) inside d270WT, showed faster degradation upon the GA stimulation than the wild
type (d270WT). The majority of d270KD bands disappeared approximately 8 h after the
stimulation with 2 mM GA, while d270WT maintained an expression level of approximately
40% even after 8 h of the GA stimulation.
We then investigated the concentrations of GA that induced the rapid degradationof d270KD. The GA stimulation for 6 h resulted in significant degradation from a concentration of 1.5 mM (Figure 2b). These results indicate that d270KD was rapidly degradedupon intracellular GA-AGE modifications by a short-term GA stimulation. In contrast
to d270WT, no GA-stimulated high-molecular-weight complex bands were observed in
d270KD (Figure 2a), suggesting that an inactivating mutation in the wild-type form changed
the structure to be less susceptible to glycation modifications by GA. We then performed
GA-AGE conversion experiments on d270KD in vitro in the absence of a degradation system. Flag-d270KD was expressed in cells, and the recombinant protein was purified byimmunoprecipitation with a FLAG antibody and then reacted with GA in a tube for 20 h.
The results obtained showed that the GA stimulation resulted in the appearance of highmolecular-weight complexes, which were detected by anti-TAGE antibodies (Figure 2c).
Therefore, the d270KD mutant was considered to be TAGE-modified by GA as well as
d270WT; presumably, high-molecular-weight complexes were not detected in these cells
because GA-stimulated degradation was accelerated.

Cells 2023, 12, 2838

appearance of high-molecular-weight complexes, which were detected by anti-TAGE an
tibodies (Figure 2c). Therefore, the d270KD mutant was considered to be TAGE-modified
by GA as well as d270WT; presumably, high-molecular-weight complexes were
9 of 26 not detected in these cells because GA-stimulated degradation was accelerated.

Figure 2. GA-stimulated rapid protein degradation of intracellular C-terminal deletion mutants

Figure2. GA-stimulated
rapid expression
protein degradationof theintracellular
deletionmutants oof CHK1.
(a) A CHK1 mutant
vector lacking
C-terminal C-terminal
regulatory domain(FlagCHK1.
(a)
A
CHK1
mutantexpression
vectorlackingtheC-terminal
regulatory
domain(Flag
d270WT) or its constitutive kinase-dead mutant expression vector (Flag-d270KD) was transfected
d270WT)or itsconstitutive
expression
vectorwas transfected
into HeLa
cells.
Cell lysateskinase-deadwere collectedmutant
at the indicatedtimes
after 2(Flag-d270KD)
mM GA was applied
tointocells
HeLa
cells.
Cell
lysateswerecollected
attheindicated
times
after
2
mM
GA
was
applied24 h later. Lysates were subjected to a Western blot analysis using the indicated antibodies. to cell
24 hThelater.
Lysates were
subjected
a Westernvalues
blot analysis
the indicatedantibodies.
Thedensitometricquantificationof toFlag-d270KD
relative tousing
the non-treatment
group
(0 h)
densitometricquantificationof Flag-d270KD
valuesrelativeto thegroup
is shown. The
data were analyzed
using a two-way
ANOVA
followedby non-treatment
the Bonferroni post
hoc (0 h) i
shown.
The aredata
wereasanalyzed
a two-way
ANOVA followedby the* Bonferroni
post
test andshown
the meanusing± SEM
of three independent
experiments.
p < 0.001. (b)A hoc tesand Flag-d270KD
are shown as
the mean± SEM
three independent
experiments.
* p stimulated< 0.001. (b)24Ah Flag-d270KD
expression
vectorwasoftransfected
into HeLa cells
and cells were
later
expressionwastransfectedofinto
and cells
stimulated
24 h laterwith the indi
with the vector
indicatedconcentration
GA HeLafor 6 h.cells
A Western
blotwere
analysis
was performedon lysatescatedconcentration
GAasfor
6 h. A above.
Westernanalysis quantification
was performedon lysates values
collected fromcollected
from theseofcells
described
Theblot
densitometricof Flag-d270KD
thesecells asdescribed
above.group
The densitometricquantification
Flag-d270KD
relative torelativeto thenon-treatment(0 mM) is shown.
The data wereofanalyzed
using avalues
one-way
ANOVA followedgroupby Dunnett’s
postishoc
test andaredatashown
as theanalyzed
mean ± SEM
of threeindependent
the non-treatment(0 mM)
shown.
The
were
using
a one-way
ANOVA folexperiments.
* p < 0.001.
(c) Intest
vitroand
GA-AGE
modifications
the d270KD
protein. A experi
lowed
by Dunnett’s
post hoc
are shownas the tomean± SEM recombinantof three independent
Flag-d270KD
expression
vectorwas transfected
into HeLato
cells
cell lysates
were extracted
24 h A Flagments.
* p < 0.001.
(c) In vitro
GA-AGE
modifications
theandd270KD
recombinant
protein.
later. expression
The lysates were
immunoprecipitatedanti-Flag
antibody
to purifywere
the Flag-d270KD
d270KD
vectorwas transfected with
into anHeLa
cells andcell lysates
extracted 24 h later
protein,which was then reacted
4 mM GA antibody
in vitro for to
20 purifyh. GA-AGE
modifications recom
The recombinant
lysates were
immunoprecipitatedwithwith
an anti-Flag
the Flag-d270KD
tothed270KD
recombinant
proteinwere
analyzedbyWesternblotting.
binant protein, which was then reacted with 4 mM GA in vitro for 20 h. GA-AGE modifications to
the d270KD
recombinant
protein wereanalyzedby Western
blotting.
3.2. The Intracellular
Destabilizing
Propertyof d270KD
upon GA
Stimulation May Be Retained
after Fusion to Other Proteins

3.2. The We
Intracellular
Propertyof d270KD
upon GA
StimulationMay
Be Retained
examined Destabilizing
whether the rapid
degradationof d270KD
inducedby the GA
stimuis atoproperty
that will be retained after fusion to other proteins. d270KD-EGFP, aafterlation
FusionOther Proteins

fusion of EGFP to the C terminus of d270KD, was expressed in COS-7 cells and then stimuWe examined whether the rapid degradation of d270KD induced by the GA stimula
lated with 4 mM GA. A Western blot analysis showed that d270KD-EGFP was markedly
tiondegradedis a property
will be retained
after fusion
otherproteins. d270KD-EGFP, aafter thethat
GA stimulation
for approximately
6 h to(Figure3a).

fusion of EGFP to the C terminus of d270KD, was expressed in COS-7 cells and then stim
ulated with 4 mM GA. A Western blot analysis showed that d270KD-EGFP was markedly
degraded after the GA stimulation for approximately 6 h (Figure 3a).

Cells 2023, 12, x FOR PEER REVIEW
Cells 2023, 12, 2838

10 of 26

10 of 26

FigureGA-stimulatedrapid
rapid intracellular
intracellular degradation
of ofd270KD-EGFP
fusionprotein.
(a) TheFigure3. 3.
GA-stimulateddegradation
d270KD-EGFP
fusionprotein.
(a) Theexpression
vectorofd270KD
fused
to
EGFP
(d270KD-EGFP)
wastransfected
into
COS-7
cells
and
expression vector of d270KD fused EGFP (d270KD-EGFP) was transfected into COS-7 cells andstimulated
with
4 mMGA
GA48
48hhlater.
later. Cell
Cell lysatesat theindicatedtimes
and subjected
stimulated
with
4 mM
lysateswere
werecollectedcollected
at theindicatedtimes
and subjecteda Western
blot
analysis.The
The densitometricdensitometric quantification
of ofd270KD-EGFP
valuesrelativeto theto theto atoWestern
blot
analysis.
quantification
d270KD-EGFP
valuesrelative
non-treatment
group
shown.The
Thedata
datawere
were analyzed
analyzed using
followed
non-treatment
group(0(0h)h)isisshown.
usingaaone-way
one-wayANOVA
ANOVA
followed byby Dunnett’s
posttest
hocandtest areand shownare shownas the
mean
± SEM
three
independentexperiments.
experiments. * p <Dunnett’s
post hocas the
mean
± SEM
of of
three
independent*p<0.001.
(b)cells
COS-7
cells
were transfected
the d270KD-EGFP
expression
24 h,0.001.
(b)
COS-7
were
transfectedwith thewith
d270KD-EGFP
expression
vectorvector
for 24 forh, stimulated
stimulatedwithphosphatebuffer(control)
or
4
mM
GA
for
6
h,
and
then
fixed.
Nucleiwerewith phosphate buﬀer (control) or 4 mM GA for 6 h, and then fixed. Nuclei were counterstained
counterstainedwith DAPI
analyzed byfluorescenceScale
microscopy.
bars = 50 µm.
with
DAPI and analyzed
byand
fluorescencemicroscopy.
bars = Scale
50 µm.

Slightly different from the degradation pattern of Flag-d270KD, high-molecular-weight
Slightly
diﬀerent from the degradation pattern of Flag-d270KD, high-molecularcomplex bands were clearly observed in the EGFP fusion 2 h after the GA stimulation.
weight complex bands were clearly observed in the EGFP fusion 2 h after the GA stimuA fluorescence imaging analysis was performed using cells expressing d270KD-EGFP.
lation.
A fluorescenceimagingthat
analysiswasperformed
using cellstheirexpressing
d270KDThe results
obtained showed
when thecells
were not stimulated,
fluorescence
EGFP.
The
results
obtained
showed
that
when
the
cells
were
not
stimulated,theirfluoreswas extensively observed in the cytoplasm, whereas after the GA stimulation for 6 h, thecence
was extensively
observed
thesmallcytoplasm,
whereasafter
thenoted
GA stimulation
fluorescentarea decreased
and inonly
granule-likesignals
were(Figure 3b). for 6
Furthermore,when
a luciferase
fusedto the C terminus
d270KD,h, thefluorescentaread270KD-Luc,decreased andonly small
granule-likesignalsofwere
notedwas(Figureexpressedin
HeLa
cells,
rapid
proteolysis
wasobservedwiththeappearanceof
high3b). Furthermore, when d270KD-Luc, a luciferase fused to the C terminus of d270KD, wasmolecular-weightbandsrapid
uponproteolysis
the GA stimulation,
similarwith
to d270KD-EGFP(Figure4a).
expressed
in HeLa cells,
was observed
the appearanceof high-moConsistent withthe uponproteolysisof stimulation,
d270KD-Luc,similar
its luciferase
activity also decreasedin Conlecular-weightbands
the GAto d270KD-EGFP(Figure 4a).
concentration- and time-dependent manners upon GA stimulation (Figure 4b). We alsosistent with the proteolysis of d270KD-Luc, its luciferase activity also decreased in coninvestigated the effects of AG, a known inhibitor of AGE formation, on the GA-stimulated
centrationand time-dependentGA
stimulation(Figure4b).
also invesrapid degradationof 270KD-Luc.manners
We foundupon
that theAG
pretreatment
inhibitedtheWedecreasetigatedtheeﬀects
of
AG,
a
known
inhibitorofAGE
formation,onthe
GA-stimulated
stimulated by GA in luciferase activity in a concentration-dependent manner (Figure 4c).rapid
degradation
ofblot
270KD-Luc.
We foundthat
AGwas
pretreatment
inhibitedofthe
decrease
The Western
analysis confirmedthatthistheeffectdue to the inhibition
the GAinduced proteolysisof d270KD-Luc(Figurestimulated
by GA in luciferase
activity
in a4d).
concentration-dependent manner (Figure 4c).

The Western blot analysis confirmed that this eﬀect was due to the inhibition of the GAinduced proteolysis of d270KD-Luc (Figure 4d).

Cells2023,12,
12,2838x FOR PEER REVIEW
Cells
2023,

11 of 26

11 of 26

Figure 4. Rapid proteolysis of d270KD-fused luciferase and its reduced activity upon GA stimulation.

Figure 4. Rapid proteolysis of d270KD-fused luciferase and its reduced activity upon GA stimula(a) A
fused
to to
luciferase
wastransfectedtransfectedinto
into
HeLa cells andtion.
(a)d270KD
A d270KD
fused
luciferaseexpression
expressionvector
vector (d270KD-Luc)(d270KD-Luc) was
HeLa
cells
and stimulated
mMafter
GA afterh. Celllysateswerecollectedatatthe
the indicatedindicated times
stimulatedwith 4with
mM4GA
24 h.24Celllysateswerecollected
timesandand subjectedsubjected
to a Western
blot analysis.
densitometricquantification
quantification ofofd270KD-Luc
valuesrelativeto a Western
blot analysis.
TheThedensitometricd270KD-Luc
valuesrelative to theto the non-treatment group (0 h) is shown. The data were analyzed using a one-way ANOVA folnon-treatment group (0 h) is shown. The data were analyzed using a one-way ANOVA followed
lowed by Dunnett’s post hoc test and are shown as the mean ± SEM of three independent experiby Dunnett’s
hoc
testd270KD-Luc
and are shownas the meanSEMof forthree
experiments.
ments.
* p < 0.001.post(b) Asin (a),
was transfected
into±
HeLa
cells
24 hindependentand stimulated*p<0.001.
As in (a),d270KD-Luc
transfected
into
HeLa
cells timefor 24h andstimulated withwithGA
at the(b)indicatedconcentrations.
Cellwaslysatescollected ateachindicated
were
subjected
toGA
the luciferase
assay. (c)concentrations.
HeLa cells were transfected
withd270KD-Luc
for 24h and thenpretreatedat the indicated
Cell lysatescollected
at eachindicated
time
were subjected towith phosphate buﬀer or aminoguanidine (AG) at the indicated concentrations for 2 h. The cells
the luciferase assay. (c) HeLa cells were transfected with d270KD-Luc for 24 h and then pretreated
were then stimulated with phosphate buﬀer or 2 mM GA for 6 h and lysed. Cell lysates were colwith andphosphatebufferor aminoguanidine(AG)at the asindicatedconcentrations
2 h.wasThe cells werelectedluciferase
activity
was measured andexpressed
100 for thecontrol. Eachfor
value
then stimulated
phosphatebuffer orError
2 mM
GA
for 6 SEM.
h and* lysed.
lysates
obtainedfrom three withindependent
experiments.
bars
indicate
p <0.01 Cellversus
2 mM were
GA collected
alone.
(d) Cell lysates
treatedto andthe experimentsubjected
to Eacha Western
and luciferase
activity
wassimilarly
measured
expressed inas (c)
100werefor thecontrol.
valueblot
was obtained
analysisusing independent
the antibodies indicated.
The densitometricquantification
valuesrel- GA alone.
from threeexperiments.
Error bars indicate
SEM.of* d270KD-Luc
p <0.01 versus
2 mM
ative to the non-treatment group (None) are shown. The data were analyzed using a one-way(d) Cell lysates treated similarly to the experiment in (c) were subjected to a Western blot analysis
using the antibodies indicated. The densitometric quantification of d270KD-Luc values relative to the
non-treatment group (None) are shown. The data were analyzed using a one-way ANOVA followedby Dunnett’s post hoc test and are shown as the mean ± SEM of three independent experiments.
* p < 0.01, ** p < 0.001, ns means not significant.

Cells 2023, 12, x FOR PEER REVIEW

Cells 2023, 12, 2838

1

ANOVA followed by Dunnett’s post hoc test and are shown as the mean ± SEM
of 26
three indep
12 ofexperiments. * p < 0.01, ** p < 0.001, ns means not significant.

3.3. The GA-Stimulated Pathway by Which d270KD Undergoes Ubiquitination and Degra
3.3. The GA-Stimulated
by Involve
Which d270KD
Undergoes
Ubiquitination
Does NotPathway
Appear to
Mule/HUWE1
E3 Ubiquitin
Ligaseand Degradation
Does Not Appear to Involve Mule/HUWE1 E3 Ubiquitin Ligase
In a human pancreatic beta cell line (1.4E7 cells), GA stimulation has been sug
In a human pancreatic beta cell line (1.4E7 cells), GA stimulation has been suggested
to inhibit the autophagy pathway, increase intracellular GA-AGE levels, and indu
to inhibit the autophagy pathway, increase intracellular GA-AGE levels, and induce cell
death [18]. However, the eﬀects of a GA stimulation on the ubiquitin–proteasome
death [18]. However, the effects of a GA stimulation on the ubiquitin–proteasome pathway
way have remained unclear. The cytotoxicity of HeLa cells induced by a 2 mM GA
have remained unclear. The cytotoxicity of HeLa cells induced by a 2 mM GA stimulation,
ulation, as measured by the LDH assay, was approximately 5% of the control at 12
as measured by the LDH assay, was approximately 5% of the control at 12 h, indicating
dicating that toxicity was still low (Figure S1). Flag-d270KD began to degrade withthat toxicity was still low (Figure S1). Flag-d270KD began to degrade within 2 h of the GAof theGAstimulation(Figure2a), andGA-AGE
presumably,
intracellular
stimulation (Figure
2a),andpresumably,
intracellularlevelswere low atGA-AGE
that time,levels we
at
that
time,
suggestingthat
thedegradation
systemconsistingof the ubiquiti
suggesting that the degradation system consisting of the ubiquitin–proteasome pathway
teasomepathwayandautophagy
pathwaywasfunctioning.
The
pretreatmentofand autophagy pathway was functioning. The pretreatment of HeLa cells with MG132, a
cells
with
MG132,
aproteasome
inhibitor,
clearly
produced
detectablelevelsofenproteasome inhibitor, clearly produced detectable levels of endogenous N-terminal CHK1N-terminal
CHK1-CPs(Figure5a), andwere
the only
expression
levelsof these CHK
CPs (Figure 5a),nous
and theexpression
levels of these
CHK1-CPs
marginallyaffectedwere
only
marginallyaﬀected
by
the
2
mM
GA
stimulation
until
at
least
by the 2 mM GA stimulation until at least 3 h (Figure 5b). These results suggest that3 h (Figu
These results
suggestN-terminal
CHK1-CPs are
subject to regulation
N-terminal CHK1-CPs
are subject
tothatregulation
via proteasomal
degradationin the basalvia protea
degradation
in
the
basal
state
of
cells.
state of cells.

Figure 5.ofInvolvementof thepathway
proteasome
pathwayin theofregulationof CHK1-CPs
Figure 5. Involvement
the proteasomein theregulation
CHK1-CPs
at the basalat the basa
(a)
HeLa
cells
were
transfectedwithscrambled
control
siRNA
(Con)
or
Chk1
siRNA, treatedlevel. (a) HeLa cells were transfected with scrambled control siRNA (Con) or Chk1 siRNA,
treatedmMoftheproteasome
inhibitor
MG132
(+)
or
DMSO
(−)
48
h
later,
andcell
lysateswith 5 mM of the proteasome inhibitor MG132 (+) or DMSO (−) 48 h later, and cell lysates were were coafter 5 h. The resulting cell lysates were subjected to a Western blot analysis with the indica
collected after 5 h. The resulting cell lysates were subjected to a Western blot analysis with thetibodies. The densitometric quantification of endogenous CHK1-CP values relative to th
indicated antibodies. The densitometric quantification of endogenous CHK1-CP values relative totreated control group (Con) is shown. The data were analyzed using a one-way ANOVA b
the non-treated control
group
is shown.
Theasdata
one-way ANOVA
nett’s post
hoc(Con)test andare shown
the weremeananalyzed± SEM ofusing
three aindependent
experiments. * p
by Dunnett’s post
hoc
test
and
are
shown
as
the
mean
±
SEM
of
three
independent
experiments.
(b) HeLa cells were treated with 5 mM of the proteasome inhibitor MG132
or DMSO for 4 h* p < 0.01. (b) HeLa
cells
werewithtreatedwith
5 mMof theproteasome
inhibitor
or DMSO
weretreated
2 mM
GA
and celllysates
were collected
at MG132
the indicatedtimesforfor a Westeanalysis.
The2 densitometricquantificationof collectedendogenous
CHK1-CPvalues
4 h. Cells were treatedwithmM GA and celllysates wereat theindicatedtimesrelative
for a to the DtreatedThecontrolgroup (DMSO
0 h) is shown.
The data wereanalyzedvalues
usingrelative
a two-way ANO
Western blot analysis.
densitometricquantificationof endogenous
CHK1-CP
lowed
by
the
Bonferroni
post
hoc
test
and
are
shown
as
the
mean
±
SEM
of
to the DMSO-treated control group (DMSO 0 h) is shown. The data were analyzed using athree
two-independ
periments.
*p<0.001.
way ANOVA followed by the Bonferroni post hoc test and are shown as the mean ± SEM of three
independent experiments. * p < 0.001.

Similarly, the pre-administration of MG132 completely blocked the degrada
d270KD
by GA (Figure of6a).MG132
Additionally,
stimulation
and GA
Similarly, the pre-administration
completely
blockedwith
the MG132
degradationof promot
d270KD by GA (Figure 6a). Additionally, stimulation with MG132 and GA promoted the formation of high-molecular-weight d270KD-EGFP complexes and increased their level of ubiquitination more than the stimulation with MG132 alone (Figure 6b). Collectively, these results

FOR PEER REVIEW

13 of 26of high-molecular-weight d270KD-EGFP complexes and increased their level
Cells 2023, 12,formation
2838

13 of 26of ubiquitination more than the stimulation with MG132 alone (Figure 6b). Collectively,
these results suggest that the GA stimulation induced GA-AGE modifications with therapid ubiquitinationof d270KD,
which
was mainly
through thewith
ubiquitin–prosuggestthat the GA
stimulationinduceddegraded
GA-AGE modifications
the rapid ubiquitination
teasome pathway.of d270KD, which was mainly degraded through the ubiquitin–proteasome pathway.

Figure6. Involvement
the proteasome
pathwayin the rapid
degradationof d270KD
by GA.
Figure 6. Involvementof theproteasomeof
pathwayin the rapid
degradationof d270KD
by GA.
(a)(a) HeLa cells
werewith expression
the Flag-d270KD
expression
cells were24incubated
24 h
HeLa cells were transfectedwiththetransfected
Flag-d270KD
vectorand cellsvector
wereandincubatedhwith 5 mMinhibitorof a proteasome
inhibitor(MG132)or DMSO
4 h. treated
Cells were treatedlater with 5 mM of latera proteasome(MG132)or DMSO(control)for 4(control)h. Cellsfor
were
4 mM GA
andcollected
cell lysatescollected attimes
the indicatedtimes fora Western
blot
analysis. The
with 4 mM GA andwith
cell lysates
were
atwerethe indicatedfor a Western
blot
analysis.
The
densitometricFlag-d270KD
valuesrelativeto the non-treatment
densitometric quantificationof quantification
Flag-d270KDof values
relativeto thenon-treatmentgroup group(0 h) (0ish) is shown.
The analyzed
data were using
analyzedusing a two-way
followed
the Bonferroni
shown. The data werea two-way
ANOVAANOVA
followedby theby
Bonferroni
postpost
hoc hoc
testtest and areshown
as±themean± SEMof three independent
experiments.
* p <(b)0.001.
GA stimulation
and are shown as themean
SEMof threeindependent
experiments.
* p < 0.001.
The(b)
GAThe
stimulation increased theincreased
ubiquitinationof d270KD-fused
EGFP. The
expression
vectorfor thed270KDthe ubiquitinationof d270KD-fused
EGFP.
The expression
vectorfor thed270KD fused to
fused to EGFP (d270KD-EGFP)
expression
vector vector
was transfected
into into
HeLa
cells
forfor4848
h handEGFP (d270KD-EGFP)
expression
was transfected
HeLa
cells
andthethe cells were
cells were then treated
DMSO(control)or 5 µM
for for3 h.3 h.
Cells
werethestimulation
then withtreatedwith DMSO(control)or 5 MG132µM MG132
Cells
werecollectedcollected after
after thestimulation with phosphatebuﬀerbuffer
or 4 mM
h. Celllysateswereextracted
underwith phosphateor 4 GA
mMfor
GA3for3 h. Cell
lysateswereextracted
underdenaturing
denaturing conditions,conditions, diluted diluted
in lysisinbuﬀer,
and immunoprecipitated(IP)(IP)withanananti-V5
lysis buffer,and immunoprecipitatedwithanti-V5antibody.
antibody. Whole
Whole cell lysatescell lysates (WCL) (WCL)and IPand
samples
werewere
subjected
to toa aWestern
analysisusing
using
indicated
IP samples
subjected
Western blot
blot analysis
thetheindicated
antibodies.
antibodies.
The E3 ubiquitin ligase that acts in the ubiquitination of d270KD has not yet been
identified.
Mule/HUWE1,
a Homologous
to E6-AP
Carboxyl
Terminus
The E3 ubiquitin ligasethat acts in theubiquitinationof d270KD
has
not yet(HECT)
been E3 ubiquitin ligase, was previously shown to be involved in regulating the basal level of theidentified. Mule/HUWE1, a Homologous to E6-AP Carboxyl Terminus (HECT) E3 ubiqCHK1 protein [29]. In the experimental system of HeLa cells used in the present study,
uitin ligase, was previously
shown to be involved in regulating the basal level of the CHK1
when Mule/HUWE1 was knocked down with siRNA, an increase in endogenous CHK1
protein [29]. In theexperimental
systemof HeLa
cells
used ininthe
study, whenlevelsand a decreasein turnover
were
observed
the present
chase experimentwith cyclohexMule/HUWE1 was
knockeddown
withsiRNA,
anincreaseinendogenous
CHK1
levelsimide (Figure 7a). Similar results were obtained with the transiently expressed Flag-fused
and a decrease inCHK1
turnoverwereobservedin thenthe chaseexperimentwithcycloheximide
protein(Figure7b). We
investigatedwhetherMule/HUWE1
played a role
in results
the GA-stimulateddegradation
d270KD. The
knockdownof Mule/HUWE1(Figure 7a). Similar
were obtainedwith theoftransiently
expressed
Flag-fused
CHK1 did not
suppressthe investigateddegradation ofwhether
d270KD Mule/HUWE1(Figure 7c). It also
did notaaffectthetheformation
protein (Figure 7b).
We thenplayedrole inGA- of highmolecular-weightcomplexes.
Therefore,
anotherubiquitinligasebesidesMule/HUWE1
stimulated degradation of d270KD. The knockdown of Mule/HUWE1 did not suppress
may be involved in the GA-stimulated d270KD degradation pathway. Alternatively, thethe degradation of
d270KD (Figure 7c). It also did not aﬀect the formation of high-molecGA-stimulated degradation of d270KD may involve a different pathway from that reguular-weight complexes.
Therefore, another ubiquitin ligase besides Mule/HUWE1 may belated at the basal level of CHK1WT because GA-stimulated d270KD undergoes GA-AGE
involved in the GA-stimulated
d270KD
modifications aswell as degradationubiquitination.pathway. Alternatively, the GA-stim-

ulated degradation of d270KD may involve a diﬀerent pathway from that regulated at thebasal level of CHK1WT because GA-stimulated d270KD undergoes GA-AGE modifications as well as ubiquitination.

x FOR PEER REVIEW
Cells 2023, 12, 2838

1414ofof 26

Figure 7. Effects of the elimination of Mule/HUWE1 E3 ubiquitin ligase on the rapid degradation of
Figure 7. Eﬀects of the elimination of Mule/HUWE1 E3 ubiquitin ligase on the rapid degradation of
d270KD stimulated by GA (a) HeLa cells were transfected with scrambled control siRNA (control)
d270KD stimulated by GA (a) HeLa cells were transfected with scrambled control siRNA (control)or Mule/HUWE1siRNA, treatedtreated with
with cycloheximide
cycloheximide (CHX;(CHX; 2020 µg/mL)µg/mL)48
48hh later,
later, andand cellcell lysatesorMule/HUWE1 siRNA,
lysates
were
The resulting
resulting cell
cell lysates
lysates were
were subjectedsubjected to
to aa Western
Western blot
were collectedcollected at
at thethe indicatedindicated times.
times. The
blot
with thetheindicatedindicatedantibodies.
antibodies.(b,c)(b,c)
Scrambled
controlsiRNAor Mule/HUWE1
siRNA
analysis with
Scrambled
controlsiRNAor Mule/HUWE1
siRNA
was
was
transfectedin and(a), andtransfectedh latera Flag-CHK1WTtransfected
into into
HeLaHeLa
cellscells
as inas(a),cellscells
wereweretransfected
24 h24later
withwitha Flag-CHK1WT(b)or d270KD-EGFP
expression
vector(c). (c).
Cells
were
treated24 h24laterwithcycloheximide
(CHX;
20
(b)
or d270KD-EGFP
expression
vectorCells
weretreatedh laterwithcycloheximide(CHX;µg/mL)(b) ormM
GA GA(c) fortimes,
and andcell lysates
werewerecollected
for aforWesternblot
20 µg/mL)(b)2or2 mM(c)thefor indicatedthe indicatedtimes,
cell lysatescollected
a Western
analysis.
The densitometricquantificationof endogenous
CHK1 (a),Flag-CHK1WT(b), or d270KDblot analysis.
The densitometricquantificationof endogenous
CHK1(a), Flag-CHK1WT(b), or
EGFP (c) values relative to the non-treatment group (0 h) is shown. The data were analyzed using ad270KD-EGFP (c) values relative to the non-treatment group (0 h) is shown. The data were analyzed
two-way ANOVA followed by the Bonferroni post hoc test and are shown as the mean values ± SEM
using a two-way ANOVA followed by the Bonferroni post hoc test and are shown as the meanof three independent experiments. * p < 0.01, ** p < 0.001, n.s and n.d mean not significant and notvalues± SEMof three independent experiments. * p < 0.01, ** p < 0.001, n.s and n.d mean notdetected,respectively.
significant and not detected, respectively.

3.4. GA-Stimulated High-Molecular-Weight Protein Complexes and GA-AGE-Modified Protein
Complexes Are Resistant to DetergentsThe d270KD protein expressed in HeLa cells appeared exclusively as high-molecularweight complexes in the insoluble fraction of RIPA buffer over time after the GA stimulation(Figure 8a). Based on this result, it was assumed that not only the d270KD-derived GAAGE-modified protein, but also many other GA-AGEs were abundant in the RIPA-insoluble
fraction. As expected, a Western blot analysis of the RIPA-insoluble fractions of cells treatedComplexes Are Resistant to Detergents

Cells 2023, 12, 2838

The d270KD protein expressed in HeLa cells appeared exclusively as high-molecularweight complexes in the insoluble fraction of RIPA buﬀer over time after the GA stimulation (Figure 8a). Based on this result, it was assumed that not only the d270KD-derived
15 of 26
GA-AGE-modified protein, but also many other GA-AGEs were abundant in the RIPAinsoluble fraction. As expected, a Western blot analysis of the RIPA-insoluble fractions ofcells treated with or without 2 mM GA for 10 h revealed that the GA-treated samples wereenriched
with high-molecular-weightcomplexesthat were strongly
detectedby antiwith or without
2 mM GA for 10 h revealed
that the GA-treatedsamples wereenriched
with
TAGE
antibodies
inatime-dependent
mannerupon
GAstimulation(Figure8b).
Therehigh-molecular-weight complexes that were strongly detected by anti-TAGE antibodies
in
fore,
when intracellular
proteins
arestimulation
modified with
GA-AGEs
by GA, theyto aca time-dependent
mannerupon GA(Figure8b). Therefore,
whenappear
intracellular
cumulate
intracellularly
as GA-AGEs
aggregatesbythat
resistantdetergents,
such asproteins are
modified with
GA,arethey
appearto
tocommonaccumulate
intracellularly
asSDS.
aggregates that are resistant to common detergents, such as SDS.

FigureFigure 8.
8. AllAll high-molecular-weighthigh-molecular-weight complexes
complexes ofof d270KD
d270KD formed
formed by
by aa GAGA stimulation
stimulation accumulate
accumulateinFlag-d270KD expression
in thethe insoluble
insoluble fraction
fraction ofof RIPA.
RIPA. (a)(a) A
A Flag-d270KD
expression vector
vector was
was transfectedtransfected into
into HeLa
HeLa
cells,
2424
h later
with
2 mM
GA GA
and andcollected
at theatindicatedtimes. times.
Cells
cells, andandcells
cellswere
werestimulated
stimulated
h laterwith
2 mMcollected
the indicatedCells were lysed in RIPA buffer and separated into supernatant (RIPA-soluble fraction) and pellet(RIPA-insoluble fraction) by centrifugation. After the addition of SDS sample buffer to each fraction,
samples from both fractions were analyzed by Western blotting using the antibodies indicated. The
densitometric quantification of Flag-d270KD high-molecular-weight complex, p62 (phosphor-Ser403),
and p62 (total) values relative to the non-treatment RIPA-soluble fraction group are shown. The data
were analyzed using a two-way ANOVA followed by the Bonferroni post hoc test and are shown asCells 2023, 12, 2838

16 of 26

the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs. 0 h. (b) HeLa cells weretreated with phosphate buffer (control) or 2 mM GA. Cells were collected at the indicated times andlysed in RIPA buffer. Each cell lysate was separated into RIPA-soluble/-insoluble fractions in thesame manner as in (a). Samples of RIPA-insoluble fractions were prepared by three independent experiments and subjected to a Western blot analysis using the indicated antibodies. The densitometricquantification of TAGE values relative to the non-treatment fraction group (0 h) is shown. The data
were analyzed using a one-way ANOVA followed by Dunnett’s post hoc test and are shown as themean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

3.5. p62 Is Involved in the GA-Induced Formation of High-Molecular-Weight d270KD Complexes
We hypothesized that the bulk degradation mechanism by the autophagy pathway,rather than the proteasome pathway, may function in the degradation of high-molecularweight aggregates. Therefore, we investigated changes in the GA-stimulated behavior of
p62, which plays an important role in selective autophagy by binding to ubiquitinatedproteins and transporting them to autophagosomes. A single band of p62 was detectedin the soluble and insoluble fractions of RIPA; in the RIPA-soluble fraction, p62 levels
decreased over time upon GA stimulation, whereas a slight increase was observed in its
levels in the RIPA-insoluble fraction (Figure 8a). On the other hand, the phosphorylated
form of serine 403, which increases binding affinity to ubiquitinated proteins [30,31],
appeared to increase in the RIPA-insoluble fraction over time upon GA stimulation. This
result suggested that the GA stimulation induced the binding of p62 to GA-AGE-modified
high-molecular-weight complexes that were abundant in the RIPA-insoluble fraction.
We also examined the effects of the p62 siRNA knockdown on the GA-induced rapid
degradation of d270KD. Contrary to our expectations, the p62 knockdown did not affectthe degradation of d270KD in the RIPA-soluble fraction (Figure 9a), while a similar study
on the RIPA-insoluble fraction showed a slight decrease in the high-molecular-weightcomplexes of d270KD in cells in which p62 was knocked down (Figure 9b). We theninvestigated whether p62 bound to the high-molecular-weight complexes of d270KD
formed by the GA stimulation. To achieve this, we generated a new protein expression
vector (d270KD-ZsGreen) with ZsGreen1 fused to the C terminus of d270KD and confirmedthat the high-molecular-weight complexes of this fusion protein were formed within a shorttime after the GA stimulation and were rapidly degraded in cells (Figure S2). HeLa cells
were transfected with the d270KD-ZsGreen expression vector and 48 h later, the cells were
stimulated with 2 mM GA for 3 h to form the high-molecular-weight complexes of d270KDZsGreen. The RIPA-soluble and RIPA-insoluble fractions were then immunoprecipitatedwith the anti-FLAG antibody to purify the d270KD-ZsGreen complexes. As a control, thesame immunoprecipitation was performed with normal rabbit IgG.
The Western blot analysis showed that in the RIPA-soluble and insoluble fractions, p62
bands were only detected by immunoprecipitation with the anti-FLAG antibody (Figure 10).
In the RIPA-insoluble fraction, not only the monomer, but also a smeared band of highmolecular-weight p62 were observed. This result indicates that GA-modified p62 waspresent as a component of the high-molecular-weight complex of d270KD-ZsGreen. Furthermore, the immunoprecipitation products of d270KD-ZsGreen with the FLAG antibody
showed a band of high-molecular-weight complexes detected with the anti-TAGE (GAAGE) antibody only in the RIPA-insoluble fraction. These results suggest that p62 was
one of the components of the high-molecular-weight complexes of d270KD modified with
GA-AGEs by the GA stimulation.

Cells 2023,Cells
12, x2023,
FOR
PEER REVIEW
12, 2838

17 of 26

17 of 26

Figure
9. Effectsofof theknockdownon the rapid
GA-stimulateddegradation of degradation
d270KD.
Figure9. Eﬀects
thep62/SQSTM1
p62/SQSTM1
knockdownon therapid GA-stimulatedof
HeLaHeLa
cells weretransfectedwith scrambled
siRNAcontrol
or p62/SQSTM1
siRNA
and transfected
d270KD.
cells
were transfectedwithcontrol
scrambledsiRNA or
p62/SQSTM1
siRNA and24 h latera Flag-d270KD
expression vector.
Cells vector.
were stimulated
24 hstimulated
later with 224
mM
GA with 2transfected
24withh laterwith a Flag-d270KD
expression
Cells wereh laterfortheindicatedtimes.
RIPA
buffer
was
added
to
cells
and
cell
lysateswere
separated
into
RIPAmM GA for the indicated times. RIPA buﬀer was added to cells and cell lysates were separated intosoluble (a) (a)andandRIPA-insoluble(b) fractions
by centrifugation.
Each fractionEach
was analyzed
Western
RIPA-soluble
RIPA-insoluble(b) fractions
by centrifugation.
fractionby
was
analyzed byblotting
using using
the indicatedantibodies.
The densitometricquantificationquantificationof Flag-d270KDofandp62
Westernblotting
the indicatedantibodies.
The densitometricFlag-d270KD(total)valuesrelativetothe
non-treatment
group
(0
h)
is
shown.
The
data
were
analyzedusing
and p62 (total) values relative to the non-treatment group (0 h) is shown. The data were aanalyzed
ANOVA
followedby the Bonferroni
post hoc test
andhocare shownas the± SEMof mean ±usingtwo-way
a two-way
ANOVA
followedby the Bonferroni
post
test andare meanshown
as thethree
independent
experiments.
*
p
<
0.05,
**
p
<
0.01,
***
p
<
0.001.
SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Cells 2023, 12, x FOR PEER REVIEW
Cells 2023, 12, 2838

18 of 26

18 of 26

Figurea componentof GA-AGE-modifiedd270KD
d270KDhigh-molecular-weighthigh-molecular-weight complexes
Figure10.10.
p62p62is aiscomponentof GA-AGE-modifiedcomplexes prepresent
inthe
RIPA-insoluble
fractionafterGA
stimulation.
The
expression
vectorfor
Flagsent in the RIPA-insoluble fraction after GA stimulation. The expression vector for the theFlag-d270KD
d270KD
fused to (d270KD-ZsGreen)
ZsGreen1 (d270KD-ZsGreen)
expression
was transfected
into HeLa
forh andfused
to ZsGreen1
expression
vector vector
was transfected
into HeLa
cellscells
for 48h andthencells treated
were thentreatedwith
2 mM
3 h.buﬀer
RIPA buffer
was added
to cells
the cells
cell
cells48were
with
2 mM
GA
for 3GAh. forRIPA
was added
to theandandthethecelllysateslysates
were separated
into RIPA-soluble(Sol.)RIPA-insoluble
and RIPA-insoluble(Ins.)
fractionscentrifugation. Eachwereseparated
into RIPA-soluble(Sol.) and(Ins.)
fractions
bybycentrifugation.
Each fraction
was immunoprecipitated(IP) withan anti-FLAG
antibody(Flag)(Flag)oror normal
normal rabbit
fraction
was immunoprecipitated(IP) withan anti-FLAG
antibody
rabbitIgG
IgG (C).
(C). Wholecell lysates
andIP samples
subjected
a Westernblot
blotanalysis
analysis using
using thethe indiWholecell lysates(WCL)(WCL)and IP
samples
werewere
subjected
to atoWestern
indicated
antibodies.
M, protein
marker.
catedantibodies.
M, protein
size size
marker.

3.6. The Inhibition of Autophagy Does Not Affect the Rapid Degradation of d270KD due to GA

3.6.Stimulation,
The Inhibitionof Autophagy
Does Not
the Rapid Degradationof d270KD due to GA
but Inhibits
the Formationof ItsAﬀect
High-Molecular-Weight
Complexes
Stimulation,
but Inhibits whether
the Formationof Its High-Molecular-Weight
Complexes
We investigated
the treatment
with the autophagy inhibitor
chloroquine
We investigatedwhetherthedegradation.
treatment withthe autophagy
inhibitor
chloroquine
affected
GA-stimulated
d270KDAs shown
in Figure 11,
after thetransient afexpressionofd270KDin
HeLa
cells,
thepretreatment
with
50
µM
chloroquine
did not exfected GA-stimulated d270KD degradation. As shown in Figure 11, after the transient
affect GA-stimulated
d270KDdegradation.
The administrationof chloroquine
chloroquine increased
pressionof d270KD in HeLa
cells,
the pretreatment
with 50 µM
did not aﬀect
theLC3-II/LC3-I
ratio,
suggestingthatchloroquine
inhibitedthefusionoflysosomes
with theGA-stimulated d270KD degradation. The administration of chloroquine increased
autophagosomes, thereby suppressing the degradation of LC3-II in the phagophore memLC3-II/LC3-I ratio, suggesting that chloroquine inhibited the fusion of lysosomes with aubrane. In addition, an increase in the basal level of p62 and the concomitant polymerization
tophagosomes,
thereby(Figure
suppressing
degradation
LC3-IIin thephagophoreof p62 were observed11). Thisthe
resultis consistentofwith
previous
findings
showingmembrane.
In
addition,anincrease
in
the
basal
level
of
p62
and
the
concomitant
polymerizathat p62 bound to ubiquitinated proteins which were degraded together in a selective
autiontophagyof p62mechanism,
were observed(FigureThis result
is consistent
with previous
findings
resultingin the11).
accumulationof p62
when autophagy
was inhibited.
showingp62
bound to ubiquitinatedproteins
which
were degraded
togetherin a seSimilarthat
to thechloroquine-insensitive
results
obtainedfor d270KD
degradation
by GA, nosignificant
changesin the expression
levelsof endogenous
CHK1-CPs
were noted
upon waslective
autophagymechanism,
resultingin theaccumulationof p62 when
autophagychloroquine
treatment
Figure S3).results obtained for d270KD degradation
inhibited.
Similarto the (Supplementarychloroquine-insensitiveTo
examinetheeffectofchloroquine
treatmenton d270KD
degradationin more wereby GA, no significant changes in the expression
levelsof endogenous
CHK1-CPsdetail, samples were separated into RIPA-soluble and RIPA-insoluble fractions for analysis.
noted upon chloroquine treatment (Supplementary Figure S3).
The results showed that in the RIPA-soluble fraction, a slight increase in the basal level ofTo examine the eﬀect of chloroquine treatment on d270KD degradation in more ded270KD was observed upon chloroquine treatment in the untreated GA fraction (Figure 12a,tail,0 samples
werenoseparated
into RIPA-soluble
and RIPA-insoluble
fractionsfor analysis.
h). However,effect of chloroquine
was observedin GA-stimulateddegradationof
Thed270KD.
results showed
that
in
the
RIPA-soluble
fraction,aslightincrease
in
the
basal
level ofOn the other hand, an analysis of the RIPA-insoluble fraction showed a slightd270KDwasupon chloroquine
treatment
the untreated
GA fraction(Figuredecreasein observed
the high-molecular-weightcomplexlevel ofind270KD
by chloroquine
treatment
8 hHowever,of GA stimulation12b). Interestingly,
p62, whichbecomes high-molecular12a,after
0 h).
no eﬀect(Figureof chloroquine
was observedin GA-stimulateddegradation
weight uponlevels in the RIPA-insoluble
fractionof d270KD.
On GA
the stimulation,other hand,accumulated
an analysisat
ofhigherthe RIPA-insoluble
fraction showeda slight
uponchloroquine
treatment.
These
resultssuggestthattheautophagy
pathwayispartially
decrease in the high-molecular-weight complex level of d270KD by chloroquine treatment
involved in the regulation of the basal level of d270KD, but its inhibition has little effectafter 8 h of GA stimulation (Figure 12b). Interestingly, p62, which becomes high-molecuon GA-stimulated degradation. It is conceivable that when autophagy is inhibited bylar-weight upon GA stimulation, accumulated at higher levels in the RIPA-insoluble fraction upon chloroquine treatment. These results suggest that the autophagy pathway ispartially involved in the regulation of the basal level of d270KD, but its inhibition has little
eﬀect on GA-stimulated degradation. It is conceivable that when autophagy is inhibitedby chloroquine and yet stimulated by GA, p62 abnormally accumulates in the insoluble

Cells 2023, 12, 2838

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Cells 2023, 12, x FOR PEER REVIEW

19 of 26

chloroquine and yet stimulated by GA, p62 abnormally accumulates in the insoluble
fraction,in aaslightslightdecrease
decrease
formationof high-molecular-weightthe high-molecular-weightfraction, resulting inin inthetheformationof thecomcomplexesof d270KD.
plexes of d270KD.

Figure11.
11.Effects
Eﬀectsof
ofchloroquine,
chloroquine,ananautophagy
autophagyinhibitor,
inhibitor,ononthetherapid
rapidGA-stimulated
GA-stimulateddegradation
degradation
Figureofd270KD.
d270KD.(a)(a)HeLa
HeLacells
cellswere
weretransfectedtransfectedwith
withaaFlag-d270KD
Flag-d270KDexpression
expressionvector.
vector.The
Thecells
cellswere
wereofstimulated
24
h
later
with
50
µM
chloroquine
for
20
h.
The
cells
were
collected
afterthestimulation
stimulated 24 h later with 50 µM chloroquine for 20 h. The cells were collected after the stimulation
with phosphate buﬀer or 2 mM GA for the indicated times. The resulting cell lysates were subjectedwith phosphate buffer or 2 mM GA for the indicated times. The resulting cell lysates were subjected
to a Western blot analysis with the indicated antibodies. (b) The densitometric quantification of Flagto a Western blot analysis with the indicated antibodies. (b) The densitometric quantification of
d270KD and total p62 values relative to the non-treatment control group (control 0 h) are shown.
andtotal p62using
valuesrelative toANOVA
the non-treatment
controlgroup (control0 h) test
are20shown.
Cells 2023, 12, x FOR PEER REVIEW Flag-d270KD
of 26
The data wereanalyzed
a two-wayfollowed by
the Bonferroni
post hoc
and
are
The
data
were
analyzedusingatwo-way
ANOVA
followedby
the
Bonferroni
post
hoc
test
and
are
shown as the mean ± SEM of three independent experiments. * p < 0.001.
shown as the mean ± SEM of three independent experiments. * p < 0.001.

Figure 12. Cont.

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20 of 26

Figure 12.
12. Eﬀects
Effectsofof chloroquine
chloroquineon
onthe
therapid
rapidGA-stimulated
GA-stimulateddegradation
degradationof
ofd270KD
d270KDininthe
theRIPARIPAFiguresolublesoluble andand RIPA-insoluble
RIPA-insoluble fractions.
fractions. HeLa
HeLacells
cellswere
were transfectedtransfected with
with aa Flag-d270KD
Flag-d270KD expression
expression
vector.
vector. The
The cells
cells were
were stimulated
stimulated 24
24 hh later
later with
with 50
50µMµM chloroquine
chloroquine forfor 2020 h.
h. TheThecells
cellswere
werecollectedcollected
afterthestimulation
withphosphate
buﬀer
or
2
mM
GA
for
the
indicatedtimes.
RIPA
after the stimulation with phosphate buffer or 2 mM GA for the indicated times. RIPA buﬀer
buffer was
was
added
addedtotocells
cellsandandcell
celllysates
lysateswere
wereseparated
separatedinto
intoRIPA-soluble
RIPA-soluble(a)(a)andandRIPA-insoluble
RIPA-insoluble(b)(b)fractions
fractionsby centrifugation. Each fraction was analyzed by Western blotting using the indicated antibodies.
by centrifugation. Each fraction was analyzed by Western blotting using the indicated antibodies.
The densitometric quantification of Flag-d270KD and total p62 values relative to the non-treatment
The densitometric quantification of Flag-d270KD and total p62 values relative to the non-treatment
control group (control 0 h) is shown. The data were analyzed using a two-way ANOVA followed bycontrolgroup (controlh) isandshown.
The data
wereanalyzed
a two-way
ANOVA
followed by
the
Bonferroni
post hoc0test
are shownas themean± SEMusingof threeindependent
experiments.
*
theBonferroni
post
hoc
test
and
are
shown
as
the
mean
±
SEM
of
three
independent
experiments.
p < 0.001 (a); * p < 0.05, ** p < 0.01 (b).
* p < 0.001 (a); * p < 0.05, ** p < 0.01 (b).

4. Discussion
4. Discussion
Under hyperglycemic conditions, glucose, fructose, and its metabolic intermediates
Under hyperglycemic conditions, glucose, fructose, and its metabolic intermediates
bind non-enzymatically to intracellular proteins to produce AGEs. Among these AGEs,
bind non-enzymatically to intracellular proteins to produce AGEs. Among these AGEs,
GA-AGEs, also known as TAGE, are highly cytotoxic and have been implicated in various
pathological conditions [3], such as diabetic complications, insulin resistance, heart disease,
Alzheimer’s disease, hypertension, nonalcoholic steatohepatitis, obesity, and cancer. GAAGEs predominantly form intracellularly as a result of glycation reactions between GA andintracellular proteins, resulting in a wide range of GA-AGE molecules with different sizes
and properties. The mechanisms by which GA-AGEs exert their cytotoxic effects have notyet been elucidated in detail; however, previous studies suggested that GA-AGE toxicity
was attributed to oxidative stress damage, the loss of protein functions due to glycation
modifications, and the aggregation and accumulation of GA-AGEs [3].
To the best of our knowledge, this is the first study to show the rapid degradation (lessthan 2 h) of a protein in an experimental model in which intracellular AGEs were formedby the administration of glycating agents, such as GA and methylglyoxal (MGO), to cells.
Even when d270KD was fused with relatively stable proteins, such as EGFP, ZsGreen1, or
luciferase, the GA-responsive rapid degradation property was retained (Figures 3, 4 and S2),
indicating that d270KD functions as a degradation element induced by glycated substances.
Furthermore, a reporter assay using luciferase activity in d270KD-Luc-transfected cells
allowed the degradation process to be monitored without a Western blot analysis, and GAinduced reductions in luciferase activity were attenuated by inhibitors of AGE formation ina concentration-dependent manner (Figure 4c). Therefore, this reporter assay using d270KD-

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Luc-transfected cells may be a useful tool for the rapid screening of natural compounds
that inhibit the formation of AGEs.
The etiology of GA-AGE toxicity is attributed to the functional impairment of theproteins themselves as a result of GA-AGE modifications. Previous studies demonstrated that chaperone molecules, such as Hsc70, and a mediator of apoptosis, caspase-3,
formed high-molecular-weight complexes that led to a loss of activity due to GA-AGE
modifications [3]. In the present study, we observed the similar formation and accumulation of high-molecular-weight complexes of CHK1 after the GA stimulation, as shown in
Figure 1. CHK1 is a well-known key player in the cellular stress response to DNA damage
and the normal progression of the cell cycle under non-stress conditions [27]. Therefore,
CHK1 may be a target of GA-AGE modifications, and its functional impairment may be
responsible for the induction of GA-AGE-mediated cell death. Notably, when HeLa cells expressing Flag-CHK1 were stimulated with GA, a decrease in CHK1-CP levels was observed,
suggesting that N-terminal CHK1 kinase fragments were susceptible to glycation modifications by GA (Figure 1b). Consistent with this result, d270WT (a kinase fragment containing
the N-terminal 270-amino acid region of wild-type CHK1), which mimics CHK1-CPs, also
showed time-dependent proteolysis upon GA treatment, but more slowly than d270KD(Figure 2a). In the course of our research, d270KD demonstrated accelerated degradationin response to GA compared to d270WT. However, the precise underlying reasons for thisphenomenon remain unclear at present. One plausible explanation is that the structuralfeatures of d270KD make it more susceptible to ubiquitin modification than its wild-type
counterpart, potentially leading to rapid degradation through the proteasome pathway.
As Halder et al. previously reported [28], CHK1-CPs are formed through cleavage by
SPRTN metalloprotease, and in our cell model, the knockdown of SPRTN by the transfection of a specific siRNA reduced the basal expression levels of Flag-CHK1-CPs (Figure 1c).
While Flag-CHK1-CPs were easily detectable in basal state cells, the expression levels ofendogenous CHK1-CPs were so low that only a few of their protein bands were detectable
after prolonged exposure in our Western blot analysis. The pretreatment with the proteasome inhibitor MG132 increased multiple bands of CHK1-CPs to readily detectable levels(Figure 5a), suggesting that the ubiquitin–proteasome pathway is involved in regulating
the basal expression levels of CHK1-CPs. The current findings, which reveal an increase inubiquitin modification of d270KD upon GA stimulation (Figure 6b), emphasize the importance of identifying the ubiquitin ligase involved in this modification for a comprehensive
understanding of the degradation mechanism of d270KD.
A previous study reported that Mule/HUWE1, a HECT-type ubiquitin E3 ligase,ubiquitinated CHK1 and played a critical role in regulating the basal level of CHK1 [29].
In our HeLa cell model, we noted a decrease in endogenously and transiently expressed
CHK1 turnover after the elimination of Mule, as shown in Figure 7a,b. However, theknockdown of Mule did not affect the degradation of d270KD in response to the GA
stimulation or suppress the formation of high-molecular-weight complexes, as shown in
Figure 7c. These results suggest that the degradation mechanism of d270KD, which isdependent on glycation modifications, involves a pathway distinct from the steady-statedegradation mechanism of CHK1.
An important result from our Western blot analysis is that Mule was also affectedby GA-induced glycation, as shown in Figure 7c (Control siRNA). Specifically, the GA
stimulation for 6 h resulted in a conspicuous smear of the putative monomer form of Mule.
However, it remains unclear whether the decrease in single bands was a consequence of
GA-AGE-modified Mule degradation induced by the GA stimulation or the formation ofhigh-molecular-weight complexes. This uncertainty is attributed to the technical challenges
associated with analyzing the behavior of GA-AGE-modified Mule over time because
the transfer efficiency of high-molecular-weight proteins (more than 460 kDa) to a PVDF
membrane is very low in a conventional Western blot analysis. The loss of function ofubiquitin ligases due to GA-AGE modifications may lead to the accumulation of proteinsthat need to be degraded, further exacerbating non-enzymatic glycation reactions andCells 2023, 12, 2838

22 of 26

disrupting protein homeostasis. Therefore, a more comprehensive and detailed analysis of
the effects of GA-induced glycation on ubiquitin ligases is warranted in the future.
Another possible cause of cellular damage due to GA-AGEs is proteopathy, a broadterm that refers to the crosslinking of glycated proteins between molecules over time, resulting in the formation of larger high-molecular-weight complexes that gradually aggregate
and accumulate, disrupting normal protein function [3]. The proper folding of intracellular
proteins is crucial for their biological functions, while aberrantly folded proteins may
accumulate and aggregate in cells, leading to cellular stress. In response to this stress, cells
activate mechanisms to monitor protein folding, such as the unfolded protein response in
the endoplasmic reticulum [30], which induces autophagy, an intracellular degradation
mechanism that recycles damaged proteins and protects cells [31–34]. Non-enzymatic
glycation modifications to intracellular proteins may lead to structural mutations and theformation of abnormal proteins, which may be recognized as aberrant by intracellular
protein quality control mechanisms.
Extracellular AGEs have also been shown to activate intracellular autophagy pathways,
which often have a cytoprotective role [30–34]. Intracellularly formed AGEs were previously
found to induce autophagy [17]. For example, in a model in which cells were treatedwith MGO, a glycating substance, to accumulate intracellular AGEs (MG-H1), autophagy
was induced through a pathway that involved p62. Importantly, while the loss of p62
promoted the accumulation of AGEs, p62 itself appeared to be affected by MGO-induced
glycation over time, forming high-molecular-weight complexes and losing its function. In
our experimental model of the formation of AGEs, in the RIPA-insoluble fraction, a slow,
time-dependent increase in the expression level of a single band of p62 was observed in
the Western blot analysis (Figure 8a). In contrast, in the RIPA-soluble fraction, a gradualdecrease was noted in the level of p62 starting 8 h after the GA stimulation. These results
suggest that p62, which was present in the RIPA-soluble fraction, bound to the protein
aggregates that gradually formed due to the GA stimulation and then migrated with theminto the RIPA-insoluble fraction. In other words, the autophagy–lysosome pathway (andpossibly the ubiquitin–proteasome degradation pathway) remains functional in the earlystages of intracellular AGE formation, allowing for the proper clearance of AGEs. However,as glycation reactions continue, degradation pathways and other potential protective
factors gradually transform into AGEs, resulting in the dysfunction of the AGE clearance
pathway. This late stage of AGE formation may eventually induce apoptosis or passive,
unprogrammed cell death by necrosis in affected cells [3]. To the best of our knowledge,
the induction of autophagy in the early stages of GA-AGE formation in cells has not yet
been demonstrated.
We previously reported that the stimulation of a human pancreatic beta cell line
(1.4E7 cells) with GA resulted in prominent cell death, accompanied by decreases in
the autophagosome markers LC3-I and LC3-II, as well as p62 [18]. These findings wereobserved after a prolonged stimulation (24 h) with 2 mM GA, at which stage, cell death
was already induced. Therefore, important factors involved in various vital functions
were modified by GA-AGEs, aggregated, and accumulated, suggesting that the GA-AGE
degradation pathway was already disrupted in the late stage. To confirm the existence ofan endogenous degradation and removal mechanism for GA-AGEs, a detailed analysis of
the behavior of degradation-related factors from the initial stage of a glycation stimulationis needed.
In the present study, we investigated the behavior of p62, a known selective autophagy
receptor, over 10 h, starting 2 h after stimulation with 2 mM GA. The results obtained
from the RIPA-insoluble fraction revealed a slight increase in total endogenous p62 levels
10 h after the administration of GA (Figure 8a). Since autophagy is responsible for the
degradation of p62 when degrading its substrates, the accumulation of p62 is often observed
when autophagy is impaired [17]. However, total p62 levels remained unchanged inboth the RIPA-soluble and insoluble fractions after 6 h of the stimulation with 2 mM
GA; therefore, the complete failure of autophagy had not yet occurred 6 h after the GA

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stimulation. p62 plays a key role in substrate degradation by recruiting ubiquitinated cargo
to autophagosomes [35,36]. The phosphorylation of the 403rd serine (Ser403) in the UBA
domain of p62 by casein kinase-2 or TANK-binding kinase 1 increased its binding affinity
to the ubiquitin chain, thereby facilitating selective autophagy [37,38].
Interestingly, after GA stimulation for 2 h, a gradual decrease in the phosphorylated
form of p62 Ser403 was observed in the RIPA-soluble fraction, in contrast to an increase overtime in the RIPA-insoluble fraction (Figure 8a). Most of the d270KD high-molecular-weightcomplexes induced by the GA stimulation were present in the RIPA-insoluble fraction, andthe level of d270KD high-molecular-weight complexes in this fraction peaked at approximately 6 h of GA stimulation, followed by a gradual decrease (Figure 8a). Furthermore, the
RIPA-insoluble fraction of the cells that did not express d270KD but were stimulated after
10 h of GA treatment and also showed an abundance of high-molecular-weight complexes
that were positive for anti-TAGE antibodies (Figure 8b). This result suggests that intracellular proteins modified with TAGEs (GA-AGEs) due to GA exposure form aggregatesthat are resistance to detergents, such as SDS, and are more likely to become insoluble
intracellularly. Furthermore, the binding of phosphorylated p62 to ubiquitinated GA-AGEs
may have been increased in the insoluble fraction, suggesting that the degradation systemthat uses selective autophagy was still partially functional during the initial stage of the GA
stimulation, despite the formation of GA-AGE-modified aggregates. Importantly, immunoprecipitation experiments on the RIPA-insoluble fraction after the GA stimulation showedthat the high-molecular-weight complexes of d270KD and p62 coprecipitated, and thesecomplexes were also positive for anti-TAGE antibodies (Figure 10). These results suggestthat the high-molecular-weight complexes of d270KD in the RIPA-insoluble fraction weremodified by GA-AGEs and also that p62 was one of its components.
Overall, the present results highlight the importance of analyzing the behavior of
degradation-related factors, such as p62, from the initial stage of glycation stimulation to
elucidate the endogenous GA-AGE degradation and removal mechanisms. Further researchis needed to clarify the complex interplay between GA-AGE formation, autophagy, andcellular responses to proteopathy, which will provide a more detailed understanding of thepathogenesis of GA-AGE-related cellular damage. Since proteasome inhibitors completely
suppressed the GA-stimulated rapid degradation of d270KD (Figure 6a), the ubiquitin–
proteasome pathway may also work in conjunction with the autophagy pathway during theearly stages of GA-AGE formation to maintain intracellular protein homeostasis, which is
disrupted by glycation modifications. The knockdown of p62 did not affect the degradationof d270KD induced by GA, but modestly reduced the formation of high-molecular-weightcomplexes derived from d270KD in the insoluble fraction (Figure 9). Therefore, p62 may
play a partial role in the formation of GA-AGE aggregates in the insoluble fraction.
We also investigated the effects of chloroquine on autophagy. The results obtained revealed that the chloroquine treatment increased the basal level of p62 in the RIPA-insoluble
fraction (Figure 12b), indicating the potential inhibition of autophagy. However, the chloroquine treatment did not affect the GA-stimulated degradation of d270KD, but slightly
suppressed the formation of high-molecular-weight complexes of d270KD in the RIPAinsoluble fraction, similar to the knockdown effect of p62 (Figures 11 and 12). These results
suggest that chloroquine interfered with the proper formation of high-molecular-weightcomplexes of d270KD, which is consistent with the knockdown effect of p62. One hypothesis is that p62 may be involved in sequestering randomly formed GA-AGEs throughout
the cell by aggregating them, thereby preventing interference with the function of normal
proteins and potentially protecting the cell. A more detailed analysis is needed on thesubcellular localization of ubiquitinated and insoluble GA-AGE aggregates. However, it
is important to note that the complete inhibition of autophagy by chloroquine was not
fully confirmed in the present study. Therefore, the exact biological significance of theinvolvement of p62 in the formation of high-molecular-weight complexes of d270KD induced by GA stimulation remains to be elucidated. Further investigations are warranted toCells 2023, 12, 2838

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obtain a more detailed understanding of the autophagy-related effects of chloroquine in
our experimental context.
5. Conclusions
Our study provides new insights into the intracellular degradation mechanism of
GA-AGEs, offering potential therapeutic strategies to prevent or alleviate GA-AGE-relateddiseases. The suggestion of a specific ubiquitin ligase that recognizes the structural changes
induced by GA glycation modification adds complexity to our understanding. Future
investigations should not only focus on identifying this unidentified ligase but also on
unraveling its differential binding affinity for d270WT and d270KD. Further explorationin this field promises to advance the development of effective interventions for diseases
associated with protein aggregation and the accumulation of AGEs, spanning conditions
such as diabetes, neurodegenerative diseases, and cardiovascular disorders.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/cells12242838/s1, Figure S1: Lactate dehydrogenase (LDH) cytotoxicity
assay. Figure S2: GA-stimulated rapid intracellular degradation of d270KD fusion proteins to
ZsGreen1. Figure S3: Effects of chloroquine on the GA-stimulated degradation of endogenous
CHK1-CPs.
Author Contributions: Conceptualization, K.T. and M.T.; methodology, K.T.; investigation, K.T.;
writing—original draft preparation, K.T.; writing—review and editing, A.S.-S., M.T. and K.T.; project
administration, A.S.-S., K.K. and M.T.; funding acquisition, K.T., A.S.-S. and M.T. All authors haveread and agreed to the published version of the manuscript.
Funding: This research was funded by JSPS KAKENHI grant numbers 20K08410 (K.T.) and21H04865 (M.T.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data for this study can be found in the main article or in the supplementary material.
Acknowledgments: The authors would like to thank Sumiyo Akazawa for generously providing uswith HeLa cells.
Conflicts of Interest: The authors declare that they have no conflict of interest. The funders had norole in the design of the study; in the collection, analyses, or interpretation of data; in the writing of
the manuscript; or in the decision to publish the results.

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