Autorepression of yeast Hsp70 cochaperones by intramolecular interactions involving their J-domains.

The 70 kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s to trigger ATP hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1's ATPase, indicate the presence of yet uncharacterized intramolecular dynamic interactions between the J-domains and the remaining C-terminal segments of these proteins. Taken together, the data suggest an autoregulatory role to these intramolecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol.

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins.

The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

Design of an Arabidopsis thaliana reporter line to detect heat-sensing and signaling mutants.

BACKGROUND: Global warming is a major challenge for plant survival and growth. Understanding the molecular mechanisms by which higher plants sense and adapt to upsurges in the ambient temperature is essential for developing strategies to enhance plant tolerance to heat stress. Here, we designed a heat-responsive Arabidopsis thaliana reporter line that allows an in-depth investigation of the mechanisms underlying the accumulation of protective heat-shock proteins (HSPs) in response to high temperature. METHODS: A transgenic Arabidopsis thaliana reporter line named "Heat-Inducible Bioluminescence And Toxicity" (HIBAT) was designed to express from a conditional heat-inducible promoter, a fusion gene encoding for nanoluciferase and D-amino acid oxidase, whose expression is toxic in the presence of D-valine. HIBAT seedlings were exposed to different heat treatments in presence or absence of D-valine and analyzed for survival rate, bioluminescence and HSP gene expression. RESULTS: Whereas at 22 °C, HIBAT seedlings grew unaffected by D-valine, and all survived iterative heat treatments without D-valine, 98% died following heat treatments on D-valine. The HSP17.3B promoter was highly specific to heat as it remained unresponsive to various plant hormones, Flagellin, H2O2, osmotic stress and high salt. RNAseq analysis of heat-treated HIBAT seedlings showed a strong correlation with expression profiles of two wild type lines, confirming that HIBAT does not significantly differ from its Col-0 parent. Using HIBAT, a forward genetic screen revealed candidate loss-of-function mutants, apparently defective either at accumulating HSPs at high temperature or at repressing HSP accumulation at non-heat-shock temperatures. CONCLUSION: HIBAT is a valuable candidate tool to identify Arabidopsis mutants defective in the response to high temperature stress. It opens new avenues for future research on the regulation of HSP expression and for understanding the mechanisms of plant acquired thermotolerance.

How do plants feel the heat and survive?

Climate change is increasingly affecting the quality of life of organisms on Earth. More frequent, extreme, and lengthy heat waves are contributing to the sixth mass extinction of complex life forms in the Earth's history. From an anthropocentric point of view, global warming is a major threat to human health because it also compromises crop yields and food security. Thus, achieving agricultural productivity under climate change calls for closer examination of the molecular mechanisms of heat-stress resistance in model and crop plants. This requires a better understanding of the mechanisms by which plant cells can sense rising temperatures and establish effective molecular defenses, such as molecular chaperones and thermoprotective metabolites, as reviewed here, to survive extreme diurnal variations in temperature and seasonal heat waves.

How do humans and plants feel the heat?

The 2021 Nobel prize was awarded for the discovery of the animal thermosensory channel TRPV1. We highlight notable shared features with the higher plant thermosensory channel CNGC2/4. Both channels respond to temperature-induced changes in plasma membrane fluidity, leading to hyperphosphorylation of the HSF1 transcription factor via a specific heat-signaling cascade.

Repair or Degrade: the Thermodynamic Dilemma of Cellular Protein Quality-Control.

Life is a non-equilibrium phenomenon. Owing to their high free energy content, the macromolecules of life tend to spontaneously react with ambient oxygen and water and turn into more stable inorganic molecules. A similar thermodynamic picture applies to the complex shapes of proteins: While a polypeptide is emerging unfolded from the ribosome, it may spontaneously acquire secondary structures and collapse into its functional native conformation. The spontaneity of this process is evidence that the free energy of the unstructured state is higher than that of the structured native state. Yet, under stress or because of mutations, complex polypeptides may fail to reach their native conformation and form instead thermodynamically stable aggregates devoid of biological activity. Cells have evolved molecular chaperones to actively counteract the misfolding of stress-labile proteins dictated by equilibrium thermodynamics. HSP60, HSP70 and HSP100 can inject energy from ATP hydrolysis into the forceful unfolding of stable misfolded structures in proteins and convert them into unstable intermediates that can collapse into the native state, even under conditions inauspicious for that state. Aggregates and misfolded proteins may also be forcefully unfolded and degraded by chaperone-gated endo-cellular proteases, and in eukaryotes also by chaperone-mediated autophagy, paving the way for their replacement by new, unaltered functional proteins. The greater energy cost of degrading and replacing a polypeptide, with respect to the cost of its chaperone-mediated repair represents a thermodynamic dilemma: some easily repairable proteins are better to be processed by chaperones, while it can be wasteful to uselessly try recover overly compromised molecules, which should instead be degraded and replaced. Evolution has solved this conundrum by creating a host of unfolding chaperones and degradation machines and by tuning their cellular amounts and activity rates.

On the evolution of chaperones and cochaperones and the expansion of proteomes across the Tree of Life.

Across the Tree of Life (ToL), the complexity of proteomes varies widely. Our systematic analysis depicts that from the simplest archaea to mammals, the total number of proteins per proteome expanded ∼200-fold. Individual proteins also became larger, and multidomain proteins expanded ∼50-fold. Apart from duplication and divergence of existing proteins, completely new proteins were born. Along the ToL, the number of different folds expanded ∼5-fold and fold combinations ∼20-fold. Proteins prone to misfolding and aggregation, such as repeat and beta-rich proteins, proliferated ∼600-fold and, accordingly, proteins predicted as aggregation-prone became 6-fold more frequent in mammalian compared with bacterial proteomes. To control the quality of these expanding proteomes, core chaperones, ranging from heat shock proteins 20 (HSP20s) that prevent aggregation to HSP60, HSP70, HSP90, and HSP100 acting as adenosine triphosphate (ATP)-fueled unfolding and refolding machines, also evolved. However, these core chaperones were already available in prokaryotes, and they comprise ∼0.3% of all genes from archaea to mammals. This challenge-roughly the same number of core chaperones supporting a massive expansion of proteomes-was met by 1) elevation of messenger RNA (mRNA) and protein abundances of the ancient generalist core chaperones in the cell, and 2) continuous emergence of new substrate-binding and nucleotide-exchange factor cochaperones that function cooperatively with core chaperones as a network.

Moderate Fever Cycles as a Potential Mechanism to Protect the Respiratory System in COVID-19 Patients.

Mortality in COVID-19 patients predominantly results from an acute respiratory distress syndrome (ARDS), in which lungs alveolar cells undergo programmed cell death. Mortality in a sepsis-induced ARDS rat model is reduced by adenovirus over-expression of the HSP70 chaperone. A natural rise of body temperature during mild fever can naturally accumulate high cellular levels of HSP70 that can arrest apoptosis and protect alveolar lung cells from inflammatory damages. However, beyond 1-2 h of fever, no HSP70 is being further produced and a decreased in body temperature required to the restore cell's ability to produce more HSP70 in a subsequent fever cycle. We suggest that antipyretics may be beneficial in COVID-19 patients subsequent to several hours of mild (<38.8°C) advantageous fever, allowing lung cells to accumulate protective HSP70 against damages from the inflammatory response to the virus SARS-CoV-2. With age, the ability to develop fever and accumulate HSP70 decreases. This could be ameliorated, when advisable to do so, by thermotherapies and/or physical training.

Interdomain communication suppressing high intrinsic ATPase activity of Sse1 is essential for its co-disaggregase activity with Ssa1.

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70-like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single-molecule FRET, small angle X-ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70-like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open-lid substrate-binding domain (SBD) in close contact with its nucleotide-binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP-hydrolysis-independent exhaustive interaction between two domains of Hsp110s, NBD-SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1-NBD compared to wild-type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1-mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co-chaperoning activity in disaggregase machinery, and stability of the protein.