This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract: In a previous collaboration with the Yates lab through the YRC (Chiranand et al, 2008), mass spectrometry was used to help identify Cta4 as a DNA binding transcription factor that is required for nitric oxide (NO)-induced gene expression in Candida albicans. We have since shown that NO induces the S-nitrosylation of Cta4 in cells, but that Cta4 is not sufficient for NO signaling when expressed in a heterologous system. These results suggest Cta4 acts as a NO sensor in combination with another protein. To identify the nitrosylated cysteines in Cta4 and other proteins in NO-treated cells, we request a collaboration in which S-nitrosylated peptides will first be purified by the Gustin lab using a modified version of the biotin switch technique from Candida cell extracts and then be identified using mass spectrometry at the YRC. The functional significance of these cysteines on Cta4 or other proteins to the NO response will be determined genetically. A second part of this requested collaboration will be to identify Cta4-interacting proteins. Using a strain expressing a functional Cta4 with tandem affinity tags, the tagged Cta4 will be selectively extracted by the Gustin lab together with interacting proteins whose identity will be determined using mass spectrometry at the YRC. The function of these proteins will be determined using appropriate deletion strains.
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