Coordination between aminoacylation and editing to protect against proteotoxicity.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.

A short-term method to evaluate anti-leishmania drugs by inhibition of stage differentiation in Leishmania mexicana using flow cytometry.

Leishmaniasis is a vector-borne neglected tropical disease caused by the Leishmania spp. Parasite. The disease is transmitted to humans and animals by the bite of infected female sandflies during the ingestion of bloodmeal. Because current drug treatments induce toxicity and parasite resistance, there is an urgent need to evaluate new drugs. Most therapeutics target the differentiation of promastigotes to amastigotes, which is necessary to maintain Leishmania infection. However, in vitro assays are laborious, time-consuming, and depend on the experience of the technician. In this study, we aimed to establish a short-term method to assess the differentiation status of Leishmania mexicana (L. mexicana) using flow cytometry. Here, we showed that flow cytometry provides a rapid means to quantify parasite differentiation in cell culture as reliably as light microscopy. Interestingly, we found using flow cytometry that miltefosine reduced promastigote-to-amastigote differentiation of L. mexicana. We conclude that flow cytometry provides a means to rapidly assay the efficacy of small molecules or natural compounds as potential anti-leishmanials.

Atomic structure of the Leishmania spp. Hsp100 N-domain.

Hsp100 is an ATP-dependent unfoldase that promotes protein disaggregation or facilitates the unfolding of aggregation-prone polypeptides marked for degradation. Recently, new Hsp100 functions are emerging. In Plasmodium, an Hsp100 drives malaria protein export, presenting a novel drug target. Whether Hsp100 has a similar function in other protists is unknown. We present the 1.06 Å resolution crystal structure of the Hsp100 N-domain from Leishmania spp., the causative agent of leishmaniasis in humans. Our structure reveals a network of methionines and aromatic amino acids that define the putative substrate-binding site and likely evolved to protect Hsp100 from oxidative damage in host immune cells.

Deciphering the mechanism and function of Hsp100 unfoldases from protein structure.

Hsp100 chaperones, also known as Clp proteins, constitute a family of ring-forming ATPases that differ in 3D structure and cellular function from other stress-inducible molecular chaperones. While the vast majority of ATP-dependent molecular chaperones promote the folding of either the nascent chain or a newly imported polypeptide to reach its native conformation, Hsp100 chaperones harness metabolic energy to perform the reverse and facilitate the unfolding of a misfolded polypeptide or protein aggregate. It is now known that inside cells and organelles, different Hsp100 members are involved in rescuing stress-damaged proteins from a previously aggregated state or in recycling polypeptides marked for degradation. Protein degradation is mediated by a barrel-shaped peptidase that physically associates with the Hsp100 hexamer to form a two-component system. Notable examples include the ClpA:ClpP (ClpAP) and ClpX:ClpP (ClpXP) proteases that resemble the ring-forming FtsH and Lon proteases, which unlike ClpAP and ClpXP, feature the ATP-binding and proteolytic domains in a single polypeptide chain. Recent advances in electron cryomicroscopy (cryoEM) together with single-molecule biophysical studies have now provided new mechanistic insight into the structure and function of this remarkable group of macromolecular machines.

The mitochondrial HSP90 paralog TRAP1 forms an OXPHOS-regulated tetramer and is involved in mitochondrial metabolic homeostasis.

BACKGROUND: The molecular chaperone TRAP1, the mitochondrial isoform of cytosolic HSP90, remains poorly understood with respect to its pivotal role in the regulation of mitochondrial metabolism. Most studies have found it to be an inhibitor of mitochondrial oxidative phosphorylation (OXPHOS) and an inducer of the Warburg phenotype of cancer cells. However, others have reported the opposite, and there is no consensus on the relevant TRAP1 interactors. This calls for a more comprehensive analysis of the TRAP1 interactome and of how TRAP1 and mitochondrial metabolism mutually affect each other. RESULTS: We show that the disruption of the gene for TRAP1 in a panel of cell lines dysregulates OXPHOS by a metabolic rewiring that induces the anaplerotic utilization of glutamine metabolism to replenish TCA cycle intermediates. Restoration of wild-type levels of OXPHOS requires full-length TRAP1. Whereas the TRAP1 ATPase activity is dispensable for this function, it modulates the interactions of TRAP1 with various mitochondrial proteins. Quantitatively by far, the major interactors of TRAP1 are the mitochondrial chaperones mtHSP70 and HSP60. However, we find that the most stable stoichiometric TRAP1 complex is a TRAP1 tetramer, whose levels change in response to both a decline and an increase in OXPHOS. CONCLUSIONS: Our work provides a roadmap for further investigations of how TRAP1 and its interactors such as the ATP synthase regulate cellular energy metabolism. Our results highlight that TRAP1 function in metabolism and cancer cannot be understood without a focus on TRAP1 tetramers as potentially the most relevant functional entity.

Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate.

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding.

An intersubunit signaling network coordinates ATP hydrolysis by m-AAA proteases.

Ring-shaped AAA+ ATPases control a variety of cellular processes by substrate unfolding and remodeling of macromolecular structures. However, how ATP hydrolysis within AAA+ rings is regulated and coupled to mechanical work is poorly understood. Here we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes, conserved AAA+ machines in the inner membrane of mitochondria. ATP binding to one AAA subunit inhibits ATP hydrolysis by the neighboring subunit, leading to coordinated rather than stochastic ATP hydrolysis within the AAA ring. Unbiased genetic screens define an intersubunit signaling pathway involving conserved AAA motifs and reveal an intimate coupling of ATPase activities to central AAA pore loops. Coordinated ATP hydrolysis between adjacent subunits is required for membrane dislocation of substrates, but not for substrate processing. These findings provide insight into how AAA+ proteins convert energy derived from ATP hydrolysis into mechanical work.

Pharmacogenetic characterization of naturally occurring germline NT5C1A variants to chemotherapeutic nucleoside analogs.

BACKGROUND: Mutations or alterations in expression of the 5' nucleotidase gene family can lead to altered responses to treatment with nucleoside analogs. While investigating leukemia susceptibility genes, we discovered a very rare p.L254P NT5C1A missense variant in the substrate recognition motif. Given the paucity of cellular drug response data from the NT5C1A germline variation, we characterized p.L254P and eight rare variants of NT5C1A from genomic databases. MATERIALS AND METHODS: Through lentiviral infection, we created HEK293 cell lines that stably overexpress wild-type NT5C1A, p.L254P, or eight NT5C1A variants reported in the National Heart Lung and Blood Institute Exome Variant Server (one truncating and seven missense). IC50 values were determined by cytotoxicity assays after exposure to chemotherapeutic nucleoside analogs (cladribine, gemcitabine, 5-fluorouracil). In addition, we used structure-based homology modeling to generate a three-dimensional model for the C-terminal region of NT5C1A. RESULTS: The p.R180X (truncating), p.A214T, and p.L254P missense changes were the only variants that significantly impaired protein function across all nucleotide analogs tested (>5-fold difference vs. wild-type; P<0.05). Several of the remaining variants individually showed differential effects (both more and less resistant) across the analogs tested. The homology model provided a structural framework to understand the impact of NT5C1A mutants on catalysis and drug processing. The model predicted active site residues within NT5C1A motif III and we experimentally confirmed that p.K314 (not p.K320) is required for NT5C1A activity. CONCLUSION: We characterized germline variation and predicted protein structures of NT5C1A. Individual missense changes showed considerable variation in response to the different nucleoside analogs tested, which may impact patients' responses to treatment.

Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the species-specific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.

Structural basis for intersubunit signaling in a protein disaggregating machine.

ClpB is a ring-forming, ATP-dependent protein disaggregase that cooperates with the cognate Hsp70 system to recover functional protein from aggregates. How ClpB harnesses the energy of ATP binding and hydrolysis to facilitate the mechanical unfolding of previously aggregated, stress-damaged proteins remains unclear. Here, we present crystal structures of the ClpB D2 domain in the nucleotide-bound and -free states, and the fitted cryoEM structure of the D2 hexamer ring, which provide a structural understanding of the ATP power stroke that drives protein translocation through the ClpB hexamer. We demonstrate that the conformation of the substrate-translocating pore loop is coupled to the nucleotide state of the cis subunit, which is transmitted to the neighboring subunit via a conserved but structurally distinct intersubunit-signaling pathway common to diverse AAA+ machines. Furthermore, we found that an engineered, disulfide cross-linked ClpB hexamer is fully functional biochemically, suggesting that ClpB deoligomerization is not required for protein disaggregation.

Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing.

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation.

Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. Whereas the ability to disaggregate proteins is dependent on the Hsp104 M-domain, the location of the M-domain is controversial and its exact function remains unknown. Here we present cryoEM structures of two Hsp104 variants in both crosslinked and noncrosslinked form, in addition to the structure of a functional Hsp104 chimera harboring T4 lysozyme within the M-domain helix L2. Unexpectedly, we found that our Hsp104 chimera has gained function and can solubilize heat-aggregated beta-galactosidase (beta-gal) in the absence of the Hsp70 system. Our fitted structures confirm that the subunit arrangement of Hsp104 is similar to other AAA+ machines, and place the M-domains on the Hsp104 exterior, where they can potentially interact with large, aggregated proteins.

Structural basis of impaired disaggregase function in the oxidation-sensitive SKD3 mutant causing 3-methylglutaconic aciduria.

Mitochondria are critical to cellular and organismal health. To prevent damage, mitochondria have evolved protein quality control machines to survey and maintain the mitochondrial proteome. SKD3, also known as CLPB, is a ring-forming, ATP-fueled protein disaggregase essential for preserving mitochondrial integrity and structure. SKD3 deficiency causes 3-methylglutaconic aciduria type VII (MGCA7) and early death in infants, while mutations in the ATPase domain impair protein disaggregation with the observed loss-of-function correlating with disease severity. How mutations in the non-catalytic N-domain cause disease is unknown. Here, we show that the disease-associated N-domain mutation, Y272C, forms an intramolecular disulfide bond with Cys267 and severely impairs SKD3Y272C function under oxidizing conditions and in living cells. While Cys267 and Tyr272 are found in all SKD3 isoforms, isoform-1 features an additional α-helix that may compete with substrate-binding as suggested by crystal structure analyses and in silico modeling, underscoring the importance of the N-domain to SKD3 function.

Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer.

Low response rates and immune-related adverse events limit the remarkable impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) and less than optimal dosing of these inhibitors limited their clinical efficacy as monotherapies. We discovered that the natural product Enniatin A (EnnA) targets Hsp90 and destabilizes its client oncoproteins without inducing an HSR. EnnA triggers immunogenic cell death in triple-negative breast cancer (TNBC) syngeneic mouse models and exhibits superior antitumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment (TME) to promote CD8+ T cell-dependent antitumor immunity by reducing PD-L1 levels and activating the chemokine receptor CX3CR1 pathway. These findings provide strong evidence for transforming the immunosuppressive TME into a more tumor-hostile milieu by engaging Hsp90 with therapeutic agents involving novel mechanisms of action.

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8-9, 2022.

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8-9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.

Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine.

Hsp104 is a double ring-forming AAA+ ATPase, which harnesses the energy of ATP binding and hydrolysis to rescue proteins from a previously aggregated state. Like other AAA+ machines, Hsp104 features conserved cis- and trans-acting elements, which are hallmarks of AAA+ members and are essential to Hsp104 function. Despite these similarities, it was recently proposed that Hsp104 is an atypical AAA+ ATPase, which markedly differs in 3D structure from other AAA+ machines. Consequently, it was proposed that arginines found in the non-conserved M-domain, but not the predicted Arg-fingers, serve the role of the critical trans-acting element in Hsp104. While the structural discrepancy has been resolved, the role of the Arg-finger residues in Hsp104 remains controversial. Here, we exploited the ability of Hsp104 variants featuring mutations in one ring to retain ATPase and chaperone activities, to elucidate the functional role of the predicted Arg-finger residues. We found that the evolutionarily conserved Arg-fingers are absolutely essential for ATP hydrolysis but are dispensable for hexamer assembly in Hsp104. On the other hand, M-domain arginines are not strictly required for ATP hydrolysis and affect the ATPase and chaperone activities in a complex manner. Our results confirm that Hsp104 is not an atypical AAA+ ATPase, and uses conserved structural elements common to diverse AAA+ machines to drive the mechanical unfolding of aggregated proteins.

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

The Mitochondrial Protease LonP1 Promotes Proteasome Inhibitor Resistance in Multiple Myeloma.

Multiple myeloma and its precursor plasma cell dyscrasias affect 3% of the elderly population in the US. Proteasome inhibitors are an essential part of several standard drug combinations used to treat this incurable cancer. These drugs interfere with the main pathway of protein degradation and lead to the accumulation of damaged proteins inside cells. Despite promising initial responses, multiple myeloma cells eventually become drug resistant in most patients. The biology behind relapsed/refractory multiple myeloma is complex and poorly understood. Several studies provide evidence that in addition to the proteasome, mitochondrial proteases can also contribute to protein quality control outside of mitochondria. We therefore hypothesized that mitochondrial proteases might counterbalance protein degradation in cancer cells treated with proteasome inhibitors. Using clinical and experimental data, we found that overexpression of the mitochondrial matrix protease LonP1 (Lon Peptidase 1) reduces the efficacy of proteasome inhibitors. Some proteasome inhibitors partially crossinhibit LonP1. However, we show that the resistance effect of LonP1 also occurs when using drugs that do not block this protease, suggesting that LonP1 can compensate for loss of proteasome activity. These results indicate that targeting both the proteasome and mitochondrial proteases such as LonP1 could be beneficial for treatment of multiple myeloma.

Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex.

A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone-protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP.

Crystallization and preliminary X-ray crystallographic analysis of a GroEL1 fragment from Mycobacterium tuberculosis H37Rv.

Full-length GroEL1 from Mycobacterium tuberculosis H37Rv was cloned, overexpressed and purified. Crystals were obtained by the hanging-drop vapor-diffusion method and contained a 23 kDa GroEL1 fragment. A complete native data set was collected from a single frozen crystal that belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 75.47, b = 78.67, c = 34.89 A, alpha = beta = gamma = 90 degrees , and diffracted to 2.2 A resolution on a home X-ray source.