A cataract is a pathological condition characterized by the clouding of the normally clear eye lens brought about by deposition of crystallin proteins in the lens fiber cells. These protein aggregates reduce visual acuity by scattering or blocking incoming light. Chemical damage to proteins of the crystallin family, accumulated over a lifetime, leads to age-related cataract, whereas inherited mutations are associated with congenital or early-onset cataract. The V75D mutant of γD-crystallin is associated with congenital cataract in mice and was previously shown to un/fold via a partially folded intermediate. Here, we structurally characterized the stable equilibrium urea unfolding intermediate of V75D at the ensemble level using solution NMR and small-angle x-ray scattering. Our data show that, in the intermediate, the C-terminal domain retains a folded conformation that is similar to the native wild-type protein, whereas the N-terminal domain is unfolded and comprises an ensemble of random conformers, without any detectable residual structural propensities.
Regulated nuclear-cytoplasmic trafficking is a well-established mechanism utilized by cells to regulate adaptive and maladaptive responses to acute oxidant stress. Commonly associated with endoplasmic reticulum stress, the bZIP transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP/DDIT3) mediates the cellular response to redox stress with effects on cellular growth, differentiation, and survival. We show through functional analyses that CHOP contains a conserved, compound pat4/bipartite nuclear localization signal within the basic DNAbinding domain. Using phylogenetic analyses and mass spectrometry, we now show that Ser107 located within the linker region of the bipartite NLS domain is a substrate for phosphorylation under standard culture conditions. Studies using the S107E phospho-mimic of CHOP indicate that changes in the charge properties at this residue regulate CHOP's nuclear-to-cytoplasmic ratio. And while co-stimulation with the SERCA inhibitor thapsigargin induced injury in cells expressing wild-type CHOP, the S107A point-mutant blocked this response. These findings indicate that phosphorylation within the bipartite NLS exerts regulatory effects on both the subcellular localization and toxic potential of DDIT3/CHOP. Future studies geared towards defining the relevant kinase/phosphatase networks that converge on the phosphorylation-regulated NLS (prNLS) phosphoepitope may provide an opportunity to constrain cellular damage in the context of acute ER stress.
The bZIP transcription factor CCAAT‐enhancer‐binding protein β (C/EBPβ) exhibits neurogenic, neuritogenic, and pro‐survival effects in the central nervous system. Here, we show that C/EBPβ regulates neural stem cell (NSC) expansion and vascular endothelial growth factor A (VEGF‐A) level by acting on a C/EBPβ‐responsive element within the Vegf‐a promoter. As predicted, C/EBPβ depletion reduced VEGF‐A production, NSC number, and average neurosphere size in proliferating cultures. Conversely, deletion of the C/EBPβ repressor CHOP‐10 induced C/EBPβ and VEGF‐A expression, while stimulating NSC expansion. These data highlight the role of C/EBPβ in regulating VEGF‐A production and the growth of NSCs and suggest CHOP‐dependent antagonism of C/EBPβ may function as a transcriptional rheostat linking stress‐associated cues with stem cell quiescence among other pathological responses affecting the neurogenic niche.
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