The Bacterial Hsp90 Chaperone: Cellular Functions and Mechanism of Action.

Heat shock protein 90 (Hsp90) is a molecular chaperone that folds and remodels proteins, thereby regulating the activity of numerous substrate proteins. Hsp90 is widely conserved across species and is essential in all eukaryotes and in some bacteria under stress conditions. To facilitate protein remodeling, bacterial Hsp90 collaborates with the Hsp70 molecular chaperone and its cochaperones. In contrast, the mechanism of protein remodeling performed by eukaryotic Hsp90 is more complex, involving more than 20 Hsp90 cochaperones in addition to Hsp70 and its cochaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of bacterial Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70. We describe the universally conserved structure and conformational dynamics of these chaperones and their interactions with one another and with client proteins. The physiological roles of Hsp90 in Escherichia coli and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide a framework for understanding the more complex eukaryotic Hsp90 system.

Escherichia coli small heat shock protein IbpA plays a role in regulating the heat shock response by controlling the translation of σ32.

Small heat shock proteins (sHsps) act as ATP-independent chaperones that prevent irreversible aggregate formation by sequestering denatured proteins. IbpA, an Escherichia coli sHsp, functions not only as a chaperone but also as a suppressor of its own expression through posttranscriptional regulation, contributing to negative feedback regulation. IbpA also regulates the expression of its paralog, IbpB, in a similar manner, but the extent to which IbpA regulates other protein expressions is unclear. We have identified that IbpA down-regulates the expression of many Hsps by repressing the translation of the heat shock transcription factor σ32. The IbpA regulation not only controls the σ32 level but also contributes to the shutoff of the heat shock response. These results revealed an unexplored role of IbpA to regulate heat shock response at a translational level, which adds an alternative layer for tightly controlled and rapid expression of σ32 on demand.

Identification of a chaperone-code responsible for Rad51-mediated genome repair.

Posttranslational modifications of Hsp90 are known to regulate its in vivo chaperone functions. Here, we demonstrate that the lysine acetylation-deacetylation dynamics of Hsp82 is a major determinant in DNA repair mediated by Rad51. We uncover that the deacetylated lysine 27 in Hsp82 dictates the formation of the Hsp82-Aha1-Rad51 complex, which is crucial for client maturation. Intriguingly, Aha1-Rad51 complex formation is not dependent on Hsp82 or its acetylation status; implying that Aha1-Rad51 association precedes the interaction with Hsp82. The DNA damage sensitivity of Hsp82 (K27Q/K27R) mutants are epistatic to the loss of the (de)acetylase hda1Δ; reinforcing the importance of the reversible acetylation of Hsp82 at the K27 position. These findings underscore the significance of the cross talk between a specific Hsp82 chaperone modification code and the cognate cochaperones in a client-specific manner. Given the pivotal role that Rad51 plays during DNA repair in eukaryotes and particularly in cancer cells, targeting the Hda1-Hsp90 axis could be explored as a new therapeutic approach against cancer.

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE.

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.

Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

Elongation factor MdEF-Tu coordinates with heat shock protein MdHsp70 to enhance apple thermotolerance.

High-temperature stress results in protein misfolding/unfolding and subsequently promotes the accumulation of cytotoxic protein aggregates that can compromise cell survival. Heat shock proteins (HSPs) function as molecular chaperones that coordinate the refolding and degradation of aggregated proteins to mitigate the detrimental effects of high temperatures. However, the relationship between HSPs and protein aggregates in apples under high temperatures remains unclear. Here, we show that an apple (Malus domestica) chloroplast-localized, heat-sensitive elongation factor Tu (MdEF-Tu), positively regulates apple thermotolerance when it is overexpressed. Transgenic apple plants exhibited higher photosynthetic capacity and better integrity of chloroplasts during heat stress. Under high temperatures, MdEF-Tu formed insoluble aggregates accompanied by ubiquitination modifications. Furthermore, we identified a chaperone heat shock protein (MdHsp70), as an interacting protein of MdEF-Tu. Moreover, we observed obviously elevated MdHsp70 levels in 35S: MdEF-Tu apple plants that prevented the accumulation of ubiquitinated MdEF-Tu aggregates, which positively contributes to the thermotolerance of the transgenic plants. Overall, our results provide new insights into the molecular chaperone function of MdHsp70, which mediates the homeostasis of thermosensitive proteins under high temperatures.

Dual roles of HSP70 chaperone HSPA1 in quality control of nascent and newly synthesized proteins.

Exposure to heat stress triggers a well-defined acute response marked by HSF1-dependent transcriptional upregulation of heat shock proteins. Cells allowed to recover acquire thermotolerance, but this adaptation is poorly understood. By quantitative proteomics, we discovered selective upregulation of HSP70-family chaperone HSPA1 and its co-factors, HSPH1 and DNAJB1, in MCF7 breast cancer cells acquiring thermotolerance. HSPA1 was found to have dual function during heat stress response: (i) During acute stress, it promotes the recruitment of the 26S proteasome to translating ribosomes, thus poising cells for rapid protein degradation and resumption of protein synthesis upon recovery; (ii) during thermotolerance, HSPA1 together with HSPH1 maintains ubiquitylated nascent/newly synthesized proteins in a soluble state required for their efficient proteasomal clearance. Consistently, deletion of HSPH1 impedes thermotolerance and esophageal tumor growth in mice, thus providing a potential explanation for the poor prognosis of digestive tract cancers with high HSPH1 and nominating HSPH1 as a cancer drug target. We propose dual roles of HSPA1 either alone or in complex with HSPH1 and DNAJB1 in promoting quality control of nascent/newly synthesized proteins and cellular thermotolerance.

Histone H3.3 chaperone HIRA renders stress-responsive genes poised for prospective lethal stresses in acquired tolerance.

Appropriate responses to environmental challenges are imperative for the survival of all living organisms. Exposure to low-dose stresses is recognized to yield increased cellular fitness, a phenomenon termed hormesis. However, our molecular understanding of how cells respond to low-dose stress remains profoundly limited. Here we report that histone variant H3.3-specific chaperone, HIRA, is required for acquired tolerance, where low-dose heat stress exposure confers resistance to subsequent lethal heat stress. We found that human HIRA activates stress-responsive genes, including HSP70, by depositing histone H3.3 following low-dose stresses. These genes are also marked with histone H3 Lys-4 trimethylation and H3 Lys-9 acetylation, both active chromatin markers. Moreover, depletion of HIRA greatly diminished acquired tolerance, both in normal diploid fibroblasts and in HeLa cells. Collectively, our study revealed that HIRA is required for eliciting adaptive stress responses under environmental fluctuations and is a master regulator of stress tolerance.

TRIM26 restricts Epstein-Barr virus infection in nasopharyngeal epithelial cells through K48-linked ubiquitination of HSP-90ß.

The tripartite interaction motif (TRIM) family of proteins is known for their antiviral activity through different mechanisms, such as interfering with viral components, regulating immune responses, and participating in autophagy-mediated defense pathways. In this study, we investigated the role of tripartite interaction motif 26 (TRIM26), which is encoded by a major histocompatibility complex (MHC) gene, in regulating Epstein-Barr virus (EBV) infection of nasopharyngeal epithelial cells. We found that TRIM26 expression was induced upon EBV infection and that it indirectly targeted EphA2, a crucial epithelial receptor for EBV entry. Our results showed that TRIM26 interacted with heat shock protein 90-beta (HSP-90ß) and promoted its polyubiquitination, which led to its degradation via the proteasome pathway. This, in turn, affected EphA2 integrity and suppressed EBV infection. These findings suggest that TRIM26 could be a valuable target for developing therapeutic interventions against EBV infection and its associated pathogenesis.

Native Size-Exclusion Chromatography-Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes.

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.

Investigation of Cellular Response to the HSP90 Inhibition in Human Cells Through Thermal Proteome Profiling.

Heat shock proteins are chaperones, and they are responsible for protein folding in cells. Heat shock protein 90 (HSP90) is one of the most important chaperones in human cells, and its inhibition is promising for cancer therapy. However, despite the development of multiple HSP90 inhibitors, none of them has been approved for disease treatment due to unexpected cellular toxicity and side effects. Hence, a more comprehensive investigation of cellular response to HSP90 inhibitors can aid in a better understanding of the molecular mechanisms of the cytotoxicity and side effects of these inhibitors. The thermal stability shifts of proteins, which represent protein structure and interaction alterations, can provide valuable information complementary to the results obtained from commonly used abundance-based proteomics analysis. Here, we systematically investigated cell response to different HSP90 inhibitors through global quantification of protein thermal stability changes using thermal proteome profiling, together with the measurement of protein abundance changes. Besides the targets and potential off-targets of the drugs, proteins with significant thermal stability changes under the HSP90 inhibition are found to be involved in cell stress responses and the translation process. Moreover, proteins with thermal stability shifts under the inhibition are upstream of those with altered expression. These findings indicate that the HSP90 inhibition perturbs cell transcription and translation processes. The current study provides a different perspective for achieving a better understanding of cellular response to chaperone inhibition.

TRITHORAX-dependent arginine methylation of HSP68 mediates circadian repression by PERIOD in the monarch butterfly.

Transcriptional repression drives feedback loops that are central to the generation of circadian (∼24-h) rhythms. In mammals, circadian repression of circadian locomotor output cycles kaput, and brain and muscle ARNT-like 1 (CLOCK:BMAL1)-mediated transcription is provided by a complex formed by PERIOD (PER) and CRYPTOCHROME (CRY) proteins. PER initiates transcriptional repression by binding CLK:BMAL1, which ultimately results in their removal from DNA. Although PER's ability to repress transcription is widely recognized, how PER binding triggers repression by removing CLK:BMAL1 from DNA is not known. Here, we use the monarch butterfly as a model system to address this problem because it harbors a simplified version of the CLK:BMAL1-activated circadian clock present in mammals. We report that an intact CLOCK mouse exon 19 homologous region (CLKe19r) and the histone methyltransferase TRITHORAX (TRX) are both necessary for monarch CLK:BMAL1-mediated transcriptional activation, CLK-PER interaction, and PER repression. Our results show that TRX catalytic activity is essential for CLK-PER interaction and PER repression via the methylation of a single arginine methylation site (R45) on heat shock protein 68 (HSP68). Our study reveals TRX and HSP68 as essential links between circadian activation and PER-mediated repression and suggests a potential conserved clock function for HSPs in eukaryotes.

Targeting heat shock protein 47 alleviated doxorubicin-induced cardiotoxicity and remodeling in mice through suppression of the NLRP3 inflammasome.

AIM: Doxorubicin-induced cardiotoxicity (DIC) is an increasing problem, occurring in many cancer patients receiving anthracycline chemotherapy, ultimately leading to heart failure (HF). Unfortunately, DIC remains difficult to manage due to an ignorance regarding pathophysiological mechanisms. Our work aimed to evaluate the role of HSP47 in doxorubicin-induced HF, and to explore the molecular mechanisms. METHODS AND RESULTS: Mice were exposed to multi-intraperitoneal injection of doxorubicin (DOX, 4mg/kg/week, for 6 weeks continuously) to produce DIC. HSP47 expression was significantly upregulated in serum and in heart tissue in DOX-treated mice and in isolated cardiomyocytes. Mice with cardiac-specific HSP47 overexpression and knockdown were generated using recombinant adeno-associated virus (rAVV9) injection. Importantly, cardiac-specific HSP47 overexpression exacerbated cardiac dysfunction in DIC, while HSP47 knockdown prevented DOX-induced cardiac dysfunction, cardiac atrophy and fibrosis in vivo and in vitro. Mechanistically, we identified that HSP47 directly interacted with IRE1α in cardiomyocytes. Furthermore, we provided powerful evidence that HSP47-IRE1α complex promoted TXNIP/NLRP3 inflammasome and reinforced USP1-mediated NLRP3 ubiquitination. Moreover, NLRP3 deficiency in vivo conspicuously abolished HSP47-mediated cardiac atrophy and fibrogenesis under DOX condition. CONCLUSION: HSP47 was highly expressed in serum and cardiac tissue after doxorubicin administration. HSP47 contributed to long-term anthracycline chemotherapy-associated cardiac dysfunction in an NLRP3-dependent manner. HSP47 therefore represents a plausible target for future therapy of doxorubicin-induced HF.

The mRNA binding-mediated self-regulatory function of small heat shock protein IbpA in γ-proteobacteria is conferred by a conserved arginine.

Bacterial small heat shock proteins, such as inclusion body-associated protein A (IbpA) and IbpB, coaggregate with denatured proteins and recruit other chaperones for the processing of aggregates thereby assisting in protein refolding. In addition, as a recently revealed uncommon feature, Escherichia coli IbpA self-represses its own translation through interaction with the 5'-untranslated region of the ibpA mRNA, enabling IbpA to act as a mediator of negative feedback regulation. Although IbpA also suppresses the expression of IbpB, IbpB does not have this self-repression activity despite the two Ibps being highly homologous. In this study, we demonstrate that the self-repression function of IbpA is conserved in other γ-proteobacterial IbpAs. Moreover, we show a cationic residue-rich region in the α-crystallin domain of IbpA, which is not conserved in IbpB, is critical for the self-suppression activity. Notably, we found arginine 93 (R93) located within the α-crystallin domain is an essential residue that cannot be replaced by any of the other 19 amino acids including lysine. We observed that IbpA-R93 mutants completely lost the interaction with the 5' untranslated region of the ibpA mRNA, but retained almost all chaperone activity and were able to sequester denatured proteins. Taken together, we propose the conserved Arg93-mediated translational control of IbpA through RNA binding would be beneficial for a rapid and massive supply of the chaperone on demand.

Saturated fatty acid- and/or monounsaturated fatty acid-containing phosphatidic acids selectively interact with heat shock protein 27.

Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T-cell activity attenuation, preferentially produces saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs), such as 16:0/16:0-, 16:0/18:0-, and 16:1/16:1-PA, in melanoma cells. In the present study, we searched for the target proteins of 16:0/16:0-PA in melanoma cells and identified heat shock protein (HSP) 27, which acts as a molecular chaperone and contributes to cancer progression. HSP27 more strongly interacted with PA than other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate. Moreover, HSP27 is more preferentially bound to SFA- and/or MUFA-containing PAs, including 16:0/16:0- and 16:0/18:1-PAs, than PUFA-containing PAs, including 18:0/20:4- and 18:0/22:6-PA. Furthermore, HSP27 and constitutively active DGKα expressed in COS-7 cells colocalized in a DGK activity-dependent manner. Notably, 16:0/16:0-PA, but not phosphatidylcholine or 16:0/16:0-phosphatidylserine, induced oligomer dissociation of HSP27, which enhances its chaperone activity. Intriguingly, HSP27 protein was barely detectable in Jurkat T cells, while the protein band was intensely detected in AKI melanoma cells. Taken together, these results strongly suggest that SFA- and/or MUFA-containing PAs produced by DGKα selectively target HSP27 and regulate its cancer-progressive function in melanoma cells but not in T cells.

The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions.

Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.

Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos.

Reactive sulfur species (RSS) have emerged as key regulators of protein quality control. However, the mechanisms by which RSS contribute to cellular processes are not fully understood. In this study, we identified a novel function of RSS in preventing parthanatos, a nonapoptotic form of cell death that is induced by poly (ADP-ribose) polymerase-1 and mediated by the aggresome-like induced structures (ALIS) composed of SQSTM1/p62. We found that sodium tetrasulfide (Na2S4), a donor of RSS, strongly suppressed oxidative stress-dependent ALIS formation and subsequent parthanatos. On the other hand, the inhibitors of the RSS-producing enzymes, such as 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase, clearly enhanced ALIS formation and parthanatos. Interestingly, we found that Na2S4 activated heat shock factor 1 by promoting its dissociation from heat shock protein 90, leading to accelerated transcription of HSP70. Considering that the genetic deletion of HSP70 allowed the enhanced ALIS formation, these findings suggest that RSS prevent parthanatos by specifically suppressing ALIS formation through induction of HSP70. Taken together, our results demonstrate a novel mechanism by which RSS prevent cell death, as well as a novel physiological role of RSS in contributing to protein quality control through HSP70 induction, which may lead to better understanding of the bioactivity of RSS.

HSP90α is needed for the survival of rod photoreceptors and regulates the expression of rod PDE6 subunits.

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90ß. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2 months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6 months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90ß paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.

Structural basis of aggregate binding by the AAA+ disaggregase ClpG.

Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a ß-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.

Crosstalk of protein clearance, inflammasome, and Ca2+ channels in retinal pigment epithelium derived from age-related macular degeneration patients.

Degeneration and/or dysfunction of retinal pigment epithelium (RPE) is generally detected as the formation of intracellular and extracellular protein aggregates, called lipofuscin and drusen, respectively, in patients with age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. These clinical hallmarks are linked to dysfunctional protein homeostasis and inflammation and furthermore, are both regulated by changes in intracellular Ca2+ concentration. While many other cellular mechanisms have been considered in the investigations of AMD-RPE, there has been relatively little work on understanding the interactions of protein clearance, inflammation, and Ca2+ dynamics in disease pathogenesis. Here we established induced pluripotent stem cell-derived RPE from two patients with advanced AMD and from an age- and gender-matched control subject. We studied autophagy and inflammasome activation under disturbed proteostasis in these cell lines and investigated changes in their intracellular Ca2+ concentration and L-type voltage-gated Ca2+ channels. Our work demonstrated dysregulated autophagy and inflammasome activation in AMD-RPE accompanied by reduced intracellular free Ca2+ levels. Interestingly, we found currents through L-type voltage-gated Ca2+ channels to be diminished and showed these channels to be significantly localized to intracellular compartments in AMD-RPE. Taken together, the alterations in Ca2+ dynamics in AMD-RPE together with dysregulated autophagy and inflammasome activation indicate an important role for Ca2+ signaling in AMD pathogenesis, providing new avenues for the development of therapeutic approaches.