With the increase of antibiotic drug resistance, alternative antibacterial treatment strategies are needed. Copper is a well-known antimicrobial and antiviral agent; however, the underlying molecular mechanisms by which copper causes cell death are not yet fully understood.

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe Grx1-roGFP2. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach, we defined a redox-dependent heterogeneity of yeast cells and characterized growth, as well as proteomic and transcriptomic profiles of distinctive redox subpopulations. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

By modifying the Hsp33's sequence, we reveal that the metastable region has evolved to abolish redox-dependent chaperone activity, rather than enhance binding affinity for client proteins. The intrinsically disordered region of Hsp33 serves as an anchor for the reduced, inactive state of Hsp33, and it dramatically affects the crosstalk with the synergetic chaperone system, DnaK/J. Using mass spectrometry, we describe the role that the metastable region plays in determining client specificity during normal and oxidative stress conditions in the cell. Innovation and Conclusion: We uncover a new role of protein plasticity in Hsp33's inactivation, client specificity, crosstalk with the synergistic chaperone system DnaK/J, and oxidative stress-specific interactions in bacteria. Our results also suggest that Hsp33 might serve as a member of the house-keeping proteostasis machinery, tasked with maintaining a "healthy" proteome during normal conditions, and that this function does not depend on the metastable linker region. Antioxid. Redox Signal. 27, 1252-1267.

Bacteria possess the ability to adapt to changing environments. To enable this, cells use reversible post-translational modifications on key proteins to modulate their behavior, metabolism, defense mechanisms and adaptation of bacteria to stress. In this review, we focus on bacterial protein switches that are activated during exposure to oxidative stress. Such protein switches are triggered by either exogenous reactive oxygen species (ROS) or endogenous ROS generated as by-products of the aerobic lifestyle. Both thiol switches and metal centers have been shown to be the primary targets of ROS. Cells take advantage of such reactivity to use these reactive sites as redox sensors to detect and combat oxidative stress conditions. This in turn may induce expression of genes involved in antioxidant strategies and thus protect the proteome against stress conditions. We further describe the well-characterized mechanism of selected proteins that are regulated by redox switches. We highlight the diversity of mechanisms and functions (as well as common features) across different switches, while also presenting integrative methodologies used in discovering new members of this family. Finally, we point to future challenges in this field, both in uncovering new types of switches, as well as defining novel additional functions.