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The increased expression of CaV1.3 calcium channels is a crucial feature of Parkinson’s disease (PD) pathophysiology. However, the mechanisms that determine this increase are not yet defined. Parkin, an enzyme of the ubiquitin-proteasome system, is known to interact with neuronal channels promoting their ubiquitin-mediated degradation. Interestingly, Parkin mutations also play a role in PD. Here, the degradation mechanisms of CaV1.3 channels and their relationship with the pathophysiology of PD are studied in detail.

Young women with breast cancer represent 15% of cancer cases in Latin America. Genomic studies have found that early-onset breast-cancer cases exhibit a higher genetic susceptibility and a specific genomic signature as compared to their older counterparts. The aim of this study was to describe clinically relevant germline mutations in a cohort of young women with breast cancer. To achieve this, we analyzed hereditary-cancer genes from whole-exome sequencing data in 108 unrelated women with an extreme phenotype of breast cancer (≤40 years of age), diagnosed and treated at the National Cancer Institute of Mexico; 11% of the patients carried a pathogenic variant. BRCA2 comprised 46% of the mutations, followed by BRCA1 with 23%; PALB2 with 15%; and TP53 and RAD51C with 8 % each. This article describes a novel pathogenic mutation in RAD51C c.519dupT. The median age at diagnosis was 35 years overall; however, it was six years younger in patients with mutations. Age at diagnosis (OR=0.82, CI 95% 0.71-0.94; P= 0.008) and first-degree family history of cancer (OR=8.26, CI95% 1.35-50; P= 0.022) were the only epidemiological variables associated with mutational status. We found no differences in disease-free survival (p=0.403) or overall survival (p=0.735) among mutational status subgroups.

Breast cancer (BC) is one of the most frequent cancer types in women worldwide. About 7% is diagnosed in young women (YBC) less than 40 years old. In Mexico, however, YBC reaches 15% suggesting a higher genetic susceptibility. There have been some reports of germline variants in YBC across the world. However, there is only one report from a Mexican population, which is not restricted by age and limited to a panel of 143 genes resulting in 15% of patients carrying putatively pathogenic variants. Nevertheless, expanding the analysis to whole exome involves using more complex tools to determine which genes and variants could be pathogenic. We used germline whole exome sequencing combined with the PeCanPie tool to analyze exome variants in 115 YBC patients. Our results showed that we were able to identify 49 high likely pathogenic variants involving 40 genes on 34% of patients. We noted many genes already reported in BC and YBC worldwide, such as BRCA1, BRCA2, ATM, CHEK2, PALB2, and POLQ, but also others not commonly reported in YBC in Latin America, such as CLTCL1, DDX3X, ERCC6, FANCE, and NFKBIE. We show further supporting and controversial evidence for some of these genes. We conclude that exome sequencing combined with robust annotation tools and further analysis, can identify more genes and more patients affected by germline mutations in cancer.

N-type calcium (CaV2.2) channels are widely expressed in the brain and the peripheral nervous system, where they play important roles in the regulation of transmitter release. Although CaV2.2 channel expression levels are precisely regulated, presently little is known regarding the molecules that mediate its synthesis and degradation. Previously, by using a combination of biochemical and functional analyses, we showed that the complex formed by the light chain 1 of the microtubule-associated protein 1B (LC1-MAP1B) and the ubiquitin-proteasome system (UPS) E2 enzyme UBE2L3, may interact with the CaV2.2 channels promoting ubiquitin-mediated degradation. The present report aims to gain further insights into the possible mechanism of degradation of the neuronal CaV2.2 channel by the UPS. First, we identified the enzymes UBE3A and Parkin, members of the UPS E3 ubiquitin ligase family, as novel CaV2.2 channel binding partners, although evidence to support a direct protein-protein interaction is not yet available. Immunoprecipitation assays confirmed the interaction between UBE3A and Parkin with CaV2.2 channels heterologously expressed in HEK-293 cells and in neural tissues. Parkin, but not UBE3A, overexpression led to a reduced CaV2.2 protein level and decreased current density. Electrophysiological recordings performed in the presence of MG132 prevented the actions of Parkin suggesting enhanced channel proteasomal degradation. Together these results unveil a novel functional coupling between Parkin and the CaV2.2 channels and provide a novel insight into the basic mechanisms of CaV channels protein quality control and functional expression.

Microtubule-associated protein B is a cytoskeleton protein consisting of heavy and light (LC) chains that play important roles in the regulation of neuronal morphogenesis and function. LC1 is also well known to interact with diverse ionotropic receptors at postsynapse. Much less is known, however, regarding the role of LC1 at presynaptic level where voltage-gated N-type Ca(2+) channels couple membrane depolarization to neurotransmitter release. Here, we investigated whether LC1 interacts with the N-type channels. Co-localization analysis revealed spatial proximity of the two proteins in hippocampal neurons. The interaction between LC1 and the N-type channel was demonstrated using co-immunoprecipitation experiments and in vitro pull-down assays. Detailed biochemical analysis suggested that the interaction occurs through the N-terminal of LC1 and the C-terminal of the pore-forming CaVα1 subunit of the channels. Patch-clamp studies in HEK-293 cells revealed a significant decrease in N-type currents upon LC1 expression, without apparent changes in kinetics. Recordings performed in the presence of MG132 prevented the actions of LC1 suggesting enhanced channel proteasomal degradation. Interestingly, using the yeast two-hybrid system and immunoprecipitation assays in HEK-293 cells, we revealed an interaction between LC1 and the ubiquitin-conjugating enzyme UBE2L3. Furthermore, we found that the LC1/UBE2L3 complex could interact with the N-type channels, suggesting that LC1 may act as a scaffold protein to increase UBE2L3-mediated channel ubiquitination. Together these results revealed a novel functional coupling between LC1 and the N-type channels.