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An elongated tract of polyQ in the carboxyl?terminus of human ?1A calcium channel induces cell apoptosis by nuclear translocation.

ABSTRACT: An aberrant elongated tract of glutamine residues (polyQ) in proteins induces multiple diseases treated in the clinic. In our previous study of progressive myoclonic epilepsy (PME), using whole?exome sequencing, a mutant Cav2.1 protein with an aberrant elongated polyQ tract was identified in PME patients. To investigate the molecular mechanism and cell biology of this aberrant elongated polyQ tract, wild?type Cav2.1 with 13 polyQ repeats (Cav2.1 wt?Q13) and mutant?type Cav2.1 with 26 polyQ repeats (Cav2.1 mt?Q26) were prepared and introduced into human SH?SY5Y neuroblastoma cells. Using a WST?1 assay, it was revealed that Cav2.1 mt?Q26 markedly suppressed the proliferation of the SH?SY5Y cells, a result not observed for the Cav2.1 wt?Q13?transfected cells. It was also revealed that Cav2.1 mt and its truncated molecules suppressed cell proliferation by inducing apoptosis rather than arresting the cell cycle. Further investigations indicated a nuclear translocation phenomenon associated with the Cav2.1 mt molecules. Mechanistically, it was revealed that the Cav2.1 mt molecules activated the Bcl?2/Bax, caspase?3 and poly ADP?ribose polymerase (PARP) apoptotic pathways. The present study may provide new insights for interpreting the pathogenesis of PME and the relationship among polyQ, CACNA1A gene mutations and PME.

SUBMITTER: Sun J

PROVIDER: S-EPMC7251683 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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An elongated tract of polyQ in the carboxyl‑terminus of human α1A calcium channel induces cell apoptosis by nuclear translocation.

Sun Ji J Sun Xiguang X Li Zhuo Z Ma Dihui D Lv Yudan Y

Oncology reports 20200422 1

An aberrant elongated tract of glutamine residues (polyQ) in proteins induces multiple diseases treated in the clinic. In our previous study of progressive myoclonic epilepsy (PME), using whole‑exome sequencing, a mutant Cav2.1 protein with an aberrant elongated polyQ tract was identified in PME patients. To investigate the molecular mechanism and cell biology of this aberrant elongated polyQ tract, wild‑type Cav2.1 with 13 polyQ repeats (Cav2.1 wt‑Q13) and mutant‑type Cav2.1 with 26 polyQ repea ...[more]

PMID: 32626992

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Project description:Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. The guiding hypothesis of this study is that a carboxyl-terminal cleavage product of the cardiac L-type calcium channel (Ca(V)1.2) autoregulates expression. First, we confirmed that the Ca(V)1.2 C terminus (CCt) is cleaved in murine cardiac myocytes from mature and developing ventricle. Overexpression of full-length CCt caused a 34+/-8% decrease of Ca(V)1.2 promoter activity, and truncated CCt caused an 80+/-3% decrease of Ca(V)1.2 promoter (n=12). The full-length CCt distributes into cytosol and nucleus. A deletion mutant of CCt has a greater relative affinity for the nucleus than full-length CCt, and this is consistent with increased repression of Ca(V)1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation analysis revealed that CCt interacts with the Ca(V)1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing Nkx2.5/Mef2, C/EBp, and a cis regulatory module. The next hypothesis tested was that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We explored whether fetal cardiac myocyte Ca(V)1.2 was regulated by serum in vitro. We tested atrial natriuretic factor promoter activity as a positive control and measured the serum response of Ca(V)1.2 promoter, protein, and L-type current (I(Ca,L)) from fetal mouse ventricular myocytes. Serum increased atrial natriuretic factor promoter activity and cell size as expected. Serum withdrawal increased Ca(V)1.2 promoter activity, mRNA, and I(Ca,L). Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analyses, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca(V)1.2, autoregulates Ca(V)1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca(V)1.2 links Ca handling to nuclear signaling.

Project description:Background ? Carboxyl terminus 1 (?CT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that ?CT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase C? phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of ?CT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study ?CT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase C? phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the ?CT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an ?-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that ?CT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of ?CT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of ?CT1 (?CT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of ?CT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.

Dataset Information

An elongated tract of polyQ in the carboxyl?terminus of human ?1A calcium channel induces cell apoptosis by nuclear translocation.

Publications

An elongated tract of polyQ in the carboxyl‑terminus of human α1A calcium channel induces cell apoptosis by nuclear translocation.

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