Phosphorylation within the bipartite NLS alters the localization and toxicity of the ER stress response factor DDIT3/CHOP.

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 DNA-binding 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.

A Combined NMR and SAXS Analysis of the Partially Folded Cataract-Associated V75D γD-Crystallin.

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.

Identification, quantification, and evolutionary analysis of a novel isoform of MCM9.

The minichromosome maintenance (MCM) family of proteins is conserved from archaea to humans and is required for assembly of pre-replication complexes (pre-RCs) to initiate DNA replication. MCM9 is an uncharacterized member of the eukaryotic MCM protein family that contains conserved ATP binding and hydrolysis motifs. We have identified a novel alternatively spliced isoform of HsMCM9 that results in a medium length protein product (MCM9M) that eliminates a long C-terminal extension of the fully spliced product (MCM9L). Quantitative real-time reverse transcriptase PCR (qRT-PCR) separated and measured the relative mRNA isoform expression levels across a variety of cell lines. Although there is some variability in expression levels, the full length MCM9L transcript is more abundant than the MCM9M variant in all cell lines tested. The expression of both MCM9 isoforms is cell cycle regulated: induced in S-phase, decreases through G2/M, and becomes constant through G1. Consistent with recent reports suggesting MCM9 participates in repair or prevention of double strand breaks, mitomycin C significantly induces the specific expression of MCM9L, while the replication fork inhibitor, hydroxyurea, has no effect. Evolutionary analysis indicates that the MCM9M isoform is a conserved variant, whereas the addition of the terminal exon producing MCM9L appears to be a more recent event present only in the highest order of eukaryotes.