The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

Abstract

The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signaling molecules is poorly characterized. The transcription repressor NmrA of Aspergillus nidulans discriminates between oxidized and reduced dinucleotides, however, dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelium grown under nitrogen sufficient conditions but abundant in mycelium starved of nitrogen. One of the proteases was purified and identified as the protein Q5BAR4 encoded by the gene AN2366.2. Fluorescence confocal microscopy showed that the nuclear levels of NmrA were reduced approximately 38% when mycelium was grown on nitrate compared to ammonium and absent when starved of nitrogen. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. NmrA digested at the C-terminal site was unable to bind to the AreA zinc finger. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site retained the ability to bind NAD 1 and showed a resistance to further digestion that was enhanced by the presence of NAD 1 . This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated.

Functional Analysis of the Nitrogen Metabolite Repression Regulator Gene nmrA in Aspergillus flavus

“…In addition, NmrA interacts directly with AreA zinc fingers (Stammers et al, 2001; Kotaka et al, 2008; Zhao et al, 2010). The A. flavus nmrA ORF consists of 1,119 bp with one introns, and encodes a putative NMR regulator NmrA with 351 amino acids.…”

“…Therefore, we suspect that the roles of Nmr1/NmrA in nitrogen regulation are regulated differently by TOR signaling in Fusarium and Aspergillus . Zhao et al (2010) reported that NmrA could discriminate between oxidized and reduced dinucleotides and was positioned close to the GATA motif in DNA, so NmrA might play a role in DNA protection.…”

“…The function of NmrA in nitrogen source metabolism in A. flavus has not been well characterized (Andrianopoulos et al, 1998; Schönig et al, 2008; Wagner et al, 2010; Zhao et al, 2010). In this study, we identified and cloned the nmrA gene from A. flavus and characterized nmrA deletion and complementation mutants.…”

The GATA factors AREA and AREB together with the co-repressor NMRA, negatively regulate arginine catabolism in Aspergillus nidulans in response to nitrogen and carbon source

“…NMRA was also shown to differentially bind nicotinamide adenine dinucleotides and may have the redox-sensing function since oxidized forms (NAD + and NADP + ) are bound preferentially (Lamb et al, 2003(Lamb et al, , 2004. NMRA is also specifically digested by a trypsin-like serine protease and oxidized dinucleotides enhance the resistant of the remained fragment to further digestion (Zhao et al, 2010). A redox-sensing function was demonstrated for HSCARG, a human homologue of NMRA, which changes its structure upon NADP + binding (Zheng et al, 2007;Zhao et al, 2008).…”

Multiple Nuclear Localization Signals Mediate Nuclear Localization of the GATA Transcription Factor AreA

“…When cells are grown under nitrogen limiting conditions, AreA activity is partially derepressed due to increased levels of areA transcription and stability of areA mRNA compared with nitrogen-sufficient conditions (37)(38)(39)(40). AreA activity is further increased during nitrogen limitation due to reduced activity of the NmrA corepressor (41)(42)(43)(44)(45). An additional level of control is observed during nitrogen starvation.…”