Search

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a major transcription factor that functions in maintaining redox homeostasis in cells. It mediates the transcription of cytoprotective genes in response to environmental and endogenous stresses to prevent oxidative damage. Thus, Nrf2 plays a significant role in chemoprevention. However, aberrant activation of Nrf2 has been shown to protect cancer cells from apoptosis and contribute to their chemoresistance. The interaction between Nrf2 and CBP is critical for the gene transcription activation. CBP and its homologue p300 interact with two transactivation domains in Nrf2, Neh4, and Neh5 domains through their TAZ1 and TAZ2 domains. To date, the molecular basis of this crucial interaction is not known, hindering a more detailed understanding of the regulation of Nrf2. To close this knowledge gap, we have used a set of biophysical experiments to dissect the Nrf2‐CBP/p300 interactions. Structural properties of Neh4 and Neh5 and their binding with the TAZ1 and TAZ2 domains of CBP/p300 were characterized. Our results show that the Neh4 and Neh5 domains of Nrf2 are intrinsically disordered, and they both can bind the TAZ1 and TAZ2 domains of CBP/p300 with micromolar affinities. The findings provide molecular insight into the regulation of Nrf2 by CBP/p300 through multi‐domain interactions. [ABSTRACT FROM AUTHOR]

Protein--metal complexes may be transferred from solution into the gas phase by electrospray ionization (ESI), such that they can be directly analyzed by mass spectrometry (MS). In principle, therefore, ESI-MS represents a simple and elegant approach for gaining insights into the binding stoichiometry and affinity of these assemblies. Unfortunately, the formation of nonspecific metal adducts during ESI can be a severe problem, often leading to binding levels that are dramatically higher than those in bulk solution. Focusing on several calcium binding proteins as test systems, this work explores the suitability of different salts to serve as metal source. Despite their widespread use in previous ESI-MS studies, calcium chloride and acetate induce extensive nonspecific adduction. In contrast, much lower levels of artifactual metal binding are observed in the presence of calcium tartrate. In the case of high and intermediate affinity proteins, the resulting ESI-MS data are in excellent agreement with the calcium binding behavior in bulk solution. The situation is more challenging when studying proteins with very low affinities, but in the presence of tartrate qualitative information on protein--metal interactions can still be obtained. The beneficial effects of tartrate also extend to zinc binding experiments. This work does not directly explore the mechanism by which tartrate suppresses nonspecific metalation. However, it seems likely that weak chelators such as tartrate sequester metal ions within rapidly shrinking droplets during the final stages of ESI, thereby reducing nonspecific metal adduction to protein carboxylates. The use of tartrate and possibly other weak chelators will greatly enhance the reliability of future ESI-MS studies on the interactions of proteins with divalent metal ions.

The change in stability between the WT drkN SH3 domain is investigated and its T22G mutant, the free-energy difference folded and unfolded states (Delta G(sub U)) for T22G mutant at 20 degree C are determined. Denaturation bhy GdmCl was monitored using intrinsic tryptophan fluorescence, and the resulting denaturation and renaturation curves could well-fit to the equation for a two-state transition.

A new set of pulse schemes for the study of methyl-containing side chain dynamics in partially folded and unfolded protein states is presented. Methyl side chain dynamics in an unfolded protein deletion mutant, Delta 131 Delta is studied.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress‐inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co‐chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in‐depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co‐chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co‐chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age‐dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age‐dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749. [ABSTRACT FROM AUTHOR]