Unique characteristics of the J-domain proximal regions of Hsp70 cochaperone Apj1 in prion propagation/elimination and its overlap with Sis1 function.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s. The Class A JDP Apj1 of the yeast cytosol has an unusually complex region between the N-terminal J-domain and the substrate binding region-often called the Grich or GF region in Class A and B JDPs because of its typical abundance of glycine. The N-terminal 161-residue Apj1 fragment is known to be sufficient for Apj1 function in prion curing, driven by the overexpression of Hsp104. Further analyzing the N-terminal segment of Apj1, we found that a 90-residue fragment that includes the 70-residue J-domain and the adjacent 12-residue glutamine/alanine (Q/A) segment is sufficient for curing. Furthermore, the 121-residue fragment that includes the Grich region was sufficient to not only sustain the growth of cells lacking the essential Class B JDP Sis1 but also enabled the maintenance of several prions normally dependent on Sis1 for propagation. A J-domain from another cytosolic JDP could substitute for the Sis1-related functions but not for Apj1 in prion curing. Together, these results separate the functions of JDPs in prion biology and underscore the diverse functionality of multi-domain cytosolic JDPs in yeast.

J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Physiologically Relevant Levels of Biliverdin Do Not Significantly Oppose Oxidative Damage in Plasma In Vitro.

AbstractAntioxidants have important physiological roles in limiting the amount of oxidative damage that an organism experiences. One putative antioxidant is biliverdin, a pigment that is most commonly associated with the blue or green colors of avian eggshells. However, despite claims that biliverdin functions as an antioxidant, neither the typical physiological concentrations of biliverdin in most species nor the ability of biliverdin to oppose oxidative damage at these concentrations has been examined. Therefore, we quantified biliverdin in the plasma of six bird species and found that they circulated levels of biliverdin between 0.02 and 0.5 µM. We then used a pool of plasma from northern bobwhite quail (Colinus virginianus) and spiked it with one of seven different concentrations of biliverdin, creating plasma-based solutions ranging from 0.09 to 231 µM biliverdin. We then compared each solution's ability to oppose oxidative damage in response to hydrogen peroxide relative to a control addition of water. We found that hydrogen peroxide consistently induced moderate amounts of oxidative damage (quantified as reactive oxygen metabolites) but that no concentration of biliverdin ameliorated this damage. However, biliverdin and hydrogen peroxide interacted, as the amount of biliverdin in hydrogen peroxide-treated samples was reduced to approximately zero, unless the initial concentration was over 100 µM biliverdin. These preliminary findings based on in vitro work indicate that while biliverdin may have important links to metabolism and immune function, at physiologically relevant concentrations it does not detectably oppose hydrogen peroxide-induced oxidative damage in plasma.

Identification of subfunctionalized aggregate-remodeling J-domain proteins in Arabidopsis thaliana.

J-domain proteins (JDPs) are critical components of the cellular protein quality control machinery, playing crucial roles in preventing the formation and, solubilization of cytotoxic protein aggregates. Bacteria, yeast, and plants additionally have large, multimeric heat shock protein 100 (Hsp100)-class disaggregases that resolubilize protein aggregates. JDPs interact with aggregated proteins and specify the aggregate-remodeling activities of Hsp70s and Hsp100s. However, the aggregate-remodeling properties of plant JDPs are not well understood. Here we identify eight orthologs of Sis1 (an evolutionarily conserved Class II JDP of budding yeast) in Arabidopsis thaliana with distinct aggregate-remodeling functionalities. Six of these JDPs associate with heat-induced protein aggregates in vivo and co-localize with Hsp101 at heat-induced protein aggregate centers. Consistent with a role in solubilizing cytotoxic protein aggregates, an atDjB3 mutant had defects in both solubilizing heat-induced aggregates and acquired thermotolerance as compared with wild-type seedlings. Next, we used yeast prions as protein aggregate models to show that the six JDPs have distinct aggregate-remodeling properties. Results presented in this study, as well as findings from phylogenetic analysis, demonstrate that plants harbor multiple, evolutionarily conserved JDPs with capacity to process a variety of protein aggregate conformers induced by heat and other stressors.

Hsp40/JDP Requirements for the Propagation of Synthetic Yeast Prions.

Yeast prions are protein-based transmissible elements, most of which are amyloids. The chaperone protein network in yeast is inexorably linked to the spreading of prions during cell division by fragmentation of amyloid prion aggregates. Specifically, the core "prion fragmentation machinery" includes the proteins Hsp104, Hsp70 and the Hsp40/J-domain protein (JDP) Sis1. Numerous novel amyloid-forming proteins have been created and examined in the yeast system and occasionally these amyloids are also capable of continuous Hsp104-dependent propagation in cell populations, forming synthetic prions. However, additional chaperone requirements, if any, have not been determined. Here, we report the first instances of a JDP-Hsp70 system requirement for the propagation of synthetic prions. We utilized constructs from a system of engineered prions with prion-forming domains (PrDs) consisting of a polyQ stretch interrupted by a single heterologous amino acid interspersed every fifth residue. These "polyQX" PrDs are fused to the MC domains of Sup35, creating chimeric proteins of which a subset forms synthetic prions in yeast. For four of these prions, we show that SIS1 repression causes prion loss in a manner consistent with Sis1's known role in prion fragmentation. PolyQX prions were sensitive to Sis1 expression levels to differing degrees, congruent with the variability observed among native prions. Our results expand the scope known Sis1 functionality, demonstrating that Sis1 acts on amyloids broadly, rather than through specific protein-protein interactions with individual yeast prion-forming proteins.

Low-dose immune challenges result in detectable levels of oxidative damage.

Infection can result in substantial costs to animals, so they frequently respond by removing infectious agents with an immune response. However, immune responses entail their own costs, including upregulation of processes that destroy pathogens (e.g. the production of reactive oxygen species) and processes that limit the extent of self-damage during the immune response (e.g. production of anti-inflammatory proteins such as haptoglobin). Here, we simulated bacterial infection across a 1000-fold range using lipopolysaccharide (LPS) administered to northern bobwhite quail (Colinus virginianus), and quantified metrics related to pro-inflammatory conditions [i.e. generation of oxidative damage (d-ROMs), depletion of antioxidant capacity], anti-inflammatory mechanisms (i.e. production of haptoglobin, expression of the enzyme heme oxygenase, production of the organic molecule biliverdin) and nutritional physiology (e.g. circulating triglyceride levels, maintenance of body mass). We detected increases in levels of haptoglobin and d-ROMs even at LPS doses that are 1/1000th the concentration of doses frequently used in ecoimmunological studies, while loss of body mass and decreases in circulating triglycerides manifested only in individuals receiving the highest dose of LPS (1 mg LPS kg-1 body mass), highlighting variation among dose-dependent responses. Additionally, individuals that lost body mass during the course of the experiment had lower levels of circulating triglycerides, and those with more oxidative damage had greater levels of heme oxygenase expression, which highlights the complex interplay between pro- and anti-inflammatory processes. Because low doses of LPS may simulate natural infection levels, variation in dose-dependent physiological responses may be particularly important in modeling how free-living animals navigate immune challenges.

Three J-proteins impact Hsp104-mediated variant-specific prion elimination: a new critical role for a low-complexity domain.

Prions are self-propagating protein isoforms that are typically amyloid. In Saccharomyces cerevisiae, amyloid prion aggregates are fragmented by a trio involving three classes of chaperone proteins: Hsp40s, also known as J-proteins, Hsp70s, and Hsp104. Hsp104, the sole Hsp100-class disaggregase in yeast, along with the Hsp70 Ssa and the J-protein Sis1, is required for the propagation of all known amyloid yeast prions. However, when Hsp104 is ectopically overexpressed, only the prion [PSI+] is efficiently eliminated from cell populations via a highly debated mechanism that also requires Sis1. Recently, we reported roles for two additional J-proteins, Apj1 and Ydj1, in this process. Deletion of Apj1, a J-protein involved in the degradation of sumoylated proteins, partially blocks Hsp104-mediated [PSI+] elimination. Apj1 and Sis1 were found to have overlapping functions, as overexpression of one compensates for loss of function of the other. In addition, overexpression of Ydj1, the most abundant J-protein in the yeast cytosol, completely blocks Hsp104-mediated curing. Yeast prions exhibit structural polymorphisms known as "variants"; most intriguingly, these J-protein effects were only observed for strong variants, suggesting variant-specific mechanisms. Here, we review these results and present new data resolving the domains of Apj1 responsible, specifically implicating the involvement of Apj1's Q/S-rich low-complexity domain.

Effects of stress-induced increases of corticosterone on circulating triglyceride levels, biliverdin concentration, and heme oxygenase expression.

When exposed to stressors, animals physiologically respond by secreting glucocorticoid hormones. Most birds, reptiles, and amphibians secrete corticosterone (CORT), which allows them to maximize short-term survival, including by modulating lipid metabolism. However, the factors regulating lipid metabolism, particularly during acute (i.e., short-term) stressors, are not well-characterized. To investigate one putative mechanism, we examined how expression of the enzyme heme oxygenase (HO), which primarily converts heme into biliverdin, changes during an acute stressor. Because HO has links to decreased levels of triglycerides, we tested the hypothesis that an acute stressor increases HO expression, which would concomitantly decrease circulating lipid levels. We compared free-living house sparrow (Passer domesticus) nestlings exposed to a one-hour stressor to control individuals, and quantified HO expression and biliverdin concentration in spleen, liver, or kidney tissue, as well as circulating CORT, triglyceride, and glycerol levels. Nestlings exposed to a stressor had reduced circulating triglycerides consistent with an increased rate of gluconeogenesis during an acute stressor. Concentrations of triglycerides were also negatively correlated with HO expression in the liver, which is consistent with mammalian studies. However, contrary to our predictions, exposure to a stressor did not affect HO expression, or biliverdin concentration in liver, spleen, or kidney. Overall, our results support links between CORT, triglyceride levels, and HO expression, though the molecular pathways connecting these metrics still need to be elucidated.

Impact of Amyloid Polymorphism on Prion-Chaperone Interactions in Yeast.

Yeast prions are protein-based genetic elements found in the baker's yeast Saccharomyces cerevisiae, most of which are amyloid aggregates that propagate by fragmentation and spreading of small, self-templating pieces called propagons. Fragmentation is carried out by molecular chaperones, specifically Hsp104, Hsp70, and Hsp40. Like other amyloid-forming proteins, amyloid-based yeast prions exhibit structural polymorphisms, termed "strains" in mammalian systems and "variants" in yeast, which demonstrate diverse phenotypes and chaperone requirements for propagation. Here, the known differential interactions between chaperone proteins and yeast prion variants are reviewed, specifically those of the yeast prions [PSI+], [RNQ+]/[PIN+], and [URE3]. For these prions, differences in variant-chaperone interactions (where known) with Hsp104, Hsp70s, Hsp40s, Sse1, and Hsp90 are summarized, as well as some interactions with chaperones of other species expressed in yeast. As amyloid structural differences greatly impact chaperone interactions, understanding and accounting for these variations may be crucial to the study of chaperones and both prion and non-prion amyloids.

The expanding world of plant J-domain proteins.

Plants maintain cellular proteostasis during different phases of growth and development despite a barrage of biotic and abiotic stressors in an ever-changing environment. This requires a collaborative effort of a cadre of molecular chaperones. Hsp70s and their obligate co-chaperones, J-domain proteins (JDPs), are arguably the most ubiquitous and formidable components of the cellular chaperone network, facilitating numerous and diverse cellular processes and allowing survival under a plethora of stressful conditions. JDPs are also among the most versatile chaperones. Compared to Hsp70s, the number of JDP-encoding genes has proliferated, suggesting the emergence of highly complex Hsp70-JDP networks, particularly in plants. Recent studies indicate that besides the increase in the number of JDP encoding genes; regulatory differences, neo- and sub-functionalization, and inter- and intra-class combinatorial interactions, is rapidly expanding the repertoire of Hsp70-JDP systems. This results in highly robust and functionally diverse chaperone networks in plants. Here, we review the current status of plant JDP research and discuss how the paradigm shift in the field can be exploited toward a better understanding of JDP function and evolution.

Using High Performance Thin Layer Chromatography-Densitometry to Study the Influence of the Prion [RNQ+] and Its Determinant Prion Protein Rnq1 on Yeast Lipid Profiles.

The baker's yeast Saccharomyces cerevisiae harbors multiple prions that allow for the creation of heterogeneity within otherwise clonal cell populations. However, in many cases, the consequences of prion infection are entirely unclear. Predictions of prion-induced changes in cell physiology are complicated by pleotropic effects, and detection is often limited to relatively insensitive cell growth assays that may obscure many physiological changes. We previously showed that silica gel high performance thin-layer chromatography-densitometry (HPTLC) can be used to empirically determine prion-induced changes in lipid content in yeast. Here, we conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-glacial acetic acid (80:20:1, v/v/v) mobile phase and phosphomolybdic acid (PMA) detection reagent]; steryl esters, methyl esters, and squalene [hexane-petroleum ether-diethyl ether-glacial acetic acid (50:20:5:1, v/v/v/v) and PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5, v/v/v) and cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([RNQ+]) and prion-free ([rnq-]) cells in both stationary- and logarithmic-growth phases. We detected multiple statistically significant differences between prion-infected and prion-free cells that varied by growth phase, confirming our pr evious observations that prions exert distinct influences on cell physiology between stationary- and log-phase growth. We also found significant differences between cells expressing or lacking the Rnq1 protein which forms the [RNQ+] prion, providing new clues to the as yet unresolved normal biological function of this prion-forming protein. This investigation further emphasizes the utility of HPTLC-densitometry to empirically determine the effects of prions and other presumed innocuous gene deletions on lipid content in yeast, and we expect that additional analyses will continue to resolve the physiological effects of prion infection.

Variant-specific and reciprocal Hsp40 functions in Hsp104-mediated prion elimination.

The amyloid-based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J-protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI+ ] by an alternative Sis1-dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J-proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well-studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J-proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J-protein complement, uncovered significant variant-dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI+ ] variants, depletion of Apj1 inhibits Hsp104-mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J-protein necessary for the propagation of at least two weak [PSI+ ] variants and no J-protein alteration, or even combination of alterations, affected the curing of weak [PSI+ ] variants, suggesting the possibility of biochemically distinct, variant-specific Hsp104-mediated curing mechanisms.

Immune challenges decrease biliverdin concentration in the spleen of northern Bobwhite quail, Colinus virginianus.

Most antioxidants have multiple functions; in addition to minimizing oxidative damage, many antioxidants have immune-modulating properties. For example, biliverdin is produced in the liver and spleen from the breakdown of heme, and has putative immune-suppressing and antioxidant properties. However, the majority of these properties have been investigated in vitro or in mammalian models, in which biliverdin reductase converts virtually all biliverdin to bilirubin. Thus, biliverdin's physiological roles remain largely untested. Here, we investigated whether biliverdin has immunomodulating roles by injecting Northern Bobwhite quail (Colinus virginianus) with either a vehicle control, lysed and rinsed pig red blood cells (pRBC) that contain erythrocyte antigens but no heme, or intact pRBC that contain both erythrocyte antigens and hemoglobin, thus increasing the amount of heme that can be converted to biliverdin. We then quantified hemagglutination and hemolysis ability, and biliverdin concentration in the liver and spleen, on 3, 6, and 9 days post-injection. We found that hemagglutination was greater in individuals that received intact pRBC, but not in those injected with heme-removed pRBC, demonstrating that biliverdin does not suppress immune function at this dosage. Biliverdin levels of liver and spleen were correlated within individuals, suggesting organism-level variation in biliverdin production. Lastly, individuals injected with intact pRBC had a reduced biliverdin concentration in the spleen, suggesting that immune challenges may reduce biliverdin production or accumulation. This initial investigation demonstrated that biliverdin may have more nuanced physiological roles than previously reported, supporting the value of further investigations into the physiology of biliverdin.

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

Prion-specific Hsp40 function: The role of the auxilin homolog Swa2.

Yeast prions are protein-based genetic elements that propagate through cell populations via cytosolic transfer from mother to daughter cell. Molecular chaperone proteins including Hsp70, the Hsp40/J-protein Sis1, and Hsp104 are required for continued prion propagation, however the specific requirements of chaperone proteins differ for various prions. We recently reported that Swa2, the yeast homolog of the mammalian protein auxilin, is specifically required for the propagation of the prion [URE3]. 1 [URE3] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain of Swa2, but does not require Swa2 clathrin binding. We concluded that the TPR domain determines the specificity of the genetic interaction between Swa2 and [URE3], and that this domain likely interacts with one or more proteins with a C-terminal EEVD motif. Here we extend that analysis to incorporate additional data that supports this hypothesis. We also present new data eliminating Hsp104 as the relevant Swa2 binding partner and discuss our findings in the context of other recent work involving Hsp90. Based on these findings, we propose a new model for Swa2's involvement in [URE3] propagation in which Swa2 and Hsp90 mediate the formation of a multi-protein complex that increases the number of sites available for Hsp104 disaggregation.

Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations.

Cannibalism, Kuru, and Mad Cows: Prion Disease As a "Choose-Your-Own-Experiment" Case Study to Simulate Scientific Inquiry in Large Lectures.

Despite significant efforts to reform undergraduate science education, students often perform worse on assessments of perceptions of science after introductory courses, demonstrating a need for new educational interventions to reverse this trend. To address this need, we created An Inexplicable Disease, an engaging, active-learning case study that is unusual because it aims to simulate scientific inquiry by allowing students to iteratively investigate the Kuru epidemic of 1957 in a choose-your-own-experiment format in large lectures. The case emphasizes the importance of specialization and communication in science and is broadly applicable to courses of any size and sub-discipline of the life sciences.

Swa2, the yeast homolog of mammalian auxilin, is specifically required for the propagation of the prion variant [URE3-1].

Yeast prions require a core set of chaperone proteins including Sis1, Hsp70 and Hsp104 to generate new amyloid templates for stable propagation, yet emerging studies indicate that propagation of some prions requires additional chaperone activities, demonstrating chaperone specificity beyond the common amyloid requirements. To comprehensively assess such prion-specific requirements for the propagation of the [URE3] prion variant [URE3-1], we screened 12 yeast cytosolic J-proteins, and here we report a novel role for the J-protein Swa2/Aux1. Swa2 is the sole yeast homolog of the mammalian protein auxilin, which, like Swa2, functions in vesicle-mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. We found that, in addition to Sis1, [URE3-1] is specifically dependent upon Swa2, but not on any of the 11 other J-proteins. Further, we show that [URE3-1] propagation requires both a functional J-domain and the tetratricopeptide repeat (TPR) domain, but surprisingly does not require Swa2-clathrin binding. Because the J-domain of Swa2 can be replaced with the J-domains of other proteins, our data strongly suggest that prion-chaperone specificity arises from the Swa2 TPR domain and supports a model where Swa2 acts through Hsp70, most likely to provide additional access points for Hsp104 to promote prion template generation.

Hsp40 function in yeast prion propagation: Amyloid diversity necessitates chaperone functional complexity.

Yeast prions are heritable protein-based elements, most of which are formed of amyloid aggregates that rely on the action of molecular chaperones for transmission to progeny. Prions can form distinct amyloid structures, known as 'strains' in mammalian systems, that dictate both pathological progression and cross-species infection barriers. In yeast these same amyloid structural polymorphisms, called 'variants', dictate the intensity of prion-associated phenotypes and stability in mitosis. We recently reported that [PSI(+)] prion variants differ in the fundamental domain requirements for one chaperone, the Hsp40/J-protein Sis1, which are mutually exclusive between 2 different yeast prions, demonstrating a functional plurality for Sis1. Here we extend that analysis to incorporate additional data that collectively support the hypothesis that Sis1 has multiple functional roles that can be accomplished by distinct sets of domains. These functions are differentially required by distinct prions and prion variants. We also present new data regarding Hsp104-mediated prion elimination and show that some Sis1 functions, but not all, are conserved in the human homolog Hdj1/DNAJB1. Importantly, of the 10 amyloid-based prions indentified to date in Saccharomyces cerevisiae, the chaperone requirements of only 4 are known, leaving a great diversity of amyloid structures, and likely modes of amyloid-chaperone interaction, largely unexplored.