VRK1 functional insufficiency due to alterations in protein stability or kinase activity of human VRK1 pathogenic variants implicated in neuromotor syndromes

Very rare polymorphisms in the human VRK1 (vaccinia-related kinase 1) gene have been identified in complex neuromotor phenotypes associated to spinal muscular atrophy (SMA), pontocerebellar hypoplasia (PCH), microcephaly, amyotrophic lateral sclerosis (ALS) and distal motor neuron dysfunctions. The mechanisms by which these VRK1 variant proteins contribute to the pathogenesis of these neurological syndromes are unknown. The syndromes are manifested when both of these rare VRK1 polymorphic alleles are implicated, either in homozygosis or compound heterozygosis. In this report, to identify the common underlying pathogenic mechanism of VRK1 polymorphisms, we have studied all human VRK1 variants identified in these neurological phenotypes from a biochemical point of view by molecular modeling, protein stability and kinase activity assays. Molecular modelling predicted that VRK1 variant proteins are either unstable or have an altered kinase activity. The stability and kinase activity of VRK1 pathogenic variants detected two groups. One composed by variants with a reduced protein stability: R133C, R358X, L195V, G135R and R321C. The other group includes VRK1variants with a reduced kinase activity tested on several substrates: histones H3 and H2AX, p53, c-Jun, coilin and 53BP1, a DNA repair protein. VRK1 variants with reduced kinase activity are H119R, R133C, G135R, V236M, R321C and R358X. The common underlying effect of VRK1 pathogenic variants with reduced protein stability or kinase activity is a functional insufficiency of VRK1 in patients with neuromotor developmental syndromes. The G135 variant cause a defective formation of 53BP1 foci in response to DNA damage, and loss Cajal bodies assembled on coilin.

Structural alterations and modelling of the human VRK1 pathogenic variants in neuromotor syndromes. To detect the possible structural effect on the stability of the VRK1 human pathogenic variants, these variants were modelled using the known three-dimensional structures of VRK1 in the Protein Data Bank using the X-crystal (2RSV) 29 and nmr (2LAV) 30 . These structures lack the C-terminal low complexity region that has alternative foldings 30 . The location of the different pathogenic variant aminoacids on the structure indicate that topologically they are in very different regions of the protein (Supplementary Fig. S1). To check whether the identified pathogenic variants of VRK1 have an effect on the structural stability of the kinase, we analyzed them using the empirical FoldX forcefield method 31 . In this method, the stability of a protein is defined by the changes in free energy (in Kcal/mol), the lower the value, the more stable is the protein. In general, a variant that brings free energy changes (ΔΔG > 0 kcal/mol) will destabilize the structure (Supplementary Table 2). The differences in free energy caused by each pathogenic variant were similar in all the structures available ( Table 3). The analysis of structural changes predicts that pathogenic variants G135R, R321C and L195V have a destabilizing effect (Table 3). These variant aminoacids alter the interaction network of these residues. In the case of the R321C variant, this R321 interacts with a neighboring helix through D163 and its folding is disrupted by its change to cysteine (Fig. 1). There are also disruptions with the G135R variant ( Supplementary Fig. S2 www.nature.com/scientificreports www.nature.com/scientificreports/ a higher energy difference, and that has novel interactions with N186, Y187 and L185. Residues R133 and G135 are located in the interaction region with the adenine moiety of ATP 30 . The R89Q ( Supplementary Fig. S3), H119R ( Supplementary Fig. S4), and R133C ( Supplementary Fig. S5) variants have no apparent effect on the stability of the kinase domain, but have a flexible organization with alternatives rotamers, which may condition their interaction or accessibility to regulatory proteins. The V236M variant does not show any detectable change in interactions within the protein, but may alter its interactions with other proteins or modulators ( Supplementary  Fig. S6). The R358X variant, is a truncated protein that lacks its C-terminal region and the nuclear localization signal, mislocalizing to the cytosol 23 . However, the folding of the C-terminal of VRK1 regulates the kinase activity 30 and thus this R358X variant has only a residual kinase activity 23 , which was confirmed in autophosphorylation and transphosphorylation assays of several substrates. The flexible VRK1 C-terminal region, which was not included in the X-ray structure, regulates the kinase activity 30 . Therefore, effects of some variant proteins may be the consequence of interactions with this low-complexity and flexible regulatory region.
Protein stability of human VRK1 pathogenic variants identified in patients. The stability of the different VRK1 pathogenic variants was determined by transfection of VRK1 variant constructs in HEK-293T cells and after addition of cycloheximide to block translation, the level of the pathogenic variant proteins was determined at different time points (Fig. 2). Two groups of variants were identified based on the stability of the protein. The stability was statistically analyzed by determining the fit by polynomic regression to detect the correlation between two variables, protein level and time. There VRK1 variants, V236M, R89Q and H119R form a group whose stability is similar to the wild type VRK1 (Fig. 2a,b), therefore the protein level does not correlate with the time. Another group includes VRK1 variants R133C, R358X, L195V, G135R and R321C, which are much more unstable and have a significantly shorter half-life (Fig. 2a,b). In these unstable variants, there is a very good correlation between the two variables of each variant, protein level as a function of time (individual data points are shown in Supplementary Fig. S7). For stable variant proteins, there is no correlation because their protein level remains constant throughout time.    Table S1).
ences in the kinase activity of VRK1 pathogenic variants, two types of assays were performed. One is autophosphorylation of the kinase 32,33 , and the other one is the phosphorylation of known specific residues in proteins that participate in different functions regulated by VRK1. All proteins in the assay, kinase and substrates, were equimolar at the low micromolar range (indicated in the methods section), and the rate limiting step of the assay is the concentration of ATP (5 µM), which is fifteen times below the VRK1 Km for ATP 32 . These permitted to perform the assays in the linear range of the activity. In the context of chromatin, the phosphorylation of two histones were studied. Histone H3 is a component of normal nucleosomes, which are remodeled based on the function of chromatin. The phosphorylation of Histone H3 in Thr3 was determined with a specific monoclonal antibody 3 . Most VRK1 variants have lost their ability to phosphorylate H3, with the exceptions or R89Q and L195V that have an increased activity (Fig. 3a).
Histone H2AX is associated to early stages in DNA damage responses 2 . The phosphorylation was determined by in vitro kinase with labeled ATP. The pattern of phosphorylation also detected a lost activity with all variants, except for R89Q and L195V that are more active (Fig. 3b). However, this reduction in activity was less noticeable regarding the autophosphorylation of VRK1, but have the same pattern.
A second group of proteins phosphorylated by VRK1 is formed by transcription factors that are implicated in DNA damage or cellular stress responses. In this context, the effect of VRK1 pathogenic variants on p53 and c-Jun was determined. VRK1 phosphorylates p53 in Thr18 5,6,34 . The phosphorylation of p53 by VRK1 pathogenic variants was determined in a kinase assay using as substrate the transactivation domain of p53 fused to GST (1-84) 5 . The phosphorylation was detected with a p53T18ph phospho specific antibody 6,35 . Only the R89Q and L195V VRK1 pathogenic variants were able to specifically phosphorylate p53, also at higher levels than the wild-type (Fig. 4a). Next, the effect of the VRK1 variants was tested on c-Jun, a transcription factor involved in cellular stress responses 36 . The phosphorylation of c-Jun regulatory domain was tested with a GST-C-Jun (1-233) fusion protein as substrate in a radioactive kinase assay. Only R89Q and L195V pathogenic variants were able to phosphorylate c-Jun regulatory region at higher levels (Fig. 4b).
The third group of substrates tested is formed by proteins associated to specific processes in which VRK1 participates, such DNA damage responses and Cajal bodies assembly. VRK1 regulates DNA damage response that is repaired by the Non-homologous end-joining route (NHEJ), where 53BP1 is a key protein that is known to be phosphorylated by VRK1 12,37 . As substrate it was used a fusion protein GST-53BP1 protein in the radioactive kinase assay. Only R89Q and L195V variants were able to phosphorylate 53BP1, also at higher levels than the wild type (Fig. 5a). www.nature.com/scientificreports www.nature.com/scientificreports/ Coilin is a scaffold protein that is necessary for the organization of Cajal bodies (CB), which are dynamically assembled and disassembled during mitosis 38 , and regulated by VRK1 19 . Coilin form complexes with ataxin-1/ SCA1 39 and SMN 40 , two proteins related to some symptoms of the clinical phenotypes. Coilin is known to be phosphorylated in several residues by VRK1 19,23 . Therefore, its phosphorylation by VRK1 was also determined (Fig. 5b). In this case, also R89Q and L195V were able to phosphorylate coilin more than double than the kinase wild type.
The phosphorylation effect depends on VRK1 pathogenic variants and not on the substrates. The differences in kinase activity might be a property either of the VRK1 pathogenic variants or of the specific phosphorylated substrate. To address this issue we reanalyzed the data from the previous autophosphorylation and trans-phosphorylation assays (Figs 3, 4 and 5). The wild-type VRK1 protein is autophosphorylated in multiple residues 5 . Therefore, the use of the radioactive kinase assay also permitted to determine the effect of VRK1 pathogenic variants on its autophosphorylation activity. This autophosphorylation assay was performed in the previous experiments for H2AX, c-Jun, 53BP1 and coilin. The results regarding VRK1 autophosphorylation and its quantification are shown in Fig. 6a. Both, the R89Q and L195V pathogenic variants are more active, and the rest of the VRK1 variants have a significant reduction in their kinase activity. To rule out that differences may be due to the substrates used, we reanalyzed the transphosphorylation data independently of the substrate based on the combined data from the six different substrates (Fig. 6b). The VRK1 pathogenic variants have a similar activity in both autophosphorylation and transphosphorylation reactions, suggesting that the alteration in VRK1 activity does not affect its specifity regarding the substrates used.

Cajal bodies are reduced by the G135R mutation. Coilin is the scaffold protein of Cajal bodies (CB)
and is required for their assembly 41,42 . Coilin forms stable complexes with three neuropathological proteins: SMN, the spinal muscular atrophy protein 43 , ataxin-1 (SCA1), associated to spinocerebellar ataxia 39 , and VCP that is associated to some rare forms of amyotrophic lateral sclerosis 44,45 . Coilin is substrate of VRK1 23 that regulates its stability and assembly in CB 19 . It is known that inactivation of the VRK1 catalytic site alters the formation of Cajal bodies 19 . Therefore, we determined whether the G135V variant could interfere with formation of Cajal bodies assembled on coilin in cell lines expressing this variant in the murine VRK1 gene. This variant was introduced in the murine VRK1 gene (mVRK1), which was expressed by a lentiviral construct used to make stable cell lines. The human endogenous VRK1 was depleted by two different human siVRK1, and the effect on Cajal bodies of the murine mVRK1-G135R variant, or the kinase-dead mVRK1-K179E as control 19 , were determined. Both the mVRK1-G135R variant and the kinase-dead mVRK1-K179E mutant resulted in defective formation of Cajal bodies (Fig. 7). www.nature.com/scientificreports www.nature.com/scientificreports/ Formation of 53BP1 foci induced by DNA damage are impaired by the G135R variant. To identify a cellular effect of the human mutations, we selected the G135R variant detected in distal muscular atrophy 46 . VRK1 is a protein involved in different aspects of DNA damage responses 2,11,12 . Therefore it was tested the effect of the response to DNA damage induced by doxorubicin by the mVRK1-G135R and the wild-type and the kinase-dead mVRK1-K179E, as controls, following depletion of the endogenous human VRK1, and treatment with doxorubicin. As marker of the DNA damage response, the formation 53BP1 foci was determined 47 . The G135R pathogenic variant and the kinase-dead K179E protein caused a loss of 53BP1 foci in response to DNA damage (Fig. 8). The statistical study of the variables, protein level and time is a covariance analysis by least-square non-linear regression to detect the correlation between the change in protein as a function of time. The assays were performed in triplicate and are represented individually as dots in the graph. Wild type VRK1 was the reference value. *p < 0.05, **p < 0.005, ***p < 0.0005. Western blots with the proteins used in kinase assays are shown in Supplementary Fig. S7.

Discussion
The implication of VRK1 in the pathogenesis of severe neuromotor developmental processes is likely to be due to alterations of cellular processes it regulates. The underlying common functional consequences of VRK1 mutations in neurodevelopmental syndrome is an impairment of neuronal functions regulated by VRK1. The common observation in either homozygous or compound heterozygous alleles indicates that the combination of two VRK1 rare pathogenic alleles leads to a functional insufficiency of the processes regulated by VRK1. Among these there are impairments in cell cycle progression 3,9,13,14,19,48 , in responses to DNA damage 11,12,37 , altered nuclear envelope assembly 20 , and also impaired neuronal differentiation by altered regulation of Sox2 48 . All these processes are critical during early neurodevelopment. Therefore, impairment of cell cycle progression can lead to microcephaly or hypoplasia 21 , which is observed in patients with VRK1 mutations 22,24,49 . The known functions of VRK1 in the context of cell cycle progression and proliferation 3,9,13,14,19,48 are consistent with this role 49 . In a murine VRK1 www.nature.com/scientificreports www.nature.com/scientificreports/ gene-trap model with a residual level of VRK1 protein 50 there is a reduction of brain mass and a motor impairment 51 , consistent with the situation in human compound heterozygous alleles.
Functionally, the most characterized pathogenic variant is R358X, which lacks its C-terminus. The C-terminal region of VRK1 is necessary for the kinase activity 23,30 because its correct folding is necessary for kinase activation 30 . Therefore truncating variants in the regulatory C-terminal region area likely to lack kinase activity, as is the case for the R358X variant that truncates the protein 23 . Furthermore, the mRNA transcript coding for the variant VRK1 (R358X) is very unstable, and its level is not detectable in cells 22 . The regulatory role of this C-terminus is supported by its regulation by autophosphorylation in Thr355 and Thr390 32 , which is inducible by DNA damage 12 , Thr355 can also be phosphorylated by PKCδ linking its regulation to other signaling pathways that induce cell death 52 .
In other cases, the variant residues directly or indirectly affects the catalytic site. The R133 residue is important for catalysis by interacting with the adenosine moiety of ATP 30 , thus this R133C variant is likely to be catalytically inactive 26 . Gly135 interacts with ATP and is required for the kinase activity 30 , and the variant G135R has lost most of its activity, and is also unstable. These two residues, R133 and G135 undergo chemical shift perturbations and affect the DRF motif 53 . It is known that the loss of VRK1 kinase activity by itself makes this protein unstable 13 . The L195 is required for the interaction of the DYG motif with ATP 30 , thus the variant L195V might facilitate the activity of this variant due to the smaller size of valine. Two of the variants, R89Q and L195V have a higher The in vitro kinase assay was performed with radiolabeled ATP. The graph shows the ratio of phosphorylated and non-phosphorylated substrates. The statistical analysis is two-sided t-test with Welch' correction. The assays were performed in triplicate and are represented individually as dots in the graph. Wild type VRK1 was the reference value. *p < 0.05, **p < 0.005, ***p < 0.0005. activity in both autophosphorylation and phosphorylation of specific substrates such as H3 (Fig. 3a) and p53 (Fig. 3b). The L195V variant has a reduced half-life that might mimic a deficiency of the active kinase.
The contribution of VRK1 to neuropathogenesis can be a consequence of a defective organization of Cajal bodies (CB) by alteration of the regulation of coilin by VRK1, which is required for CB assembly and disassembly 19 . Coilin is associated to proteins such as SMN 43,54 , scaRNPs 54 or ataxin-1 39 that are implicated in severe neurological phenotypes such as muscular atrophy 55,56 and ataxias 39 . Ataxin-1 (SCA1) is a phosphorylated protein 57,58 , and in this context, VRK1 mutations might be a pathogenic alternative to expansion of poly-Q expansion in ataxin-1 59 . Coilin also interacts with VCP, a protein associated to amyotrophic lateral sclerosis 44,60 . Therefore, an alteration in the organization and regulation of these coilin and Cajal body complexes by VRK1 variants is likely to have important neuropathological consequences, particularly since VRK1 regulates the proteasomal degradation of coilin 19 , which form complexes with several proteins associated to several neuromotor syndromes. Moreover, Cajal bodies are RNP-complexes that are altered in spinal muscular atrophy (SMA) 61 , and RNA binding proteins also play a not well known role in amyotrophic lateral sclerosis (ALS) 62 . Therefore, this function might be defective in patients with VRK1 pathogenic variants. In this context, the defective formation of Cajal bodies by the G135R variant (Fig. 7) is consistent with this interpretation. VRK1 might also contribute to neurological syndromes as a consequence of its roles on neural cell motility. A reduction in VRK1 protein levels, independent of its activity, has been associated to a downregulation of amyloid-beta precursor protein (APP) that leads to a reduction in neuronal migration, which can be rescued by overexpression of APP 49 . Moreover, an effect of VRK1 on cellular motility has also been identified as contributing to breast cancer metastasis by facilitating mesenchymal to epithelial transition 63 . Therefore, defects in neuronal migration are likely to have important consequences in size and organization of the nervous system, leading to hypoplasia or microcephaly, and consequently cause severe neuromotor and neurodevelopmental delay syndromes.
An additional pathogenic role of VRK1 variants is likely to be a consequence of its roles in chromatin reorganization 1,16 . Alterations in histone posttranslational epigenetic modifications are involved in neurodegerative disorders by not yet known mechanisms 64,65 . Another alternative mechanism might be a consequence of the role that VRK1 plays in the cellular response to DNA damage 11,12,16,37,47 . It is well known that mutations in genes coding for . The points represent six independent kinase assays. The statistical analysis is two-sided t-test with Welch' correction and are represented individually as dots in the graph. Wild type VRK1 was the reference value. *p < 0.05, **p < 0.005, ***p < 0.0005. (b) Transphosphorylation of the VRK1 substrates by determination of the ratio phosphorylated /non-phosphorylated substrate, independently of the type of substrates. Statistics on normalized combined data from assays using H2AX, c-Jun, 53BP1 and coilin as phosphorylation substrates. The points represent six independent kinase assays. The statistical analysis is two-sided t-test with Welch' correction and are represented individually as dots in the graph. Wild type VRK1 was the reference value. *p < 0.05, **p < 0.005, ***p < 0.0005. www.nature.com/scientificreports www.nature.com/scientificreports/ proteins that participate in DNA-damage responses cause neurodegerative syndromes 66,67 , such as Nijmegen 68 , Cockayne syndromes 69 and many others 67 , including amyotrophic lateral sclerosis 70 . Therefore, it is likely that alterations in DDR by a VRK1 deficiency can lead to neurodevelopmental phenotypes. There is an extensive neuropathology associated to defects in genes coding for proteins that participate in different aspects of DNA repair processes. Among them is NBS1 71,72 that is regulated by p53 72 . P53 6,73 and NBS1 are both phosphorylation targets of VRK1 11 . Hereditary ATM (ataxia-telangiectasia mutated) mutations are also associated to neurological phenotypes 74,75 , and in this context VRK1 is an upstream regulator of ATM-mediated responses to DNA damage 2,11,12 . In this context the defective formation of 53BP1 foci in response to DNA damage by the G135R variant (Fig. 8) is consistent with this interpretation.

Scientific RepoRtS
Furthermore, an additional factor that can explain the loss of stability is a consequence of the lack of data on the protein interaction of VRK1 with its substrates, as well as its recognition by regulatory proteins and active degradation mechanisms. VRK1 can be degraded in the lysosome 35 , but this requires VRK1 nuclear export and lysosomal targeting. The variants might alter interactions with other regulatory or interacting proteins, but there is no available data on any of these VRK1 protein complexes. Another factor might be due to VRK1 effects that are independent of the kinase activity. Kinase-dead VRK1 has been shown to regulate neuronal migration in murine embryo development 49 .
The heterogeneity or variability among the clinical phenotypes associated to VRK1 pathogenic variants are likely to be conditioned by additional genetic alterations present in the affected families, since all cases occurred either in consanguineous families or within a specific ethnic subgroup, which might have a higher frequency incidence of pathogenic variant alleles. In addition, genetic variations or alterations, such as copy number variations (CNVs) or pathogenic variants in other genes, which differ among patients, can significantly contribute to the www.nature.com/scientificreports www.nature.com/scientificreports/ heterogeneity of the clinical phenotypes identified. A genetic heterogeneity has already been reported in other complex neurological diseases, such as epilepsy 76,77 and neurodevelopmental delays [78][79][80] .
In this report we conclude that the common underlying effect of human VRK1 pathogenic variants identified in patients with neuromotor and neurodevelopmental syndromes is a functional insufficiency of VRK1 resulting from an altered protein stability or reduced kinase activity that impair cellular functions regulated by VRK1.

Materials and Methods
Molecular modelling of missense VRK1 mutations. Modelling of the human VRK1 pathogenic variant proteins was performed using as reference the published structures 2RSV 29 and 2LAV 30,53 available from the Protein Data Bank. FoldX program 81 (http://foldxsuite.crg.eu/) was used to predict the effect of missense mutations in the structural stability of the VRK kinase (PDB identifier 2LAV). This structure was selected, as its partial C-terminal domain is correctly located within the structure as compared to 2RSV. For each protein variant we calculated ten iterations to ensure that the algorithm reaches convergence. The reported accuracy of FoldX is 0.46 kcal/mol (i.e., the SD of the difference between ΔΔGs calculated by FoldX and the experimental values). We can bin the ΔΔG values into seven categories of stability ( Supplementary Fig. S1). Regarding the van der Waals clashes, this indicates whether the mutation impairs drastically the structure of the protein. We studied the following mutations, and represented the relative TE differences using an R custom script to generate the image files. Plasmids and mutagenesis. Human VRK1 was expressed from mammalian expression vector, pCEFL-HA-VRK1 82 , and bacterial expression pGEX-4T-VRK1 3,5,82 . These plasmids were used as targets to generate the pathogenic variants identified in patients. The primers used to generate the VRK1 mutations are listed in Supplementary Table S2. Mutations in VRK1 were performed using the Quickchange site-directed www.nature.com/scientificreports www.nature.com/scientificreports/ mutagenesis kit (Stratagene-Thermo-Fisher). Sanger sequencing was used to confirm all variants generated using the following primers VRK1-forward (5′-CCTCGTGTAAAAGCAGCTCAAGCTG-3′) and VRK1-reverse (5′-GGACTCTCTTTCTGGTTCTTGAACGG-3′). The kinase-dead VRK1, K179E mutation, has already been reported 13 . VRK1 wild type and variants (R89Q, H119R, R133C, G135R, L195V, V236M, R321C, R358X) were expressed from constructs pGEX4T-GST-VRK1 plasmid expressed in E. coli BL21 strain competent cells. The following plasmids were used to express the substrates: plasmid pGEX4T-GST-c-Jun (1-233) 7 , pGST4T-53BP1 (1-346) was a gift of J. Chen 12,83 ; GST-p53 (1-85) was from D. Meek   5,34,84 , and pGEX4T-GST-Coilin (160-214) [S184A] 19 .
All plasmids were expressed in BL21 E. coli to express and purify the fusion protein used as substrate in kinase assays. The purity of purified kinases and substrate proteins are shown in Supplementary Fig. S8. Cell lines, transfection and cell lysate. HEK-293T (ATCC-CRL-11268) and HeLa (ATCC-CCL2) validated cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, and penicillin (50 U/ml)/streptomycin(50 µg/µl) (GIBCO-life technologies) 19,48 . Cells were transfected using Lipofectin as previously described 11,19,48 . The plasmid expressing the pathogenic variants were used in an amount chosen to express a similar initial level of protein, which was completed with empty vector plasmid to reach the same final concentration of DNA in the transfection.
Cell extract were prepared by using a mild lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100 and 1 mM EDTA) supplemented with protease inhibitors (1 mM PMSF, 10 µg/mL aprotinin and 10 µg/mL leupeptin) and phosphatase inhibitors (1 mM sodium orthovanadate, 1 mM NaF) and incubating for 20 minutes. Cell lysates were centrifuged for 20 minutes at 16.100 g to remove debris and supernatants were used for the study.
Electrophoresis, antibodies and immunoblots. The size-dependent separation of proteins was performed by vertical electrophoresis in SDS-PAGE gels under denaturing conditions in a running buffer (25 mM Tris-HCl, 200 mM glycine, 1.7 mM SDS). Proteins were transferred to a PVDF membrane (Immobilon-FL, Millipore) in transfer buffer (25 mM Tris-HCl, 19.2 ml glycine, 15% methanol) as previously described 11,19,37,48 . The primary and secondary antibodies used for this work are described in Supplementary Table S3. The secondary antibodies were incubated for an hour and the fluorescence was detected with LI-COR Odyssey Infrared Imaging System or with ECL Western Blotting Detection Reagent (SIGMA-ALDRICH) if the secondary antibodies were conjugated with peroxidase (Supplementary Table S3).
Statistical analysis. Statistical analysis were performed using the IBM SPSS 28 statistics package. All assays were performed in the lineal response range and in identical conditions for all substrates. Individual quantitative experiments were repeated between three and six times, and statistical significance was analyzed using two-tailed T-test with Welch' correction 87 . In all cases, the level of significance was: *p < 0.05; **p < 0.005; and ***p < 0.0005.
In stability experiments, data was analyzed using covariance least-square polynomic regression test to detect the correlation coefficient 87 .