Understanding the molecular functions of Neuronal Calcium Sensor-1 in Neurospora crassa

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Neuronal calcium sensor-1 (NCS-1) belongs to the neuronal calcium sensor family of proteins consisting of N terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. NCS-1 is a highly conserved protein from lower to higher eukaryotes. In mammals NCS-1 is a very important protein as it is associated with synaptic transmission, memory and learning. In Neurospora crassa, NCS-1 plays an important role in vegetative growth, Ca2+ and ultraviolet stress survival. The molecular pathway of the neuronal calcium sensor-1 (NCS-1) mediated Ca2+ stress tolerance in N. crassa was studied in this research work. The expression of ncs-1 inceases with increasing Ca2+ stress condition; however, addition of the calcineurin inhibitor FK506 in the medium caused a severe downregulation of ncs-1 transcription. In addition, chromatin immunoprecipitation (ChIP) assay revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and upregulates ncs-1 expression during the Ca2+ stress condition. These results suggest the regulation of NCS-1 function through calcineurin -CRZ-1 signaling pathway. Furthermore, the electrophoretic mobility shift assay (EMSA) revealed that CRZ-1 specifically binds to an 8 bp sequence 5ʹ-CCTTCACA-3ʹ in the ncs-1 promoter, located 216 bp upstream of the ATG start codon. A physical interaction between NCS-1 and the Ca2+ permeable channel MID-1 during Ca2+ stress was also shown. Therefore, CRZ-1 binds to a unique sequence in the ncs-1 promoter for its upregulation, and NCS-1 interacts with MID-1 for Ca2+ stress survival.
Supervisors: Ranjan Tamuli and Ajaykumar B. Kunnumakkara