eIF4G as a switch for heat shock mRNA translation.

The heat shock response is crucial for cell survival. In this issue of Molecular Cell, Desroches Altamirano et al.1 demonstrate that a temperature-induced conformational change in the translation initiation factor eIF4G is a key mechanism regulating translation during the heat shock response.

Lipid Biosynthesis Coordinates a Mitochondrial-to-Cytosolic Stress Response.

Defects in mitochondrial metabolism have been increasingly linked with age-onset protein-misfolding diseases such as Alzheimer's, Parkinson's, and Huntington's. In response to protein-folding stress, compartment-specific unfolded protein responses (UPRs) within the ER, mitochondria, and cytosol work in parallel to ensure cellular protein homeostasis. While perturbation of individual compartments can make other compartments more susceptible to protein stress, the cellular conditions that trigger cross-communication between the individual UPRs remain poorly understood. We have uncovered a conserved, robust mechanism linking mitochondrial protein homeostasis and the cytosolic folding environment through changes in lipid homeostasis. Metabolic restructuring caused by mitochondrial stress or small-molecule activators trigger changes in gene expression coordinated uniquely by both the mitochondrial and cytosolic UPRs, protecting the cell from disease-associated proteins. Our data suggest an intricate and unique system of communication between UPRs in response to metabolic changes that could unveil new targets for diseases of protein misfolding.

NF-κB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria.

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1ß and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1ß-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Remembering the Past: A New Form of Protein-Based Inheritance.

A comprehensive analysis uncovered a set of yeast proteins promoting protein-based inheritance that shares many of the non-Mendelian properties of prions. Lacking any sequence or structural signatures of known prions, these proteins represent a new class of non-amyloid, protein-based epigenetic determinants that can control phenotype without impacting genotype.

Intrinsically Disordered Proteins Drive Emergence and Inheritance of Biological Traits.

Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here, we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (ORF; ∼5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal. These traits were beneficial, had prion-like patterns of inheritance, were common in wild yeasts, and could be transmitted to naive cells with protein alone. Most inducing proteins were not known prions and did not form amyloid. Instead, they are highly enriched in nucleic acid binding proteins with large intrinsically disordered domains that have been widely conserved across evolution. Thus, our data establish a common type of protein-based inheritance through which intrinsically disordered proteins can drive the emergence of new traits and adaptive opportunities.

Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

Regulation of heat shock transcription factors and their roles in physiology and disease.

The heat shock transcription factors (HSFs) were discovered over 30 years ago as direct transcriptional activators of genes regulated by thermal stress, encoding heat shock proteins. The accepted paradigm posited that HSFs exclusively activate the expression of protein chaperones in response to conditions that cause protein misfolding by recognizing a simple promoter binding site referred to as a heat shock element. However, we now realize that the mammalian family of HSFs comprises proteins that independently or in concert drive combinatorial gene regulation events that activate or repress transcription in different contexts. Advances in our understanding of HSF structure, post-translational modifications and the breadth of HSF-regulated target genes have revealed exciting new mechanisms that modulate HSFs and shed new light on their roles in physiology and pathology. For example, the ability of HSF1 to protect cells from proteotoxicity and cell death is impaired in neurodegenerative diseases but can be exploited by cancer cells to support their growth, survival and metastasis. These new insights into HSF structure, function and regulation should facilitate the development tof new disease therapeutics to manipulate this transcription factor family.

The temperature sensor TWA1 is required for thermotolerance in Arabidopsis.

Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation1,2. The mechanism that senses cellular temperature for inducing thermotolerance is still unclear3. Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.

Chaperones directly and efficiently disperse stress-triggered biomolecular condensates.

Stresses such as heat shock trigger the formation of protein aggregates and the induction of a disaggregation system composed of molecular chaperones. Recent work reveals that several cases of apparent heat-induced aggregation, long thought to be the result of toxic misfolding, instead reflect evolved, adaptive biomolecular condensation, with chaperone activity contributing to condensate regulation. Here we show that the yeast disaggregation system directly disperses heat-induced biomolecular condensates of endogenous poly(A)-binding protein (Pab1) orders of magnitude more rapidly than aggregates of the most commonly used misfolded model substrate, firefly luciferase. Beyond its efficiency, heat-induced condensate dispersal differs from heat-induced aggregate dispersal in its molecular requirements and mechanistic behavior. Our work establishes a bona fide endogenous heat-induced substrate for long-studied heat shock proteins, isolates a specific example of chaperone regulation of condensates, and underscores needed expansion of the proteotoxic interpretation of the heat shock response to encompass adaptive, chaperone-mediated regulation.

A sestrin-dependent Erk-Jnk-p38 MAPK activation complex inhibits immunity during aging.

Mitogen-activated protein kinases (MAPKs) including Erk, Jnk and p38 regulate diverse cellular functions and are thought to be controlled by independent upstream activation cascades. Here we show that the sestrins bind to and coordinate simultaneous Erk, Jnk and p38 MAPK activation in T lymphocytes within a new immune-inhibitory complex (sestrin-MAPK activation complex (sMAC)). Whereas sestrin ablation resulted in broad reconstitution of immune function in stressed T cells, inhibition of individual MAPKs allowed only partial functional recovery. T cells from old humans (>65 years old) or mice (16-20 months old) were more likely to form the sMAC, and disruption of this complex restored antigen-specific functional responses in these cells. Correspondingly, sestrin deficiency or simultaneous inhibition of all three MAPKs enhanced vaccine responsiveness in old mice. Thus, disruption of sMAC provides a foundation for rejuvenating immunity during aging.

Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events.

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.

Sestrins function as guanine nucleotide dissociation inhibitors for Rag GTPases to control mTORC1 signaling.

Mechanistic target of rapamycin complex 1 (mTORC1) integrates diverse environmental signals to control cellular growth and organismal homeostasis. In response to nutrients, Rag GTPases recruit mTORC1 to the lysosome to be activated, but how Rags are regulated remains incompletely understood. Here, we show that Sestrins bind to the heterodimeric RagA/B-RagC/D GTPases, and function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B. Sestrin overexpression inhibits amino-acid-induced Rag guanine nucleotide exchange and mTORC1 translocation to the lysosome. Mutation of the conserved GDI motif creates a dominant-negative form of Sestrin that renders mTORC1 activation insensitive to amino acid deprivation, whereas a cell-permeable peptide containing the GDI motif inhibits mTORC1 signaling. Mice deficient in all Sestrins exhibit reduced postnatal survival associated with defective mTORC1 inactivation in multiple organs during neonatal fasting. These findings reveal a nonredundant mechanism by which the Sestrin family of GDIs regulates the nutrient-sensing Rag GTPases to control mTORC1 signaling.

Visualizing GroEL/ES in the act of encapsulating a folding protein.

The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.

Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet.

Coordinated beating of cilia in the trachea generates a directional flow of mucus required to clear the airways. Each cilium originates from a barrel-shaped basal body, from the side of which protrudes a structure known as the basal foot. We generated mice in which exons 6 and 7 of Odf2, encoding a basal body and centrosome-associated protein Odf2/cenexin, are disrupted. Although Odf2(ΔEx6,7/ΔEx6,7) mice form cilia, ciliary beating is uncoordinated, and the mice display a coughing/sneezing phenotype. Whereas residual expression of the C-terminal region of Odf2 in these mice is sufficient for ciliogenesis, the resulting basal bodies lack basal feet. Loss of basal feet in ciliated epithelia disrupted the polarized organization of apical microtubule lattice without affecting planar cell polarity. The requirement for Odf2 in basal foot formation, therefore, reveals a crucial role of this structure in the polarized alignment of basal bodies and coordinated ciliary beating.

A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress.

The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

ATP-Dependent Dynamic Protein Aggregation Regulates Bacterial Dormancy Depth Critical for Antibiotic Tolerance.

Cell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and regrowth at the single-cell level and found that each individual survival cell shows different "dormancy depth," which in return regulates the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

Mutation of the polyadenylation complex subunit CstF77 reveals that mRNA 3' end formation and HSP101 levels are critical for a robust heat stress response.

Heat shock protein 101 (HSP101) in plants, and bacterial and yeast orthologs, is essential for thermotolerance. To investigate thermotolerance mechanisms involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a missense HSP101 allele (hot1-4). hot1-4 plants are sensitive to acclimation heat treatments that are otherwise permissive for HSP101 null mutants, indicating that the hot1-4 protein is toxic. We report one suppressor (shot2, suppressor of hot1-4 2) has a missense mutation of a conserved residue in CLEAVAGE STIMULATION FACTOR77 (CstF77), a subunit of the polyadenylation complex critical for mRNA 3' end maturation. We performed ribosomal RNA depletion RNA-Seq and captured transcriptional readthrough with a custom bioinformatics pipeline. Acclimation heat treatment caused transcriptional readthrough in hot1-4 shot2, with more readthrough in heat-induced genes, reducing the levels of toxic hot1-4 protein and suppressing hot1-4 heat sensitivity. Although shot2 mutants develop like the wild type in the absence of stress and survive mild heat stress, reduction of heat-induced genes and decreased HSP accumulation makes shot2 in HSP101 null and wild-type backgrounds sensitive to severe heat stress. Our study reveals the critical function of CstF77 for 3' end formation of mRNA and the dominant role of HSP101 in dictating the outcome of severe heat stress.

Comprehensive structural characterization of the human AAA+ disaggregase CLPB in the apo- and substrate-bound states reveals a unique mode of action driven by oligomerization.

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space (IMS) and functions to promote the solubilization of various mitochondrial proteins. Loss-of-function CLPB mutations are associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. How CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo-tetradecamers in apo-state and is remodeled into homo-dodecamers upon substrate binding. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of 2 amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

Substrate Insolubility Dictates Hsp104-Dependent Endoplasmic-Reticulum-Associated Degradation.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by ER-associated degradation (ERAD). Although the retrotranslocation of misfolded proteins from the ER has been reconstituted, how a polypeptide is initially selected for ERAD remains poorly defined. To address this question while controlling for the diverse nature of ERAD substrates, we constructed a series of truncations in a single ER-tethered domain. We observed that the truncated proteins exhibited variable degradation rates and discovered a positive correlation between ERAD substrate instability and detergent insolubility, which demonstrates that aggregation-prone species can be selected for ERAD. Further, Hsp104 facilitated degradation of an insoluble species, consistent with the chaperone's disaggregase activity. We also show that retrotranslocation of the ubiquitinated substrate from the ER was inhibited in the absence of Hsp104. Therefore, chaperone-mediated selection frees the ER membrane of potentially toxic, aggregation-prone species.