Loss of heat shock factor 1 promotes hepatic stellate cell activation and drives liver fibrosis.

Liver fibrosis is an aberrant wound healing response that results from chronic injury and is mediated by hepatocellular death and activation of hepatic stellate cells (HSCs). While induction of oxidative stress is well established in fibrotic livers, there is limited information on stress-mediated mechanisms of HSC activation. Cellular stress triggers an adaptive defense mechanism via master protein homeostasis regulator, heat shock factor 1 (HSF1), which induces heat shock proteins to respond to proteotoxic stress. Although the importance of HSF1 in restoring cellular homeostasis is well-established, its potential role in liver fibrosis is unknown. Here, we show that HSF1 messenger RNA is induced in human cirrhotic and murine fibrotic livers. Hepatocytes exhibit nuclear HSF1, whereas stellate cells expressing alpha smooth muscle actin do not express nuclear HSF1 in human cirrhosis. Interestingly, despite nuclear HSF1, murine fibrotic livers did not show induction of HSF1 DNA binding activity compared with controls. HSF1-deficient mice exhibit augmented HSC activation and fibrosis despite limited pro-inflammatory cytokine response and display delayed fibrosis resolution. Stellate cell and hepatocyte-specific HSF1 knockout mice exhibit higher induction of profibrogenic response, suggesting an important role for HSF1 in HSC activation and fibrosis. Stable expression of dominant negative HSF1 promotes fibrogenic activation of HSCs. Overactivation of HSF1 decreased phosphorylation of JNK and prevented HSC activation, supporting a protective role for HSF1. Our findings identify an unconventional role for HSF1 in liver fibrosis. Conclusion: Our results show that deficiency of HSF1 is associated with exacerbated HSC activation promoting liver fibrosis, whereas activation of HSF1 prevents profibrogenic HSC activation.

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope.

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

Chemical Biology Framework to Illuminate Proteostasis.

Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.

Non-antibiotic Small-Molecule Regulation of DHFR-Based Destabilizing Domains In Vivo.

The E. coli dihydrofolate reductase (DHFR) destabilizing domain (DD), which shows promise as a biologic tool and potential gene therapy approach, can be utilized to achieve spatial and temporal control of protein abundance in vivo simply by administration of its stabilizing ligand, the routinely prescribed antibiotic trimethoprim (TMP). However, chronic TMP use drives development of antibiotic resistance (increasing likelihood of subsequent infections) and disrupts the gut microbiota (linked to autoimmune and neurodegenerative diseases), tempering translational excitement of this approach in model systems and for treating human diseases. Herein, we identified a TMP-based, non-antibiotic small molecule, termed 14a (MCC8529), and tested its ability to control multiple DHFR-based reporters and signaling proteins. We found that 14a is non-toxic and can effectively stabilize DHFR DDs expressed in mammalian cells. Furthermore, 14a crosses the blood-retinal barrier and stabilizes DHFR DDs expressed in the mouse eye with kinetics comparable to that of TMP (≤6 h). Surprisingly, 14a stabilized a DHFR DD in the liver significantly better than TMP did, while having no effect on the mouse gut microbiota. Our results suggest that alternative small-molecule DHFR DD stabilizers (such as 14a) may be ideal substitutes for TMP in instances when conditional, non-antibiotic control of protein abundance is desired in the eye and beyond.

SUMOylation and the HSF1-Regulated Chaperone Network Converge to Promote Proteostasis in Response to Heat Shock.

The role of stress-induced increases in SUMO2/3 conjugation during the heat shock response (HSR) has remained enigmatic. We investigated SUMO signal transduction at the proteomic and functional level during the HSR in cells depleted of proteostasis network components via chronic heat shock factor 1 inhibition. In the recovery phase post heat shock, high SUMO2/3 conjugation was prolonged in cells lacking sufficient chaperones. Similar results were obtained upon inhibiting HSP90, indicating that increased chaperone activity during the HSR is critical for recovery to normal SUMO2/3 levels post-heat shock. Proteasome inhibition likewise prolonged SUMO2/3 conjugation, indicating that stress-induced SUMO2/3 targets are subsequently degraded by the ubiquitin-proteasome system. Functionally, we suggest that SUMOylation can enhance the solubility of target proteins upon heat shock, a phenomenon that we experimentally observed in vitro. Collectively, our results implicate SUMO2/3 as a rapid response factor that coordinates proteome degradation and assists the maintenance of proteostasis upon proteotoxic stress.