Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.The mitochondrion plays important roles in cell physiology. The mitochondrion functions as the “cellular power house” by generating most of the supply of ATP for the cell. In addition, the mitochondrion is involved in a number of critical cellular processes including the synthesis of metabolites, lipid metabolism, free radical production, and metal ion homeostasis. The mitochondrion consists of four compartments, the outer membrane, the inner membrane, the intermembrane space, and the mitochondrial matrix. The mitochondrion contains a large number of proteins (1), but only a few of these are translated within the mitochondrion (2). Therefore, the majority of the mitochondrial proteins are synthesized in the cytosol and translocated into the mitochondrion.The mitochondrial preproteins contain specific targeting signals to reach the correct compartments within the mitochondria. The mitochondrial matrix preproteins contain N-terminal targeting sequences that form the short amphipathic helices (2–6). On the other hand, some mitochondrial proteins of the inner and outer membrane contain internal targeting signals within the mature proteins (7). The mitochondrion has developed a set of delicate translocons to transport the preproteins into the mitochondrial compartments, one translocase of the outer membrane (TOM)² and two translocases of the inner membrane (TIM23 and TIM22) (4, 5, 8). The TOM complex has two surface receptors, Tom20 and Tom70 (9, 10). Tom20 recognizes the N-terminal mitochondrial targeting signals from the preproteins, whereas Tom70 binds to internal targeting sequences of preproteins such as the multi-transmembrane carrier proteins residing in the mitochondrial membranes (9–12). The crystal structure of Saccharomyces cerevisiae Tom70 revealed that Tom70 contained 11 TPR motifs, and the TPR motifs were clustered into two domains. The three TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70/Hsp90, whereas the C-terminal domain of Tom70p contains a large preprotein-binding pocket (13).Molecular chaperones Hsp70 and Hsp90 play important roles in targeting the preproteins to TOM complex (14). Hsp70 and Hsp90 can protect these preproteins from aggregation in the cytosol (15). The C-terminal EEVD motifs of Hsp70/Hsp90 may interact directly with the N-terminal domain of Tom70p to target the preproteins to TOM complex (13, 14, 16). The C-terminal EEVD motif of Hsp70/Hsp90 has been indicated to bind several proteins containing TPR motifs including Hop and CHIP. The complex structures for the Hsp70/Hsp90 EEVD motif and Hop and CHIP TPR regions have been determined (17–21).Tom71 (also known as Tom72) was identified as a homologue with Tom70 with high amino acid sequence identity (>50%) (22). Tom71 shares overlapping functions with Tom70 to transfer the preproteins and maintain the mitochondrial morphology (23, 24). In this study, we have determined the crystal structures of S. cerevisiae Tom71 and the complexes of Tom71 and Hsp70/Hsp90 C-terminal EEVD motifs. These structures suggest that the Hsp70/Hsp90 binding to Tom70/Tom71 may keep Tom70/Tom71 in the open state for receiving preproteins. The Hsp70/Hsp90 interactions may also increase the volume of the preprotein-binding pocket of Tom70/Tom71 and prepare Tom70/Tom71 for preprotein loading.     

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.Oligomeric heat shock protein 27 (Hsp27)² is a ubiquitous mammalian protein with a variety of functions in health and disease (1–8). These functions include ATP-independent chaperone activity in response to environmental stress, e.g. heat shock and oxidative stress, control of apoptosis, and regulation of actin cytoskeleton dynamics. Hsp27 is a member of the α-crystallin small heat shock protein family of which αB-crystallin is the archetype. These proteins are characterized by an α-crystallin domain of 80–90 residues consisting of roughly eight β-strands that form an intermolecular β-sheet interaction interface within a dimer, the basic building subunit of the oligomer (2, 4, 9–11).Hsp27 is in equilibrium between high molecular weight oligomers and much lower molecular weight multimers. It has been reported that unphosphorylated Hsp27 includes predominantly a distribution of high molecular species ranging in size from 12-mer to 35-mer (12–19). Phosphorylation of Hsp27 at serines 15, 78, and 82 by the p38-activated MAPKAP-2 kinase (20–22) or the use of the triple Ser-to-Asp phospho-mimic results in a major shift in the equilibrium toward much smaller multimers (23) and in an alteration of its function (1, 3, 6, 7, 24, 25). The size distribution of the smaller species has been reported to be between monomer and tetramer (12–16, 18, 19).Small heat shock proteins, including Hsp27, behave as ATP-independent molecular chaperones during cellular heat shock. They bind partially unfolded proteins and prevent their aggregation until the proteins can be refolded by larger ATP-dependent chaperones or are digested (7, 8, 26). This function includes the up-regulation and/or phosphorylation of Hsp27.It is not entirely clear what the role of Hsp27 size and phosphorylation state plays in its heat shock function because there are conflicting results in the literature. Some in vitro studies concluded that the unphosphorylated oligomeric Hsp27 (or the murine isoform Hsp25) protects proteins against aggregation better than does the phosphorylation mimic (13, 19, 27), whereas others found no difference (16, 28, 29), and still other studies found that the mimic protects better than does the unphosphorylated wild type (27, 30, 31). In-cell studies found that phosphorylation of Hsp27 was essential for thermo-protection of actin filaments (32), and the Hsp27 phosphorylation mimic decreased inclusion body formation better than did unphosphorylated Hsp27 (33). This study was undertaken to investigate the molecular chaperone function of Hsp27 by correlating chaperone activity with Hsp27 size and by comparing fully phosphorylated Hsp27 with its phospho-mimic.     

Sti1 Regulation of Hsp70 and Hsp90 Is Critical for Curing of Saccharomyces cerevisiae [PSI+] Prions by Hsp104

Although propagation of Saccharomyces cerevisiae prions requires Hsp104 protein disaggregating activity, overproducing Hsp104 “cures” cells of [PSI⁺] prions. Earlier evidence suggests that the Hsp70 mutant Ssa1-21 impairs [PSI⁺] by a related mechanism. Here, we confirm this link by finding that deletion of STI1 both suppresses Ssa1-21 impairment of [PSI⁺] and blocks Hsp104 curing of [PSI⁺]. Hsp104''s tetratricopeptide repeat (TPR) interaction motif was dispensable for curing; however, cells expressing Sti1 defective in Hsp70 or Hsp90 interaction cured less efficiently, and the Hsp90 inhibitor radicicol abolished curing, implying that Sti1 acts in curing through Hsp70 and Hsp90 interactions. Accordingly, strains lacking constitutive or inducible Hsp90 isoforms cured at reduced rates. We confirm an earlier finding that elevating free ubiquitin levels enhances curing, but it did not overcome inhibition of curing caused by Hsp90 defects, suggesting that Hsp90 machinery is important for the contribution of ubiquitin to curing. We also find curing associated with cell division. Our findings point to crucial roles of Hsp70, Sti1, and Hsp90 for efficient curing by overexpressed Hsp104 and provide evidence supporting the earlier suggestion that destruction of prions by protein disaggregation does not adequately explain the curing.Saccharomyces cerevisiae prions are self-replicating misfolded forms of normal cellular proteins. They are believed to propagate as amyloid, which is a highly ordered fibrous aggregate. What triggers prion formation is uncertain, but in order to be maintained in an expanding yeast population, prions must grow, replicate, and be transmitted to daughter cells during cell division. Growth occurs when soluble protein joins the fiber ends and is converted into the prion form (30, 52, 58). Replication is associated with fragmentation of prion polymers, which generates new prions from preexisting material (37, 50). Transmission is believed to occur by passive diffusion of prions with cytoplasm (57).Although it is uncertain to what extent cellular factors influence growth or transmission of prions, it is clear that the Hsp104 disaggregation machinery is necessary for prion replication (10, 17, 55, 70). Hsp104 is a hexameric AAA+ chaperone that protects cells from a variety of stresses by resolubilizing proteins from aggregates (24, 25, 53). With help from Hsp70 and Hsp40, it extracts monomers from aggregates and extrudes them through its central pore (24, 41, 68). This machinery could act in prion replication by extracting monomers from amyloid fibers (29, 68), which would destabilize the fibers, causing them to break into more numerous pieces that each can continue to propagate the prion.Paradoxically, overexpressing Hsp104 very efficiently “cures” cells of the [PSI⁺] prion, which is composed of the translation termination factor Sup35 (10). A widely held view of this curing is that elevating the cellular protein disaggregation activity causes complete destruction of prions. However, elevating Hsp104 has little or no effect on most other amyloidogenic prions (15, 16, 38, 47, 54, 66), although it can be inferred to cure [MCA] prions in cells also propagating a prion of an Mca1-Sup35 fusion (49). Together, these results suggest that prions of Sup35, and perhaps those of Mca1, are particularly sensitive to Hsp104 disaggregation activity. Alternatively, something in addition to or other than a simple increase in protein disaggregation is involved in the curing.Although protein disaggregation activity of Hsp104 is required for both thermotolerance and prion propagation, we and others have identified mutations in Hsp104 that affect these processes separately (27, 32, 39, 60). The ability of Hsp104 to thread proteins through its central pore, however, is required for both processes (29, 41, 68), so this distinction in Hsp104 function could be due to differences in how Hsp104 interacts with amorphous aggregates of thermally denatured proteins and highly ordered prion aggregates or with cofactors that interact with the different prions as substrates. In any scenario, efficiency and specificity of Hsp104 function are affected by interactions with other components of the disaggregation machinery, in particular the Hsp70s and Hsp40s, which are believed to interact first with substrates to facilitate action of Hsp100 family disaggregases (2, 71, 72).Increasing expression of either ubiquitin (Ub) or Ssb, an Hsp70 that has roles in protein translation and proteasome degradation, enhances Hsp104 curing of [PSI⁺] (3, 11, 12). Predictably, reducing expression of either of them reduces curing efficiency. The mechanisms underlying these effects are unknown, but the combined effects of Ssb and Ub are additive, suggesting that they act in different pathways. The role of Ub is indirect, as Sup35 is neither ubiquitylated nor degraded during curing. Whether other chaperones are involved in the effects of Ub on curing has not been investigated.Earlier we isolated a mutant of the Hsp70 Ssa1, designated Ssa1-21, that weakens and destabilizes [PSI⁺] propagation (33). We later isolated several Hsp104 mutants that suppress this antiprion effect (29). The Hsp104 mutants retain normal functions in thermotolerance, protein disaggregation, and prion propagation, but when overexpressed, they are unable to cure [PSI⁺], even in wild-type cells. These findings argue against a specific hypersensitivity of [PSI⁺] to disaggregation and support the notion that something distinct from or in addition to complete destruction of prions is involved in the curing. They also imply that Ssa1-21 and elevated Hsp104 inhibit [PSI⁺] prions by similar mechanisms. A prediction from this conclusion is that other suppressors of Ssa1-21 will also inhibit curing of [PSI⁺] by overexpressed Hsp104. Indeed, we find here that alterations that suppress Ssa1-21 inhibition of [PSI⁺] do interfere with curing of [PSI⁺] by overexpressed Hsp104. We also provide evidence that Hsp90 has a critical role in this curing and that the ability of Ub to enhance curing depends on proper function of Hsp90 machinery.     

Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.Stress-inducible phosphoprotein I (STI1)¹ is a conserved cochaperone protein that assists Hsp90 in managing client proteins, by mediating the transfer of proteins between Hsp70 and Hsp90 (1–3). STI1 contains several tetratricopeptide-repeat domains (TRP) that can serve as interaction modules with Hsp90 and Hsp70 (4). STI1 helps to drive the sequential steps involved in the Hsp90 chaperone machinery (5) and regulates the ATPase activity of Hsp90 (6, 7). STI1 is also secreted by distinct cells (8–12), using a noncanonical mechanism involving extracellular vesicles (11). Secreted STI1 can activate multiple signaling pathways in distinct cell types (8–10, 13–18).Elimination of STI1 in yeast sensitizes cells to Hsp90 inhibitors, but it is not by itself lethal (19). STI1 can also be eliminated in C. elegans, although it results in decreased life span (20). In contrast, STI1 mutant mice do not survive E10.5 and present several morphological defects, owing to decreased levels of several Hsp90-client proteins (21). Mouse embryonic fibroblasts obtained from STI1-deficient embryos also fail to thrive and present increased levels of the DNA damage marker γ-H2AX, suggestive of increased cellular stress (21). Hence, in mammals STI1 seems to play additional roles in cellular survival that are not yet fully understood.STI1 is abundantly expressed in the cytoplasm of cells, but can also be found in the Golgi (22), in vesicles and in multivesicular bodies (11). Moreover, this cochaperone has been shown to shuttle between the cytoplasm and the nucleus in cell lines (23). Cellular stress, arrest in G1/S phase of the cell cycle and phosphorylation are factors that seem to regulate STI1 nuclear localization (23, 24). Presumably nuclear STI1 can regulate chaperone activity, but whether it can interact with nuclear proteins is unknown.Previous experiments using cell lines have shown that knockdown of STI1 increases susceptibility of cells to irradiation (25). Whether changes in STI1 levels in primary differentiated cells, such as astrocytes, may affect their response to irradiation stress is unknown. This is of interest, as astrocytes, which can give rise to distinct tumor cells, are highly radioresistant (26). Indeed, astrocytes have a noncanonical DNA damage response (DDR) to irradiation (26). Here we show that STI1 undergoes nuclear translocation in astrocytes after γ-radiation-induced DNA damage. Moreover, astrocytes haploinsufficient for STI1 are more susceptible to cell death induced by irradiation. To understand potential mechanisms involved with STI1 nuclear retention, we have performed yeast-two hybrid screenings to identify STI1 nuclear partners. We identified protein inhibitor of activated STAT (PIAS1) as a direct interactor of STI1 and provide evidence that it acts as a small ubiquitin-like modifier (SUMO) E3 ligase for STI1. We show this interaction is involved with STI1 nuclear retention after irradiation. Interestingly, tissue microarray analysis demonstrated that higher PIAS1 levels are found in glioblastoma multiforme (GBM) when compared with non-neoplastic tissue. Furthermore, we uncovered a positive relationship between increased PIAS1 expression in GBMs and augmented STI1 nuclear localization. Our results reveal a novel mechanism by which increased expression of PIAS1, as observed in GBM, can increase the retention of nuclear STI1, a critical regulator of the chaperone machinery.     

A Straight Path to Circular Proteins

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23).     

Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins

The conserved eukaryotic protein SGT1 (suppressor of G₂ allele of skp1) participates in diverse physiological processes such as cell cycle progression in yeast, plant immunity against pathogens and plant hormone signalling. Recent genetic and biochemical studies suggest that SGT1 functions as a novel co-chaperone for cytosolic/nuclear HSP90 and HSP70 molecular chaperones in the folding and maturation of substrate proteins. Since proteins containing the leucine-rich repeat (LRR) protein-protein interaction motif are overrepresented in SGT1-dependent phenomena, we consider whether LRR-containing proteins are preferential substrates of an SGT1/HSP70/HSP90 complex. Such a chaperone organisation is reminiscent of the HOP/HSP70/HSP90 machinery which controls maturation and activation of glucocorticoid receptors in animals. Drawing on this parallel, we discuss the possible contribution of an SGT1-chaperone complex in the folding and maturation of LRR-containing proteins and its evolutionary consequences for the emergence of novel LRR interaction surfaces.Key words: heat shock protein, SGT1, co-chaperone, HSP90, HSP70, leucine-rich repeat, LRR, resistance, SCF, ubiquitinThe proper folding and maturation of proteins is essential for cell viability during de novo protein synthesis, translocation, complex assembly or under denaturing stress conditions. A complex machinery composed of molecular chaperones (heat-shock proteins, HSPs) and their modulators known as co-chaperones, catalyzes these protein folding events.^1,2 In animals, defects in the chaperone machinery is implicated in an increasing number of diseases such as cancers, susceptibility to viruses, neurodegenerative disease and cystic fibrosis, and thus it has become a major pharmacological target.^3,4 In plants, molecular genetic studies have identified chaperones and co-chaperones as components of various physiological responses and are now starting to yield important information on how chaperones work. Notably, processes in plant innate immunity rely on the HSP70 and HSP90^5–7 chaperones as well as two recently characterised co-chaperones, RAR1 (required for Mla12 resistance) and SGT1 (suppressor of G₂ allele of skp1).^8–11SGT1 is a highly conserved and essential co-chaperone in eukaryotes and is organized into three structural domains: a tetratricopeptide repeat (TPR), a CHORD/SGT1 (CS) and an SGT1-specific (SGS) domain (Fig. 1A). SGT1 is involved in a number of apparently unrelated physiological responses ranging from cell cycle progression and adenylyl cyclase activity in yeast to plant immunity against pathogens, heat shock tolerance and plant hormone (auxin and jasmonic acid) signalling.^7–9,12,13 Because the SGT1 TPR domain is able to interact with Skp1, SGT1 was initially believed to be a component of SCF (Skp1/Cullin/F-box) E3 ubiquitin ligases that are important for auxin/JA signalling in plants and cell cycle progression in yeast.^13,14 However, mutagenesis of SGT1 revealed that the TPR domain is dispensable for plant immunity and auxin signalling.¹⁵ Also, SGT1-Skp1 interaction was not observed in Arabidopsis.¹³ More relevant to SGT1 functions appear to be the CS and SGS domains.¹⁶ The former is necessary and sufficient for RAR1 and HSP90 binding. The latter is the most conserved of all SGT1 domains and the site of numerous disabling mutations.^14,16,17Open in a separate windowFigure 1Model for SGT1/chaperone complex functions in the folding of LRR-containing proteins. (A) The structural domains of SGT1, their sites of action (above) and respective binding partners (below) are shown. N- and C-termini are indicated. TPR, tetratricopeptide repeat; CS, CHORD/SGT1; SGS, SGT1-specific. (B) Conceptual analogy between steroid receptor folding by the HOP/chaperone machinery and LRR protein folding by the SGT1/chaperone machinery. LRR motifs are overrepresented in processes requiring SGT1 such as plant immune receptor signalling, yeast adenylyl cyclase activity and plant or yeast SCF (Skp1/Cullin/F-box) E3 ubiquitin ligase activities. (C) Opposite forces drive LRR evolution. Structure of LRRs 16 to 18 of the F-box auxin receptor TIR1 is displayed as an illustration of the LRR folds.³⁰ Leucine/isoleucine residues (side chain displayed in yellow) are under strong purifying selection and build the hydrophobic LRR backbone (Left). By contrast, solvent-exposed residues of the β-strands define a polymorphic and hydrophilic binding surface conferring substrate specificity to the LRR (Right) and are often under diversifying selection.We recently demonstrated that Arabidopsis SGT1 interacts stably through its SGS domain with cytosolic/nuclear HSP70 chaperones.⁷ The SGS domain was both necessary and sufficient for HSP70 binding and mutations affecting SGT1-HSP70 interaction compromised JA/auxin signalling and immune responses. An independent in vitro study also found interaction between human SGT1 and HSP70.¹⁸ The finding that SGT1 protein interacts directly with two chaperones (HSP90/70) and one co-chaperone (RAR1) reinforces the notion that SGT1 behaves as a co-chaperone, nucleating a larger chaperone complex that is essential for eukaryotic physiology. A future challenge will be to dissect the chaperone network at the molecular and subcellular levels. In plant cells, SGT1 localization appears to be highly dynamic with conditional nuclear localization⁷ and its association with HSP90 was recently shown to be modulated in vitro by RAR1.¹⁶A co-chaperone function suits SGT1 diverse physiological roles better than a specific contribution to SCF ubiquitin E3 ligases. Because SGT1 does not affect HSP90 ATPase activity, SGT1 was proposed rather as a scaffold protein.^16,19 In the light of our findings and earlier studies,²⁰ SGT1 is reminiscent of HOP (Hsp70/Hsp90 organizing protein) which links HSP90 and HSP70 activities and mediates optimal substrate channelling between the two chaperones (Fig. 1B).²¹ While the contribution of the HSP70/HOP/HSP90 to the maturation of glucocorticoid receptors is well established,²¹ direct substrates of an HSP70/SGT1/HSP90 complex remain elusive.It is interesting that SGT1 appears to share a functional link with leucine-rich repeat- (LRR) containing proteins although LRR domains are not so widespread in eukaryotes. For example, plant SGT1 affects the activities of the SCF^TIR1 and SCF^COI1 E3 ligase complexes whose F-box proteins contain LRRs.¹³ Moreover, plant intracellular immune receptors comprise a large group of LRR proteins that recruit SGT1.^8,9 LRRs are also found in yeast adenylyl cyclase Cyr1p and the F-box protein Grr1p which is required for SGT1-dependent cyclin destruction during G₁/S transition.^12,14 Yeast 2-hybrid interaction assays also revealed that yeast and plant SGT1 tend to associate directly or indirectly with LRR proteins.^12,22,23 We speculate that SGT1 bridges the HSP90-HSC70 chaperone machinery with LRR proteins during complex maturation and/or activation. The only other structural motif linked to SGT1 are WD40 domains found in yeast Cdc4p F-box protein and SGT1 interactors identified in yeast two-hybrid screens.¹²What mechanisms underlie a preferential SGT1-LRR interaction? HSP70/SGT1/HSP90 may have co-evolved to assist specifically in folding and maturation of LRR proteins. Alternatively, LRR structures may have an intrinsically greater need for chaperoning activity to fold compared to other motifs. These two scenarios are not mutually exclusive. The LRR domain contains multiple 20 to 29 amino acid repeats, forming an α/β horseshoe fold.²⁴ Each repeat is rich in hydrophobic leucine/isoleucine residues which are buried inside the structure and form the structural backbone of the motif (Fig. 1C, left). Such residues are under strong purifying selection to preserve structure. These hydrophobic residues would render the LRR a possible HSP70 substrate.²⁵ By contrast, hydrophilic solvent- exposed residues of the β strands build a surface which confers ligand recognition specificity of the LRRs (Fig. 1C). In many plant immune receptors for instance, these residues are under diversifying selection that is likely to favour the emergence of novel pathogen recognition specificities in response to pathogen evolution.²⁶ The LRR domain of such a protein has to survive such antagonist selection forces and yet remain functional. Under strong selection pressure, LRR proteins might need to accommodate less stable LRRs because their recognition specificities are advantageous. This could be the point at which LRRs benefit most from a chaperoning machinery such as the HSP90/SGT1/HSP70 complex. This picture is reminiscent of the genetic buffering that HSP90 exerts on many traits to mask mutations that would normally be deleterious to protein folding and/or function, as revealed in Drosophila and Arabidopsis.²⁷ It will be interesting to test whether the HSP90/SGT1/HSP70 complex acts as a buffer for genetic variation, favouring the emergence of novel LRR recognition surfaces in, for example, highly co-evolved plant-pathogen interactions.^28,29     

Engineering therapeutic protein disaggregases

Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD.We urgently need to pioneer game-changing solutions to remedy a number of increasingly prevalent and fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD; Cushman et al., 2010 ; Jackrel and Shorter, 2015 ). These disorders relentlessly erode our morale and economic resources. Aging is the major risk factor for all of these diseases, which threaten public health on a global scale and represent a severe impediment to living longer lives. A number of promising drugs have emerged to treat cancer and heart disease, but, distressingly, this is not the case for these and other neurodegenerative diseases, for which drug pipelines lie dormant and empty. This situation is unacceptable, and an impending healthcare crisis looms worldwide as population demographics inexorably shift toward older age groups.ALS, PD, AD, and related neurodegenerative disorders are unified by a common underlying theme: the misfolding and aggregation of specific proteins (characteristic for each disease) in the CNS (Cushman et al., 2010 ; Eisele et al., 2015 ). Thus, in ALS, typically an RNA-binding protein with a prion-like domain, TDP-43, mislocalizes from the nucleus to cytoplasmic inclusions in degenerating motor neurons (Neumann et al., 2006 ; Gitler and Shorter, 2011 ; King et al., 2012 ; Robberecht and Philips, 2013 ; March et al., 2016 ). In PD, α-synuclein forms toxic soluble oligomers and cytoplasmic aggregates, termed Lewy bodies, in degenerating dopaminergic neurons (Dehay et al., 2015 ). By contrast, in AD, amyloid-β (Aβ) peptides form extracellular plaques and the microtubule-binding protein, tau, forms cytoplasmic neurofibrillary tangles in afflicted brain regions (Jucker and Walker, 2011 ). Typically, these disorders are categorized into ∼80–90% sporadic cases and ∼10–20% familial cases. Familial forms of disease often have clear genetic causes, which might one day be amenable to gene editing via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 therapeutics if critical safety and ethical concerns can be successfully addressed and respected (Doudna and Charpentier, 2014 ; Baltimore et al., 2015 ; Rahdar et al., 2015 ; Callaway, 2016 ). However, the more common sporadic forms of disease often have no clear underlying genetics, and wild-type proteins misfold (Cushman et al., 2010 ; Jucker and Walker, 2011 ; Robberecht and Philips, 2013 ; Dehay et al., 2015 ). Consequently, therapeutic agents that directly target and safely reverse deleterious protein misfolding are likely to have broad utility (Eisele et al., 2015 ).There are no treatments that directly target the reversal of the protein-misfolding phenomena that underlie these disorders (Jackrel and Shorter, 2015 ). Strategies that directly reverse protein misfolding and restore proteins to native form and function could, in principle, eradicate any severely damaging loss-of-function or toxic gain-of-function phenotypes caused by misfolded conformers (Figure 1; Jackrel and Shorter, 2015 ). Moreover, therapeutic disaggregases would dismantle self-templating amyloid or prion structures, which spread pathology and nucleate formation of neurotoxic, soluble oligomers (Figure 1; Cushman et al., 2010 ; Cohen et al., 2013 ; Guo and Lee, 2014 ; Jackrel and Shorter, 2015 ). My group has endeavored to engineer and evolve Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast (DeSantis and Shorter, 2012 ; Sweeny and Shorter, 2015 ), to more effectively disaggregate misfolded proteins connected with various neurodegenerative disorders, including ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). Although wild-type Hsp104 can disaggregate diverse amyloid and prion conformers, as well as toxic soluble oligomers (Lo Bianco et al., 2008 ; DeSantis et al., 2012 ), its activity against human neurodegenerative disease proteins is suboptimal. Is it even possible to improve on existing Hsp104 disaggregase activity, which has been wrought over the course of millions of years of evolution?Open in a separate windowFIGURE 1:Therapeutic protein disaggregases. Two malicious problems are commonly associated with protein misfolding into disordered aggregates, toxic oligomers, and cross–β amyloid or prion fibrils: 1) a toxic gain of function of the protein in various misfolded states; and 2) a loss of function of the protein in the various misfolded states. These problems can contribute to the etiology of diverse neurodegenerative diseases in a combinatorial or mutually exclusive manner. A therapeutic protein disaggregase would reverse protein misfolding and recover natively folded functional proteins from disordered aggregates, toxic oligomers, and cross–β amyloid or prion fibrils. In this way, any toxic gain of function or toxic loss of function caused by protein misfolding would be simultaneously reversed. Ideally, all toxic misfolded conformers would be purged. Therapeutic protein disaggregases could thus have broad utility for various fatal neurodegenerative diseases.Remarkably, the answer to this question is yes! We used nimble yeast models of neurodegenerative proteinopathies (Outeiro and Lindquist, 2003 ; Gitler, 2008 ; Johnson et al., 2008 ; Sun et al., 2011 ; Khurana et al., 2015 ) as a platform to isolate enhanced disaggregases from large libraries of Hsp104 variants generated by error-prone PCR (Jackrel et al., 2014b ). In this way, we reprogrammed Hsp104 to yield the first disaggregases that reverse TDP-43, FUS (another RNA-binding protein with a prion-like domain connected to ALS), and α-synuclein (connected to PD) aggregation and proteotoxicity (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ; Torrente et al., 2016 ). Remarkably, a therapeutic gain of Hsp104 function could be elicited by a single missense mutation (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Under conditions in which Hsp104 was ineffective, potentiated Hsp104 variants dissolved protein inclusions, restored protein localization (e.g., TDP-43 returned to the nucleus from cytoplasmic inclusions), suppressed proteotoxicity, and attenuated dopaminergic neurodegeneration in a Caenorhabditis elegans PD model (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Remarkably, these therapeutic modalities originated from degenerate loss of amino acid identity at select positions of Hsp104 rather than specific mutation (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Some of these changes were remarkably small (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Thus, potentiated Hsp104 variants could be generated by removal of a methyl group from a single side chain or addition or removal of a single methylene bridge from a single side chain (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Thus, small molecules that bind in accessible regions of Hsp104 rich in potentiating mutations might also be able to enhance activity. However, a small-scale screen for small-molecule modulators of Hsp104 revealed only inhibitors (Torrente et al., 2014 ). Nonetheless, our work has established that disease-associated aggregates and amyloid are tractable targets and that enhanced artificial disaggregases can restore proteostasis and mitigate neurodegeneration (Jackrel and Shorter, 2015 ).One surprising aspect of this work is just how many Hsp104 variants we could isolate with potentiated activity. We now have hundreds (Jackrel et al., 2014a ; Jackrel et al., 2015 ). Typically, potentiated Hsp104 variants displayed enhanced activity against several neurodegenerative disease proteins. For example, Hsp104^A503S rescued the aggregation and toxicity of TDP-43, FUS, TAF15, and α-synuclein (Jackrel et al., 2014a ; Jackrel and Shorter, 2014 ). By contrast, some potentiated Hsp104 variants rescued only the aggregation and toxicity of a subset of disease proteins. For example, Hsp104^D498V rescued only the aggregation and toxicity of FUS and α-synuclein (Jackrel et al., 2014a ). A challenge that lies ahead is to engineer potentiated Hsp104 variants that are highly substrate specific to mitigate any potential off-target effects, should they arise (Jackrel and Shorter, 2015 ).Very small changes in primary sequence yield potentiated Hsp104 variants. However, Hsp104 has no metazoan homologue (Erives and Fassler, 2015 ). Now comes the important point. Neuroprotection could be broadly achieved by making very subtle modifications to existing human chaperones with newly appreciated disaggregase activity—for example, Hsp110, Hsp70, and Hsp40 (Torrente and Shorter, 2013 ) and HtrA1 (Poepsel et al., 2015 ).Whether Metazoa even possess a powerful protein disaggregation and reactivation machinery had been a long-standing enigma (Torrente and Shorter, 2013 ). However, it has recently emerged that two metazoan chaperone systems—1) Hsp110, Hsp70, and Hsp40 (Torrente and Shorter, 2013 ) and 2) HtrA1 (Poepsel et al., 2015 )—possess disaggregase activity that could be therapeutically harnessed or stimulated to reverse deleterious protein misfolding in neurodegenerative disease. I suspect that Metazoa harbor additional disaggregase systems that remain to be identified (Guo et al., 2014 ). Whether due to vicissitudes of aging, environment, or genetic background, these disaggregase systems fail in the context of ALS, PD, and AD. Based on the surprising precedent of our potentiated versions of Hsp104 (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ), I hypothesize that it is possible to engineer and evolve potentiated variants of these human protein disaggregases to more effectively counter deleterious misfolding events in ALS, PD, and AD (Torrente and Shorter, 2013 ; Mack and Shorter, 2016 ).Using classical biochemical reconstitution, it was discovered that one mammalian protein-disaggregase system comprises three molecular chaperones—Hsp110, Hsp70, and Hsp40—which synergize to dissolve and reactivate model proteins trapped in disordered aggregates and can even depolymerize amyloid fibrils formed by α-synuclein from their ends (Shorter, 2011 ; Duennwald et al., 2012 ; Torrente and Shorter, 2013 ). Hsp110, Hsp70, and Hsp40 isoforms are found in the cytoplasm, nucleus, and endoplasmic reticulum, which suggest that protein disaggregation and reactivation can occur in several compartments (Finka et al., 2015 ). Subsequent studies suggest that this system may be more powerful than initially anticipated (Rampelt et al., 2012 ; Mattoo et al., 2013 ; Gao et al., 2015 ; Nillegoda et al., 2015 ). Nonetheless, this system must become overwhelmed in neurodegenerative disorders. Perhaps selectively vulnerable neurons display particular deficits in this machinery. Hence, potentiating the activity of this system via engineering could be extremely valuable. It is promising that directed evolution studies yielded DnaK (Hsp70 in Escherichia coli) variants with improved ability to refold specific substrates (Aponte et al., 2010 ; Schweizer et al., 2011 ; Mack and Shorter, 2016 ), but whether this can be extended to human Hsp70 and the disaggregation of neurodegenerative disease proteins is uncertain.It is exciting that recent studies have revealed that HtrA1, a homo-oligomeric PDZ serine protease, can dissolve and degrade AD-linked tau and Aβ42 fibrils in an ATP-independent manner (Tennstaedt et al., 2012 ; Poepsel et al., 2015 ). HtrA1 first dissolves tau and Aβ42 fibrils and then degrades them, as protease-defective HtrA1 variants dissolve fibrils without degrading them (Poepsel et al., 2015 ). HtrA1 is found in the cytoplasm (∼30%) but is also secreted (∼70%; Poepsel et al., 2015 ). Indeed, HtrA1 is known to degrade substrates in both the extracellular space and the cytoplasm (Chien et al., 2009 ; Campioni et al., 2010 ; Tiaden and Richards, 2013 ). Thus HtrA1 could dissolve Aβ42 fibrils in the extracellular space and tau fibrils in the cytoplasm and simultaneously destroy the two cardinal features of AD (Poepsel et al., 2015 ). I suspect that this system becomes overwhelmed or is insufficient in AD, and thus potentiating and tailoring HtrA1 disaggregase activity could be a valuable therapeutic strategy. For example, it might be advantageous to simply degrade Aβ42 after disaggregation if the peptide has no beneficial function. Thus HtrA1 variants with enhanced disaggregation and degradation activity against Aβ42 could be extremely useful. However, Aβ42 (and related Aβ peptides) may have physiological functions that are presently underappreciated (Soscia et al., 2010 ; Fedele et al., 2015 ), in which case HtrA1 variants with enhanced disaggregase activity but reduced proteolytic activity could be vital. HtrA1 variants with enhanced disaggregase activity but reduced proteolytic activity may also be particularly important to recover functional tau from neurofibrillary tangles to reverse loss of tau function in AD and various tauopathies (Santacruz et al., 2005 ; Trojanowski and Lee, 2005 ; Dixit et al., 2008 ).I suggest that relatively small changes in primary sequence will yield large increases in disaggregase activity for these systems as they do for Hsp104 (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). If true, this would further suggest that small molecules that bind in the appropriate regions of Hsp110, Hsp70, Hsp40, or HtrA1 might also enhance disaggregase activity. Thus, isolating small-molecule enhancers of the Hsp110, Hsp70, and Hsp40 or HtrA1 disaggregase systems could yield important therapeutics. Indeed, I hypothesize that enhancing the activity of the Hsp110, Hsp70, and Hsp40 or HtrA1 disaggregase system with specific small molecules will enable dissolution of toxic oligomeric and amyloid forms of various disease proteins and confer therapeutic benefits in ALS, PD, AD, and potentially other neurodegenerative disorders.It is intriguing that several small molecules are already known to enhance various aspects of Hsp70 chaperone activity (Pratt et al., 2015 ; Shrestha et al., 2016 ). These include MKT-077, JG-98, YM-1, YM-8, and 115-7c (Wisen et al., 2010 ; Pratt et al., 2015 ). It is not known whether any of these stimulates the disaggregase activity of the Hsp110, Hsp70, and Hsp40 system. MKT-077, JG-98, YM-1, and YM-8 are rhodocyanines that bind with low micromolar affinity to the nucleotide-binding domain of ADP- but not ATP-bound Hsp70, stabilizing the ADP-bound state (Pratt et al., 2015 ). The ADP-bound state of Hsp70 engages clients with higher affinity, and consequently MKT-077, JG-98, and YM-1 activate binding of Hsp70 to misfolded proteins (Wang et al., 2013 ; Pratt et al., 2015 ). Thus, under some conditions, these small molecules can promote folding of certain Hsp70 clients (Morishima et al., 2011 ; Pratt et al., 2015 ). However, prolonged interaction of clients with Hsp70 promotes their CHIP-dependent ubiquitylation and degradation in vivo (Morishima et al., 2011 ; Wang et al., 2013 ; Pratt et al., 2015 ). Intriguingly, YM-1 promotes clearance of polyglutamine oligomers and aggregates in cells (Wang et al., 2013 ; Pratt et al., 2015 ). MKT-0777, YM-1, JG-98, and YM-8 also promote clearance of tau and confer therapeutic benefit in tauopathy models (Abisambra et al., 2013 ; Miyata et al., 2013 ; Fontaine et al., 2015 ). Of importance, YM-8 is long lived in vivo and crosses the blood–brain barrier (Miyata et al., 2013 ). The dihydropyrimidine 115-7c activates Hsp70 ATPase turnover rate, promotes Hsp70 substrate refolding, and reduces α-synuclein aggregation in cell culture (Wisen et al., 2010 ; Kilpatrick et al., 2013 ). It binds to the IIA subdomain of Hsp70 and promotes the active Hsp70–Hsp40 complex (Wisen et al., 2010 ). Small-molecule enhancers of HtrA1 protease activity have also emerged (Jo et al., 2014 ). Thus it will be important to assess whether these small molecules enhance the activity of their respective disaggregases against various neurodegenerative substrates.Although small molecules that enhance disaggregase activity of endogenous human proteins might be the most immediately translatable, gene-, mRNA-, or protein-based therapies can also be envisioned. For example, adeno-associated viruses expressing enhanced disaggregases might be used to target degenerating neurons (Dong et al., 2005 ; Lo Bianco et al., 2008 ; Deverman et al., 2016 ). Alternatively, if viral vectors are undesirable, modified mRNAs might serve as an alternative to DNA-based gene therapy (Kormann et al., 2011 ). Protein-based therapeutics could also be explored. For example, intraperitoneal injection of human Hsp70 increased lifespan, delayed symptom onset, preserved motor function, and prolonged motor neuron viability in a mouse model of ALS (Gifondorwa et al., 2007 ; Gifondorwa et al., 2012 ). Several other studies suggest that exogenous delivery of Hsp70 can have beneficial, neuroprotective effects in mice (Nagel et al., 2008 ; Bobkova et al., 2014 ; Bobkova et al., 2015 ).Ultimately, if safety and ethical concerns can be overcome in a circumspect, risk-averse manner, CRISPR-Cas9–based therapeutics might even be used to genetically alter the underlying disaggregase to a potentiated form in selectively vulnerable neuronal populations. This approach might be particularly valuable if enhanced disaggregase activity is not detrimental in the long term. Moreover, stem cell–based therapies for replacing lost neurons could also be fortified to express enhanced disaggregase systems. Thus they would be endowed with resistance to potential infection by prion-like conformers that might have accumulated during disease progression (Cushman et al., 2010 ).Enhanced disaggregase activity is likely to be highly advantageous to neurons under circumstances in which protein misfolding has overwhelmed the system (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). However, inappropriate hyperactivity of protein disaggregases might also have detrimental, off-target effects under regular conditions in which protein misfolding is not an overwhelming issue (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Thus it may be advantageous to engineer enhanced protein disaggregases to be highly substrate specific. In this way, off-target effects would be readily avoided. There is strong precedent for directed evolution or engineering of specialized chaperone or protein activity from a generalist antecedent (Wang et al., 2002 ; Farrell et al., 2007 ; Smith et al., 2015 ). Thus, engineering specialist disaggregases for each disease substrate could be achieved. Alternatively, transient or intermittent doses of enhanced disaggregases at specific times or places where they are most needed would also minimize potentially toxic side effects. For example, enhanced disaggregase activity might be applied ephemerally to clear existing misfolded conformers and then be withdrawn once the endogenous proteostasis network regains control. Similarly, it is straightforward to envision administration of small-molecule enhancers of disaggregase activity in intermittent protocols that enable facile recovery from potential side effects (Fontaine et al., 2015 ). In this way, any adverse effects of enhanced protein-disaggregase activity under normal physiological conditions would be avoided. Many barriers will need to be safely overcome to implement a successful therapeutic disaggregase, including how to deliver enhanced disaggregase activity to exactly where it is needed. However, these obstacles are not a reason to be pessimistic. On the contrary, the isolation of engineered disaggregases that efficaciously reverse deleterious misfolding of neurodegenerative disease proteins directs our attention to considerably expand the environs in which they should be sought. My closing sentences, therefore, are intended to be provocative.I suspect that neuroprotection could be broadly actualized via precise but subtle alterations to existing protein-disaggregase modalities. The engineering and evolution of protein disaggregases could yield important solutions to avert an imminent plague of neurodegenerative disorders that promises to devastate our society. I strongly suspect that cures for various neurodegenerative disorders will be realized by pioneering as-yet-uncharted regions of disaggregase sequence space or chemical space to elucidate small-molecule enhancers of disaggregase activity.     

MALDI Imaging Mass Spectrometry: STATE OF THE ART TECHNOLOGY IN CLINICAL PROTEOMICS*

A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patient''s entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.MS has become a versatile tool that we are familiar with in large part due to important electronic and informatics advancements. The ability to obtain the molecular weight is one of the first steps in the identification of a molecule. With the addition of primary structural information mass spectrometry has become a useful technique to identify molecules within complex mixtures.Biological specimens, such as tissues, urine, or plasma, are complex and highly heterogeneous, which makes them inherently difficult to analyze. Further research and developments are necessary to achieve reliable biological models for understanding and studying pathologies. Therefore, it is of primary importance to identify the constituents of these systems and subsequently understand how they function within the framework of the tissue. With regard to clinical proteomics, there is the added dimension of disease, and therefore, the main goal is to characterize the cellular circuitry with a focus on the impact of the disease and/or therapy on these cellular networks.Mass spectrometry has become a centerpiece technology predominantly in the field of proteomics. Nonetheless a more comprehensive understanding of the constituents of biological systems will be aided by determining the constituent distribution. This anatomical dimension has been added through mass spectrometry imaging (MSI)¹ especially using MALDI-MSI.MALDI is an ion source that is well compatible with the introduction of raw materials and surfaces. Shortly after its introduction, MALDI was used for direct tissue profiling. The first applications were neurobiological studies on dissected organs from the mollusk Lymnaea stagnalis (1–8), crustaceans (9), and other mollusks (10, 11). More recently, MALDI was used to generate profiles from tissue sections and ion images using a scanning method to analyze the surface (12) (Fig. 1). This led to the first MALDI MS tissue section imaging micrographs in 1997 (13–15). These studies were followed by 10 years of intense efforts to improve the sensitivity, reproducibility, data processing, tissue preservation, and preparation treatments to fully characterize the proteome leading to a clear improvement of molecular images (16–39) (Fig. 2).Open in a separate windowFig. 1.Schematic representation of the MALDI-MSI work flow. After tissue sectioning and transfer onto a conductive and transparent sample plate, the MALDI matrix is deposited, and data are acquired by recording mass spectra according to a raster of points covering the surface to be analyzed. Mass spectra recorded with their coordinates on the tissue are processed, and molecular images of the localization of molecules can be reconstructed. a.u., arbitrary units; ITO, idium tin oxide.Open in a separate windowFig. 2.Ten years'' evolution from one of the first MALDI images presented in 1999 at the 47th ASMS Conference on Mass Spectrometry and Allied Topics (left) (reprinted with permission of Caprioli and co-workers (84)) and molecular images obtained by our group for mouse stem cells injected in brain tissue sections (right) (M. Wisztorski, C. Meriaux, M. Salzet, and I. Fournier, unpublished results).These developments led to clinical studies using MALDI-MSI technology. Clinical proteomics has many objectives including 1) diagnosis based on signatures as a complement to histopathology, 2) early disease detection, 3) individualized selection of therapeutic combinations that best target the patient''s entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.Based on these key objectives, can we consider MALDI-MSI a mature technology for use in clinical studies? What is the potential impact of this technology in anatomy/pathology and disease? By reviewing each objective, do we have sufficient evidence that MALDI-MSI satisfies the criteria imposed by clinical proteomics? We will now specifically address each of these key points.