Partial Inhibition of Calcineurin Activity by Rcn2 as a Potential Remedy for Vps13 Deficiency

Abstract

Regulation of calcineurin, a Ca2+/calmodulin-regulated phosphatase, is important for the nervous system, and its abnormal activity is associated with various pathologies, including neurodegenerative disorders. In yeast cells lacking the VPS13 gene (vps13Δ), a model of VPS13-linked neurological diseases, we recently demonstrated that calcineurin is activated, and its downregulation reduces the negative effects associated with vps13Δ mutation. Here, we show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13Δ cells. We studied the molecular mechanism underlying this effect through site-directed mutagenesis of RCN2. The interaction of the resulting Rcn2 variants with a MAPK kinase, Slt2, and subunits of calcineurin was tested. We show that Rcn2 binds preferentially to Cmp2, one of two alternative catalytic subunits of calcineurin, and partially inhibits calcineurin. Rcn2 ability to bind to and reduce the activity of calcineurin was important for the suppression. The binding of Rcn2 to Cmp2 requires two motifs in Rcn2: the previously characterized C-terminal motif and a new N-terminal motif that was discovered in this study. Altogether, our findings can help to better understand calcineurin regulation and to develop new therapeutic strategies against neurodegenerative diseases based on modulation of the activity of selected calcineurin isoforms.

Yeast as a Model to Find New Drugs and Drug Targets for VPS13-Dependent Neurodegenerative Diseases

“…Moreover, FK-506 was active only in v concentration, and higher concentration caused toxicity. This is in line with experiments and shows that basal calcineurin activity is required for SDS protect [126,136].…”

“…Similar defects were observed in neurons generated from ChAc patient iPSC, and these alteration in SOCE functioning, at least partially, contrib neurodegeneration [156]. Despite enormous efforts, AD, PD and HD are still in and only symptomatic relief drugs are available; research on effective treatmen The identification of the RCN2 gene as another suppressor of vps13∆ [136] is in line with the results described above. RCN2 encodes Rcn2 protein, a negative calcineurin regulator [137,138].…”

“…Moreover, the deletion of CMP2 actually suppresses vps13∆, contrary to CNA1 and previously shown CNB1 deletions, which negatively influence vps13∆ growth in the presence of SDS. The CNA1 and CNB1 are also required for vps13∆ suppression by RCN2 [136] as for MYO3-N, indicating that vps13∆ suppression is achieved by reducing calcineurin activity related to Cmp2 catalytic subunit (Figure 4), while the activity mediated by Cna1 is crucial for SDS stress survival. Similarly, in mammals catalytic calcineurin subunits are differently expressed and regulated [139][140][141][142].…”

Yeast Models and Molecular Mechanisms of Neurodegenerative Diseases

“…Soczewka et al [10] and Wardaszka et al [11] further characterized the importance of VPS13 proteins for cell physiology by studying the yeast vps13∆ mutant devoid of the yeast Vps13 protein. A simple phenotype, i.e., the hypersensitivity of the vps13∆ mutant to the commonly used detergent sodium dodecyl sulphate (SDS), allowed the authors to use the power of yeast genetics to discover changes in copper homeostasis, in addition to the already described changes in calcium [12] and iron [13] homeostasis.…”

“…Moreover, they showed several ways to alleviate the growth defect caused by the vps13∆ mutant. The first way shown in this Special Issue is overexpression of the RCN2 gene, encoding the Rcn2 protein, which causes partial downregulation of calcineurin activity, a key calciumdependent phosphatase, by inhibiting only the subset of catalytic complexes [11]. The second way is the increase in the cellular level of copper ions via simple supplementation with copper salts, treatment with copper ionophores, or genetic manipulation, resulting in an increase in copper uptake [10].…”