Abstract
The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid l-cysteine. Addition of l-cysteine caused a significant dose-dependent decrease in the protein helicity – dropping from 69.1% to 23.8% in the presence of 1 mM of l-cysteine – and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 µM, an 8∶1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free l-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of l-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress.
Highlights
C/EBP Homologous Protein (CHOP) is a nuclear protein that acts as both a dominant-negative inhibitor of CCATT/enhancer binding proteins (C/EBPs) and a transcriptional activator of Activating Protein–1 (AP-1) [1]
Disorder analysis of CHOP Previously, we reported that CHOP is an intrinsically disordered proteins (IDPs) based on in silico amino acid sequence analyses, nuclear magnetic resonance (NMR) and Circular dichroism (CD) spectroscopy [57]
We employed several different programs (i.e., PONDR VLXT [72,73], PONDR VSL2 [74], PONDR VL3 [75], IUPred [76], TopIDP [77], Foldindex [78] and a meta predictor PONDR-FIT [79]) for the in silico analysis of the amino acid sequence (Figure 1A), which consistently predicted that CHOP is expected to be highly disordered over most of its length when looking at the disorder predicted on a per-residue basis
Summary
C/EBP Homologous Protein (CHOP) is a nuclear protein that acts as both a dominant-negative inhibitor of CCATT/enhancer binding proteins (C/EBPs) and a transcriptional activator of Activating Protein–1 (AP-1) [1]. CHOP plays an integral role in one of the branches of the unfolded protein response (UPR) which is initiated in response to endoplasmic reticulum (ER) stress [5,6,7,8], and occurs in response to factors such as nutrient starvation, calcium depletion, inflammation, glucose deprivation, and viral infection [5,9,10,11]. This response promotes cellular recovery, and leads to apoptosis if recovery is not possible. Deletion mutant analysis of CHOP revealed that bZIP domain is important for CHOP-induced apoptosis [18,19]
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