# Doxorubicin-induced senescence is modulated by the eukaryotic release factor 3a and its polyglycine expansion in HCT116 cells

Béatrice Jolles, Vérène Stierlé

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Béatrice Jolles, Vérène Stierlé. Doxorubicin-induced senescence is modulated by the eukaryotic release factor 3a and its polyglycine expansion in HCT116 cells. Biochimie, In press, 10.1016/j.biochi.2025.02.009. hal-04974259

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# Doxorubicin-induced senescence is modulated by the eukaryotic release factor 3a and its polyglycine expansion in HCT116 cells

Béatrice Jolles<sup>a</sup>, Vérène Stierlé<sup>a, b, *</sup>

## ARTICLE INFO

**Article history:**

Received 10 October 2024

Received in revised form

17 February 2025

Accepted 24 February 2025

Available online xxx

Handling Editor: O.A. Dontsova

**Keywords:**

Translation termination

eRF3a

GSPT1

Doxorubicin

p21

Senescence-associated β-galactosidase

activity

## ABSTRACT

In humans, the release factor eRF3a exists in several forms that differ in the length of the polyglycine tract (7, 10, 11 or 12 glycines) in its N-terminal domain. For the 12-Gly eRF3a, an association with cancer risk and a decreased affinity for the cytoplasmic poly (A) binding protein have already been established. In this work, HCT116 colon cancer cells were treated with low doses of doxorubicin, which is known to induce senescence in these cells with high efficiency. The expression of p21 and p53 (senescence marker proteins) as well as lysosomal β-galactosidase activity were reduced when 12-Gly-eRF3a was overexpressed or eRF3a was depleted in cells. If low activity of mTORC1 pathway might be responsible for reduced senescence onset after eRF3a depletion, its activity is maintained in cells overexpressing 12-Gly-eRF3a. In both cases, a defect in termination efficiency could be involved.

© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

In eukaryotes, at the end of translation, the eukaryotic release factor 3 (eRF3) functions as a small GTPase that forms a complex with another eukaryotic release factor, eRF1, which enhances eRF1 activity in a GTP-dependent manner and promotes the release of the nascent peptide from the ribosome [1]. In mammals, eRF3 has two distinct isoforms: eRF3a encoded by the G1 to S phase transition 1 gene (GSPT1), and eRF3b, encoded by the G1 to S phase transition 2 gene (GSPT2) [2–4]. eRF3a is the major form of eRF3 in most tissues and its abundance regulates the formation of translation termination complex. eRF3b is very poorly expressed in most tissues and is unlikely to play a major role in translation termination [5]. eRF3a and eRF3b proteins share nearly 87 % identity. A significant difference resides in their N-terminal domain: that of eRF3b lacks a polyglycine expansion, which is present in eRF3a,

encoded by a stable GGcn polymorphism in GSPT1 exon 1. GSPT1 mRNA is expressed in a proliferation-dependent manner [6]. It is overexpressed in different types of human cancers including gastric tumors [7], breast cancer [8], colon cancer [9] and liver cancer [10]. Overexpression of eRF3a has also been shown in colon and liver cancers [9,10]. eRF3a is therefore believed to be a promising target for cancer drug therapy. In addition to its role in translation termination, eRF3a has also been implicated in ribosome recycling and in the control of mRNA degradation through its association with the cytoplasmic poly (A) binding protein, PABP [11].

In the human population, there are four known alleles of the GGC repeat of GSPT1, encoding 7, 10, 11 and 12 glycines in the N-terminal domain of eRF3a [12]. The most common GSPT1 allele is the 10-GGC (about 70 %) [8], coding by convention for 10-Gly-eRF3a. (The N-terminal domain of eRF3a has an additional Glycine, which is not coded by GGC.) To our knowledge, studies concerning the proportion of the other alleles have been conducted only in the Portuguese and Iranian populations in control and cancer patients. They highlighted the particularity of the 12-GGC-GSPT1 allele. In the Portuguese population, the 12-GGC-GSPT1 allele was only detected in breast cancer patients (allelic frequency = 0.025) [8] or

<footnote><sup>a</sup> Sorbonne Université, Institute of Biology Paris-Seine, IBPS, CNRS, UMR 8256, Biological Adaptation and Ageing, B2A, F 75005, Paris, France</footnote>

<footnote><sup>b</sup> Sorbonne Université, Institute of Biology Paris-Seine, IBPS, CNRS, UMR 8263, INSERM U1345 Development Adaptation and Ageing, Dev2A, F 75005, Paris, France</footnote>

<footnote>* Corresponding author. Sorbonne Université, Institute of Biology Paris-Seine, IBPS, CNRS UMR 8263, Development Adaptation and Ageing, Dev2A, F 75005, Paris, France.</footnote>

<footnote>E-mail address: verene.stierle@sorbonne-universite.fr (V. Stierlé).</footnote>
in gastric cancer patients (allelic frequency = 0.05) [12]. Patients with the 12-GGC-GSPT1 allele had an 11-fold increased risk of breast cancer [8] and a 20-fold increased risk of gastric cancer [12]. In the case of Iranian women with breast cancer, the allelic frequency of the 12-GGC-GSPT1 allele increased from 1 % in the control population to 2.8 % [13]. A threefold increased risk of breast cancer development was determined in that case. The 12-GGC-GSPT1 allele often appeared as heterozygous 10-12-GGC or 11-12-GGC [12,13].

Till now, the potential association between the 12-GGC-GSPT1 allele and cancer remains unclear. Readthrough experiments did not reveal differences in translation termination activity between 7-, 10-, 11- and 12-Gly-eRF3a proteins [8]. The overexpression of GSPT1 mRNA level in cancer cells, probably higher for the 12-GGC-GSPT1 allele, did not result in a higher methylation level of the CpG sites within the GGC expansion [13]. In a previous paper [14], we showed that the 12-Gly-eRF3a has a more than 20 fold lower association rate with poly(A)- bound PABP compared to the 10-Gly-eRF3a. Since the glycine repeat is located close to the PABP binding site [15], its length could have an effect on the eRF3a-PABP interaction. In the case of the 12-Gly-eRF3a, the decrease in affinity of the protein for PABP had been suggested to favor the recruitment of deadenylation complexes [14].

It is now well established that low doses of DNA damaging agents, such as the DNA topoisomerase inhibitor doxorubicin, can induce senescence in human cancer cells [16]. Senescence and cancer are linked in cell fate. Senescence is a state of theoretically complete cell cycle arrest that occurs in response to many different stresses, such as telomere dysfunction, DNA damage, oncogene activation or anticancer therapies (therapy induced senescence) [17]. Senescence is beneficial in young organisms because of its involvement in wound healing, cellular plasticity or embryonic development [18]. Therefore it was initially considered as a protective mechanism against tumorigenesis [19]. This is no longer the case, especially in older organisms in which accumulation of senescent cells in tissues induces inflammation via the senescence-associated secretory phenotype (SASP) and favors tumorigenesis [20,21]. In fact, although senescent cells no longer replicate, they are still metabolically active (SASP), increase in size with an enlarged and flattened morphology, maintain an active mammalian target of rapamycin (mTORC1) complex, have increased expression of cyclin-dependent kinase (CDK) inhibitors including p16 and p21 (an effector of the tumor suppressor protein p53), increased lysosome number and lysosomal β-galactosidase activity [18]. To go further in the understanding of the differences between the GSPT1 alleles, we compared in the present paper the effect of low doses of doxorubicin on cells overexpressing these different alleles.

## 2. Materials and methods

### 2.1. Cell culture and treatment

The colon carcinoma cell line HCT116 (ATCC CCL-247) was maintained in McCoy medium (Invitrogen) supplemented with 10 % fetal bovine serum, 1 mM sodium pyruvate, 100 mg/ml streptomycin and 100 units/ml penicillin at 37 °C under 5 % CO2 atmosphere. For eRF3a depletion, a pSUPER plasmid expressing small interfering RNAs targeting GSPT1 mRNA (sh-eRF3a) has already been described [5]. A pSUPER plasmid (sh-Ctrl) expressing small interfering RNAs (5'-ATTCTCCGAACGTGTCACG-3') which have no target in human cells was used as negative control shRNA [22]. Cell transfection was performed using a Gene pulser II electroporation system (Bio-Rad) as previously described [5]. For overexpression of GSPT1 alleles, cells were harvested 72 h after eRF3a depletion and retransfected with either a pCMV-GST plasmid

or pCMV-7-, 10-, 11- and 12-Gly-eRF3a plasmids containing the entire coding sequence of the allelic 7-GGC, 10-GGC, 11-GGC and 12-GGC-GSPT1 genes [5,8]. After transfection, cells were plated into 10-cm plates for western blotting or 6-well plates for β-galactosidase activity assay in order to reach approximately 30 % cell confluence 96 h later. At this time point, senescence was induced by addition of 100 nM doxorubicin (Sigma-Aldrich) [23].

### 2.2. Western blotting

Collected cells were lysed and western blotting was performed as previously described [22]. Antibodies directed against eRF3a and eRF1 have already been described [5]. Antibodies directed against p21 (10355-1-AP), phosphorylated ribosomal protein S6 (Ser240/244) (29220-1-AP) and α-tubulin (66031-1-Ig) were purchased from Proteintech. Antibody against ribosomal protein S6 (#2217) was purchased from Cell Signaling Technology, antibody against β-actin (A2228) from Sigma and antibody against p53 (SC-47698) from Santa Cruz Biotechnology. Protein levels have been quantified using Image Lab 6.1 (Bio-Rad) or ImageJ 1.52a Gel Analyzer.

### 2.3. Senescence-associated β-galactosidase (SA-β-gal) activity assay

HCT116 cells (control versus eRF3a-depleted) or HCT116 cells (overexpressing 10- or 12-GGC-GSPT1 alleles) were seeded in 6 well plates. After 96 h, 100 nM doxorubicin was added and the SA-β-gal activity assay was performed six days later. The OZ Biosciences Cellular Senescence kit was used according to the instructions, while adjusting the pH of the staining solution to 6.

### 2.4. Statistical analysis

Statistical analysis was performed using a two-tailed Student's t-test to examine the differences between two groups. Data are expressed as means ± SD. A value of *p* ≤ 0.05 was considered statistically significant (*p* ≤ 0.05, **p* ≤ 0.01, ***p* ≤ 0.005 and ****p* ≤ 0.001). All graphs represent the mean of at least three independent experiments for western blotting and two independent experiments for SA-β-gal activity assay.

## 3. Results

### 3.1. 12-Gly-eRF3a overexpression reduces the onset of senescence

72 h after eRF3a depletion followed by 96 h overexpression of pCMV-GST or pCMV-7-, 10-, 11- or 12-Gly-eRF3a plasmids, HCT116 cells were treated with doxorubicin on day 0 (see 2-1). Doxorubicin has been widely used to induce senescence in HCT116 cells [23,24]. Protein expression was monitored for three days and representative western blots are shown in Fig. 1a and b. Fig. 1a shows the results obtained after overexpression of pCMV-GST, pCMV-10-Gly-eRF3a or pCMV-12-Gly-eRF3a plasmids. In Fig. 1b, 7-, 10-, 11- or 12-Gly-eRF3a are overexpressed. In Fig. 1a, the first lane (sh-eRF3a) corresponds to cells after 72 h of eRF3a depletion and before gene overexpression. Silencing of eRF3a in this first lane validates the efficiency of the depletion. The CDK inhibitor p21, known to be a major marker of senescence, has increased expression as early as 1 day after doxorubicin addition (Fig. 1a and b). P21 is encoded by the CDKN1a gene, the expression of which is also increased after doxorubicin addition (Supplementary Fig. S1 Supplemental Fig.S1). Note that eRF3a depletion alone does not lead to an increase in p21 expression (day 0). The increase in p21 expression after doxorubicin treatment is lower after overexpression of 12-Gly-eRF3a than after overexpression of 7-, 10- or 11-Gly-eRF3a (Fig. 1a and b). If we look
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Overexpression of 12-Gly-eRF3a inhibits p21 expression in HCT116 cells treated with 100 nM doxorubicin for 3 days. (a) Representative Western blot analysis for eRF3a and p21 in HCT116 cells depleted of eRF3a and then transfected with a pCMV-GST plasmid or pCMV-10-Gly-eRF3a or 12-Gly-eRF3a plasmids. α-tubulin was used as a loading control. (b) Representative Western blot analysis for p21, p53 and eRF1 in HCT116 cells depleted of eRF3a and then transfected with pCMV-7-Gly-, 10-Gly-, 11-Gly- or 12-Gly-eRF3a plasmids. α-tubulin or β-actin were used as loading controls. (c) Quantification data of p21 expression level (cells overexpressing 10-Gly-eRF3a versus cells overexpressing 12-Gly-eRF3a) are mean±SD from 3 independent biological replicates (*P* value < 0.05). (d) Representative Western blot analysis for phospho S6 ribosomal protein (Ser240/244) and S6 ribosomal protein in HCT116 cells depleted of eRF3a and then transfected with 10- or 12-Gly-eRF3a plasmids. °: sample problem.</figcaption></figure>

at the *CDKN1a* mRNA level after doxorubicin treatment, its increase is slower after overexpression of 12-Gly-eRF3a compared to that of 10-Gly-eRF3a (Supplemental Fig. S1). An increase in p53 expression after doxorubicin treatment is also observed and, as in the case of p21, this increase is attenuated in the case of 12-Gly-eRF3a over-expression (Fig. 1b).

We further analyzed p21 expression in the case of 10- and 12-Gly-eRF3a overexpression. Quantification data (Fig. 1c), resulting from three independent experiments, allow to demonstrate that, after doxorubicin addition, the differences between the two populations are significant (*P* values = 0.037 on day 1, 0.034 on day 2 and 0.012 on day 3).

There is apparently no change in the level of eRF3a (Fig. 1a) or eRF1 (Fig. 1b), regardless of the overexpressed plasmid. As evidenced in the case of GST overexpression (Fig. 1a), endogenous eRF3a is translated de novo along the days. This results from the 96 h plasmid overexpression following eRF3a depletion. However, we assume the effect of 10- and 12-Gly-eRF3a overexpression is predominant over de novo eRF3a synthesis because 10- and 12-Gly-eRF3a overexpression do not lead to similar results (Fig. 1a and b). Accordingly, overexpression of minor *GSPT1* alleles will only result in enrichment for that allele.

12-Gly-eRF3a overexpression does not lead to difference in the expression of the ribosomal protein S6, an effector of mTORC1 (Fig. 1d). In particular, its phosphorylation level on day 0 is the same for the 10- and 12-GGC alleles, indicating that the 12-Gly-eRF3a protein has no effect on mTORC1 activity. After the addition of

doxorubicin, a small decrease in S6 phosphorylation over the days is observed and has been described previously [25].

Lysosomal β-galactosidase activity was analyzed after 6 days of doxorubicin treatment (Fig. 2). Doxorubicin-treated cells are flat and significantly enlarged (Fig. 2b and c) compared to untreated cells (Fig. 2a). They have acquired a senescent morphology, which was observed as early as one to two days after doxorubicin treatment. Proliferation was largely arrested after 1 day of doxorubicin treatment, consistent with the expected cell growth arrest after the onset of senescence. Quantification data (Fig. 2d), obtained from two independent experiments, allow evaluation of the percentage of SA-β-gal-positive cells (blue cells): approximately 70 % in 10-Gly-eRF3a cells and 60 % in 12-Gly-eRF3a cells (*P* = 0.02). This suggests a significant deficiency in the induction of senescence for 12-Gly-eRF3a cells.

## 3.2. eRF3a depletion inhibits the onset of senescence

What would have been observed if doxorubicin had been added at the end of the first electroporation, i.e. after eRF3a depletion? 72 h after electroporation of sh-Ctrl or sh-eRF3a plasmids, doxorubicin was added on day 0 and protein expression was monitored for three days. Representative western blots are shown in Fig. 3a, b and c eRF3a depletion is very efficient (Fig. 3a). It is accompanied by a reduction in the intracellular level of eRF1, albeit moderate compared to that of eRF3a itself. This is an expected effect, due to the lower stability of eRF1 after eRF3a depletion [5]. A
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Senescence-associated β-galactosidase (SA-β-gal) staining. Representative images of (a) control HCT116 cells. (b) HCT116 cells overexpressing 10-Gly-eRF3a after 6 days of doxorubicin treatment. (c) HCT116 cells overexpressing 12-Gly-eRF3a after 6 days of doxorubicin treatment. (d) Quantification data for percentage of SA-β-gal-positive cells for 10-Gly-eRF3a and 12-Gly-eRF3a HCT116 cells are mean±SD from 2 independent biological replicates (*P* value < 0.05). Over 1500 cells were analyzed.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** eRF3a depletion inhibits p21 expression in HCT116 cells treated with 100 nM doxorubicin for 3 days. (a) Representative Western blot analysis for eRF3a and eRF1 in control and eRF3a-depleted cells. β-actin was used as a loading control. (b) Representative Western blot analysis for p21 in control and eRF3a-depleted cells. β-actin was used as a loading control. (c) Representative Western blot analysis for p53, phospho S6 (Ser240/244) ribosomal protein and S6 ribosomal protein in control and eRF3a-depleted cells. β-actin was used as a loading control. (d) Quantification data of p21 expression level (control cells versus eRF3a-depleted cells) are mean±SD from 4 independent biological replicates (**P* value < 0.01, ***P* value < 0.005).</figcaption></figure>

representative p21 Western blot is shown in Fig. 3b. P21 expression was increased as early as 1 day after doxorubicin addition and this increase was smaller in erf3a-depleted cells. (At the level of the expression of the CDKN1A gene (Supplemental Fig. S2) similar results were obtained). Quantification data resulting from four independent experiments (Fig. 3d) show a clear difference between p21 expression in control and eRF3a-depleted cells. After addition

of doxorubicin, differences between the two populations are always strongly significant (*P* values = 0.005 on day 1, 0.002 on day 2 and 0.004 on day 3).

The increase in p21 expression after doxorubicin addition is accompanied by a similar increase in p53 expression, which is significantly lower in the case eRF3a depletion (Fig. 3c). In the absence of doxorubicin, i.e. on day 0, the level of the ribosomal
protein S6 phosphorylation is lower in eRF3a-depleted cells (Fig. 3c). This is due to the inhibition of mTORC1, which has already been described to occur after eRF3a depletion [26]. The addition of doxorubicin does not cause a major change in the phosphorylation level of the ribosomal protein S6 on day 1, consistent with the fact that an active mTORC1 complex is maintained after the onset of senescence [18]. In the case of control cells, the small decrease in S6 phosphorylation over the days has already been discussed (Fig. 1d). In the case of eRF3a-depleted cells, the possible increase in S6 phosphorylation over the days (Fig. 3c) could be due to a slight de novo synthesis of eRF3a six days after its depletion, not yet detectable in Western blot and leading to increased mTORC1 activity.

Lysosomal β-galactosidase activity was analyzed after 6 days of doxorubicin treatment (Fig. 4). Control and eRF3a-depleted cells have acquired a senescent morphology, which can be observed as early as one or two days after doxorubicin treatment. The lower density of eRF3a-depleted cells (Fig. 4b), compared to that of control cells (Fig. 4a) results from the inhibition of the mTORC1 pathway, as expected after eRF3a depletion [26]. This affected cell proliferation even before the addition of doxorubicin. Proliferation was largely arrested after one day of doxorubicin treatment. Quantification data (Fig. 4c), resulting from two independent experiments, allow to evaluate the percentage of SA-β-gal-positive blue cells: approximately 85 % in control and only 60 % in eRF3a-depleted HCT116 cells (P = 0.000017). This suggests a very significant deficiency in the induction of senescence in eRF3a-depleted HCT116 cells.

4. Discussion

Are these results consistent with the possible association of the 12-GGC-GSPT1 allele with cancer? After the addition of doxorubicin, p21 expression is increased regardless of the overexpressed n-Gly-eRF3a and whether eRF3a is depleted or not. However, this increase is reduced by overexpressing 12-Gly-eRF3a and by depleting eRF3a. The same comment was made for the expression of p53. Similarly, lysosomal β-galactosidase activity was reduced when HCT116 cells overexpressing 12-Gly-eRF3a or depleted of

eRF3a were induced into senescence. We assume that the differences between control and eRF3a-depleted cells are much more significant than they were between 10- and 12-Gly-eRF3a cells because doxorubicin is added only after 72 h without the additional 96 h of plasmid overexpression, which has been shown to result in 12-Gly-eRF3a enrichment only.

Since the p16 gene is silenced in HCT 116 cells [27], the molecular pathway leading to senescence and increased p21 expression should be p53-dependent. Indeed, p21 is an effector of p53 in the ATM/ATR pathway, which is activated by the DNA lesions induced by doxorubicin treatment [28–30]. All of these results suggest that both 12-Gly-eRF3a overexpression and eRF3a depletion prevent the onset of senescence in HCT116 cells but, certainly the two cases are very different.

Depletion of eRF3a inhibits proliferation of HCT116 cells in a very efficient manner [9,26]. It inhibits mTORC1 signaling [26], leading to cell cycle arrest in the G1 phase, along with a decrease in cyclin D1 [9,26] and cyclin A [26] levels. eRF3a is strongly correlated with colon and cancer tumorigenesis [8] and its depletion is beneficial at this regard. Less active mTORC1 (less phosphorylated ribosomal protein S6 (Fig. 3c),) is expected to inhibit senescence onset, which is consistent with the reduced expression of p21 and lysosomal β-galactosidase after eRF3a depletion (Figs. 3 and 4). Since the benefit of eRF3a depletion is associated with less senescence, this is a case where senescence is detrimental and would likely lead to cancer progression through the SASP. Since depletion of eRF3a is also accompanied by a reduction in eRF1 intracellular level [5], a decrease in the translation termination activity could also occur.

In contrast, the level of eRF3a is maintained after overexpression of pCMV-GST, pCMV-10-Gly-eRF3a or pCMV-12-Gly-eRF3a plasmids (Fig. 1a). The level of eRF1, too, is maintained (Fig. 1b). The mTORC1 activity (phosphorylation of the ribosomal protein S6) is maintained at a similar level for 10- and 12-Gly-eRF3a (Fig. 1d). The singularity of the results obtained in the case of the 12-Gly-eRF3a overexpression is most likely related to its decreased affinity for PABP [14]. The length of the glycine repeat, located near the PABP binding site [15], could modulate the eRF3a-PABP interaction. Just one additional glycine (12-GGC) would make the eRF3a-PABP

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Senescence-associated β-galactosidase (SA-β-gal) staining. Representative images of (a) HCT116 cells after 6 days of doxorubicin treatment. (b) eRF3a-depleted HCT116 cells after 6 days of doxorubicin treatment. (c) Quantification data for percentage of SA-β-gal-positive cells for control and eRF3a-depleted HCT116 cells are mean±SD from 2 independent biological replicates (****P value < 0,0001). More than 1700 cells were analyzed.</figcaption></figure>
interaction less favorable. With less eRF3a associated with PABP and therefore available to be recruited by eRF1 to boost its termination activity, a defect in termination efficiency could occur. The reduced affinity of the 12-Gly-eRF3a for PABP could also favor the recruitment of deadenylation complexes and lead to the degradation of specific mRNAs [14]. The present results lead us to the assumption that these specific mRNAs should be involved in the senescence pathway. The association between 12-Gly-eRF3a, which corresponds to an increased risk of cancer development, and reduced onset of senescence would suggest that, in the case of 12-Gly-eRF3a overexpression, attempting to induce senescence would be beneficial.

In conclusion, both eRF3a depletion and 12-Gly-eRF3a over-expression are likely to result in a defect in translation termination efficiency that impedes the onset of senescence. Degradation of senescence-specific mRNAs has also been evoked in the case of 12-Gly-eRF3a overexpression. Although it would be desirable to verify these results in additional cancer cell lines overexpressing eRF3a, a link between translation termination and senescence induction has been established. After doxorubicin-induced senescence, the 12-GGC- GSPT1 allele is once more different from the other GSPT1 alleles. It is likely that the particular behavior of the 12-GGC- GSPT1 allele will only be deciphered from a set of clues.

### CRediT authorship contribution statement

**Béatrice Jolles:** Writing – original draft, Investigation, Conceptualization. **Vérène Stierlé:** Writing – original draft, Investigation, Conceptualization.

### Declaration of interest

All authors declare no conflicts of interest.

### Funding

This work was supported by institutional funding from Sorbonne Université (SU) and from Centre National de la Recherche Scientifique (CNRS) to the UMR8256/B2A

### Declaration of interest

None.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2025.02.009.

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