DNA damage repair

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. Our group has reported that STIM1 is closely related to the protection from endogenous DNA damage and replicative stress, as well as to the response to interstrand crosslinks (ICLs). We have shown that the endoplasmic reticulum Ca(2+) sensor STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. Our project unveiled a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expanded our understanding of the genes involved in DNA repair (Sanchez-Lopez, 2024).

STIM1 translocates to the nucleus to protect cell from DNA damage.tif

Cytoskeleton, cell migration and metastasis

One of our objectives is to study the molecular basis of cell migration, with special attention to the migration of tumor and highly invasive cells. Intracellular signaling mediated by Ca(2+) ions is fundamental for the regulation of this migration, as the cytoskeleton is controlled by variations in the local free Ca2+ concentration. One of the pathways of Ca(2+) entry into the cytosol, termed SOCE (store-operated calcium entry), has been described as a key pathway in this regulation. The generation of SOCE-deficient cell lines is one approach to studying how SOCE modulates cell signaling. Using STIM1-KO and ORAI1-KO cell lines, generated by genomic editing with CRISPR/Cas9, we have been able to demonstrate that these two proteins, STIM1 and ORAI1, are important modulators of the Ca(2+) entry pathway that regulates the reorganization of the cortical cytoskeleton required for lamellipodia formation at the leading edge of migrating cells (López-Guerrero et al., 2017; López-Guerrero et al., 2020). However, further studies are required to explain how the pool of phosphorylated STIM1 localizes to the leading edge.

Migrating U2OS cells ORAI1-KO U2OS cells

Neurodegeneration

Another objective is to study the involvement of STIM1 in the genesis of neurodegenerative diseases. We recently reported a decrease in the level of STIM1 protein expression with the progression of neurodegeneration in human brain tissues from patients with pathologically confirmed Alzheimer's disease. Using SH-SY5Y cells edited by CRISPR/Cas9 to knockout STIM1, we demonstrated a significant decrease in mitochondrial respiratory chain complex I activity, depolarization of the mitochondrial inner membrane, reduced mitochondrial free Ca(2+) concentration, higher levels of senescence compared to wild-type cells, and enhanced Ca(2+) influx in response to depolarization.

Our data showed that stable knockdown of CACNA1C transcripts (encoding Cav1.2 channels) restored mitochondrial function, normalized mitochondrial Ca(2+) levels, and decreased senescence to basal levels. This finding highlighted the essential role of voltage-operated Ca(2+) entry regulation through Cav1.2 channels in STIM1-deficient cell death (Pascual-Caro et al., 2018).

Furthermore, using SH-SY5Y cells, we verified that STIM1 is a positive regulator of ITPR3 gene expression. ITPR3 (or IP3R3) is a Ca(2+) channel enriched at ER-mitochondria contact sites, providing Ca(2+) for transport into the mitochondria. We presented compelling evidence for a new mode of regulation of [Ca2+]mit, mediated by the STIM1-ITPR3 axis (Pascual-Caro et al., 2020).

References:

Sanchez-Lopez I et al. (2024) STIM1 translocation to the nucleus protects cells from DNA damage. Nucleic Acids Research. DOI: 10.1093/nar/gkae001.

Pascual-Caro C et al. (2020) STIM1 deficiency leads to specific downregulation of ITPR3 in SH-SY5Y cells. Int J Mol Sci 21(18):6598.

Lopez-Guerrero AM et al. (2020) RAC1-dependent ORAI1 translocation to the leading edge supports lamellipodia formation and directional persistence. Sci Reports 10:6580.

Pascual-Caro C et al. (2018) STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca(2+) entry. J Mol Med 96(10):1061-1079.

Lopez-Guerrero A et al. (2017) Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains. Sci. Reports 7:383.

Tomas-Martin P et al. (2015) Phospho-STIM1 is a downstream effector that mediates the signaling triggered by IGF-1 in HEK293 cells. Cell Signal. 27(3):545-54.

Casas-Rua V et al. (2015) STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration. Biochim Biophys Acta 1853(1):233-43.

Pozo-Guisado E et al. (2013) Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1. J Cell Sci. 126(Pt 14):3170-80.

Pozo-Guisado E et al. (2010) Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry. J Cell Sci. 123(Pt 18):3084-93.

F.J. Martin-Romero is a signer of the San Francisco Declaration on Research Assessment (DORA).