DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network.

From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.

SUMOylation of protein phosphatase 5 regulates phosphatase activity and substrate release.

The serine/threonine protein phosphatase 5 (PP5) regulates hormone and stress-induced signaling networks. Unlike other phosphoprotein phosphatases, PP5 contains both regulatory and catalytic domains and is further regulated through post-translational modifications (PTMs). Here we identify that SUMOylation of K430 in the catalytic domain of PP5 regulates phosphatase activity. Additionally, phosphorylation of PP5-T362 is pre-requisite for SUMOylation, suggesting the ordered addition of PTMs regulates PP5 function in cells. Using the glucocorticoid receptor, a well known substrate for PP5, we demonstrate that SUMOylation results in substrate release from PP5. We harness this information to create a non-SUMOylatable K430R mutant as a 'substrate trap' and globally identified novel PP5 substrate candidates. Lastly, we generated a consensus dephosphorylation motif using known substrates, and verified its presence in the new candidate substrates. This study unravels the impact of cross talk of SUMOylation and phosphorylation on PP5 phosphatase activity and substrate release in cells.

The role of the co-chaperone HOP in plant homeostasis during development and stress.

Proteins need to acquire their native structure in order to become fully functional. In specific cases, the active conformation is obtained spontaneously; nevertheless, many proteins need the assistance of chaperones and co-chaperones to be properly folded. These proteins help to maintain protein homeostasis under control conditions and under different stresses. HOP (HSP70-HSP90 organizing protein) is a highly conserved family of co-chaperones that assist HSP70 and HSP90 in the folding of specific proteins. In the last few years, findings in mammals and yeast have revealed novel functions of HOP and re-defined the role of HOP in protein folding. Here, we provide an overview of the most important aspects of HOP regulation and function in other eukaryotes and analyse whether these aspects are conserved in plants. In addition, we highlight the HOP clients described in plants and the role of HOP in plant development and stress response.

Functions of the Hsp90-Binding FKBP Immunophilins.

The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is abundantly clear that the client protein influences, and is often influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members.

Impact of Co-chaperones and Posttranslational Modifications Toward Hsp90 Drug Sensitivity.

Posttranslational modifications (PTMs) regulate myriad cellular processes by modulating protein function and protein-protein interaction. Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone whose activity is responsible for the stabilization and maturation of more than 300 client proteins. Hsp90 is a substrate for numerous PTMs, which have diverse effects on Hsp90 function. Interestingly, many Hsp90 clients are enzymes that catalyze PTM, demonstrating one of the several modes of regulation of Hsp90 activity. Approximately 25 co-chaperone regulatory proteins of Hsp90 impact structural rearrangements, ATP hydrolysis, and client interaction, representing a second layer of influence on Hsp90 activity. A growing body of literature has also established that PTM of these co-chaperones fine-tune their activity toward Hsp90; however, many of the identified PTMs remain uncharacterized. Given the critical role of Hsp90 in supporting signaling in cancer, clinical evaluation of Hsp90 inhibitors is an area of great interest. Interestingly, differential PTM and co-chaperone interaction have been shown to impact Hsp90 binding to its inhibitors. Therefore, understanding these layers of Hsp90 regulation will provide a more complete understanding of the chaperone code, facilitating the development of new biomarkers and combination therapies.

Congenital Hyperinsulinism in Humans and Insulin Secretory Dysfunction in Mice Caused by Biallelic DNAJC3 Variants.

The BiP co-chaperone DNAJC3 protects cells during ER stress. In mice, the deficiency of DNAJC3 leads to beta-cell apoptosis and the gradual onset of hyperglycemia. In humans, biallelic DNAJC3 variants cause a multisystem disease, including early-onset diabetes mellitus. Recently, hyperinsulinemic hypoglycemia (HH) has been recognized as part of this syndrome. This report presents a case study of an individual with HH caused by DNAJC3 variants and provides an overview of the metabolic phenotype of individuals with HH and DNAJC3 variants. The study demonstrates that HH may be a primary symptom of DNAJC3 deficiency and can persist until adolescence. Additionally, glycemia and insulin release were analyzed in young DNACJ3 knockout (K.O.) mice, which are equivalent to human infants. In the youngest experimentally accessible age group of 4-week-old mice, the in vivo glycemic phenotype was already dominated by a reduced total insulin secretion capacity. However, on a cellular level, the degree of insulin release of DNAJC3 K.O. islets was higher during periods of increased synthetic activity (high-glucose stimulation). We propose that calcium leakage from the ER into the cytosol, due to disrupted DNAJC3-controlled gating of the Sec61 channel, is the most likely mechanism for HH. This is the first genetic mechanism explaining HH solely by the disruption of intracellular calcium homeostasis. Clinicians should screen for HH in DNAJC3 deficiency and consider DNAJC3 variants in the differential diagnosis of congenital hyperinsulinism.

WGS-based screening of the co-chaperone protein DjlA-induced curved DNA binding protein A (CbpA) from a new multidrug-resistant zoonotic mastitis-causing Klebsiella pneumoniae strain: a novel molecular target of selective flavonoids.

The research aimed to establish a multidrug-resistant Klebsiella pneumoniae-induced genetic model for mastitis considering the alternative mechanisms of the DjlA-mediated CbpA protein regulation. The Whole Genome Sequencing of the newly isolated K. pneumoniae strain was conducted to annotate the frequently occurring antibiotic resistance and virulence factors following PCR and MALDI-TOF mass-spectrophotometry. Co-chaperon DjlA was identified and extracted via restriction digestion on PAGE. Based on the molecular string property analysis of different DnaJ and DnaK type genes, CbpA was identified to be regulated most by the DjlA protein during mastitis. Based on the quantum tunnel-cluster profiles, CbpA was modeled as a novel target for diversified biosynthetic, and chemosynthetic compounds. Pharmacokinetic and pharmacodynamic analyses were conducted to determine the maximal point-specificity of selective flavonoids in complexing with the CbpA macromolecule at molecular docking. The molecular dynamic simulation (100 ns) of each of the flavonoid-protein complexes was studied regarding the parameters RMSD, RMSF, Rg, SASA, MMGBSA, and intramolecular hydrogen bonds; where all of them resulted significantly. To ratify all the molecular dynamic simulation outputs, the potential stability of the flavonoids in complexing with CbpA can be remarked as Quercetin > Biochanin A > Kaempherol > Myricetin, which were all significant in comparison to the control Galangin. Finally, a comprehensive drug-gene interaction pathway for each of the flavonoids was developed to determine the simultaneous and quantitative-synergistic effects of different operons belonging to the DnaJ-type proteins on the metabolism of the tested pharmacophores in CbpA. Considering all the in vitro and in silico parameters, DjlA-mediated CbpA can be a novel target for the tested flavonoids as the potential therapeutics of mastitis as futuristic drugs.

TTT (Tel2-Tti1-Tti2) Complex, the Co-Chaperone of PIKKs and a Potential Target for Cancer Chemotherapy.

The heterotrimeric Tel2-Tti1-Tti2 or TTT complex is essential for cell viability and highly observed in eukaryotes. As the co-chaperone of ATR, ATM, DNA-PKcs, mTOR, SMG1, and TRRAP, the phosphatidylinositol 3-kinase-related kinases (PIKKs) and a group of large proteins of 300-500 kDa, the TTT plays crucial roles in genome stability, cell proliferation, telomere maintenance, and aging. Most of the protein kinases in the kinome are targeted by co-chaperone Cdc37 for proper folding and stability. Like Cdc37, accumulating evidence has established the mechanism by which the TTT interacts with chaperone Hsp90 via R2TP (Rvb1-Rvb2-Tah1-Pih1) complex or other proteins for co-translational maturation of the PIKKs. Recent structural studies have revealed the α-solenoid structure of the TTT and its interactions with the R2TP complex, which shed new light on the co-chaperone mechanism and provide new research opportunities. A series of mutations of the TTT have been identified that cause disease syndrome with neurodevelopmental defects, and misregulation of the TTT has been shown to contribute to myeloma, colorectal, and non-small-cell lung cancers. Surprisingly, Tel2 in the TTT complex has recently been found to be a target of ivermectin, an antiparasitic drug that has been used by millions of patients. This discovery provides mechanistic insight into the anti-cancer effect of ivermectin and thus promotes the repurposing of this Nobel-prize-winning medicine for cancer chemotherapy. Here, we briefly review the discovery of the TTT complex, discuss the recent studies, and describe the perspectives for future investigation.

The mTOR Independent Function of Tsc1 and FNIPs.

New roles for Tsc1 and FNIP1/2 as regulators of the molecular chaperone Hsp90 were recently identified, demonstrating a broader cellular impact outside of AMPK-mTOR signaling. In studying the function of these proteins we must take a holistic view of the cell, instead of maintaining our focus on a single pathway.

Genome-wide comparative analysis of DNAJ genes and their co-expression patterns with HSP70s in aestivation of the sea cucumber Apostichopus japonicus.

DNAJ proteins function as co-chaperones of HSP70 and play key roles in cell physiology to promote protein folding and degradation, especially under environmental stress. Based on our previous study on HSP70, a systematic study of DNAJ was performed in sea cucumber Apostichopus japonicus using the transcriptomic and genomic data, identifying 43 AjDNAJ genes, including six AjDNAJA genes, eight AjDNAJB genes, and 29 AjDNAJC genes. Slight expansion and conserved genomic structure were observed using the phylogenetic and syntenic analysis. Differential period-specific and tissue-specific expression patterns of AjDNAJs were observed between adult and juvenile individuals during aestivation. Strong tissue-specific expression correlations between AjDNAJ and AjHSP70 genes were found, indicating that the involvements of AjHSP70IVAs in the aestivation of sea cucumbers were regulated by AjDNAJs. Several key genes with significant expression correlations, such as AjDNAJB4L and AjHSP70IVAs, were suggested to function together under heat stress. Together, these findings provide early insight into the involvement of AjDNAJs in the aestivation and their roles as co-chaperones of AjHSP70s.

Orchardgrass ACTIVATOR OF HSP90 ATPASE possesses autonomous chaperone properties and activates Hsp90 transcription to enhance thermotolerance.

High temperature stress is an environmental factor that negatively affects the growth and development of crops. Hsp90 (90 kDa heat shock protein) is a major molecular chaperone in eukaryotic cells, contributing to the maintenance of cell homeostasis through interaction with co-chaperones. Aha1 (activator of Hsp90 ATPase) is well known as a co-chaperone that activates ATPase activity of Hsp90 in mammals. However, biochemical and physiological evidence relating to Aha has not yet been identified in plants. In this study, we investigated the heat-tolerance function of orchardgrass (Dactylis glomerata L.) Aha (DgAha). Recombinant DgAha interacted with cytosolic DgHsp90s and efficiently protected substrates from thermal denaturation. Furthermore, heterologous expression of DgAha in yeast (Saccharomyces cerevisiae) cells and Arabidopsis (Arabidopsis thaliana) plants conferred thermotolerance in vivo. Enhanced expression of DgAha in Arabidopsis stimulates the transcription of Hsp90 under heat stress. Our data demonstrate that plant Aha plays a positive role in heat stress tolerance via chaperone properties and/or activation of Hsp90 to protect substrate proteins in plants from thermal injury.

HOP1 and HOP2 are involved in salt tolerance by facilitating the brassinosteroid-related nucleo-cytoplasmic partitioning of the HSP90-BIN2 complex.

The co-chaperone heat shock protein (HSP)70-HSP90 organizing protein (HOP) is involved in plant thermotolerance. However, its function in plant salinity tolerance was not yet studied. We found that Arabidopsis HOP1 and HOP2 play critical roles in salt tolerance by affecting the nucleo-cytoplasmic partitioning of HSP90 and brassinosteroid-insensitive 2 (BIN2). A hop1/2 double mutant was hypersensitive to salt-stress. Interestingly, this sensitivity was remedied by exogenous brassinolide application, while the application of brassinazole impeded growth of both wild-type (WT) and hop1/2 plants under normal and salt stress conditions. This suggested that the insufficient brassinosteroid (BR) content was responsible for the salt-sensitivity of hop1/2. After WT was transferred to salt stress conditions, HOP1/2, BIN2 and HSP90 accumulated in the nucleus, brassinazole-resistant 1 (BZR1) was phosphorylated and accumulated in the cytoplasm, and BR content significantly increased. This initial response resulted in dephosphorylation of BZR1 and BR response. This dynamic regulation of BR content was impeded in salt-stressed hop1/2. Thus, we propose that HOP1 and HOP2 are involved in salt tolerance by affecting BR signalling.

The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients.

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

Post-translational modifications of Hsp90 and translating the chaperone code.

Cells have a remarkable ability to synthesize large amounts of protein in a very short period of time. Under these conditions, many hydrophobic surfaces on proteins may be transiently exposed, and the likelihood of deleterious interactions is quite high. To counter this threat to cell viability, molecular chaperones have evolved to help nascent polypeptides fold correctly and multimeric protein complexes assemble productively, while minimizing the danger of protein aggregation. Heat shock protein 90 (Hsp90) is an evolutionarily conserved molecular chaperone that is involved in the stability and activation of at least 300 proteins, also known as clients, under normal cellular conditions. The Hsp90 clients participate in the full breadth of cellular processes, including cell growth and cell cycle control, signal transduction, DNA repair, transcription, and many others. Hsp90 chaperone function is coupled to its ability to bind and hydrolyze ATP, which is tightly regulated both by co-chaperone proteins and post-translational modifications (PTMs). Many reported PTMs of Hsp90 alter chaperone function and consequently affect myriad cellular processes. Here, we review the contributions of PTMs, such as phosphorylation, acetylation, SUMOylation, methylation, O-GlcNAcylation, ubiquitination, and others, toward regulation of Hsp90 function. We also discuss how the Hsp90 modification state affects cellular sensitivity to Hsp90-targeted therapeutics that specifically bind and inhibit its chaperone activity. The ultimate challenge is to decipher the comprehensive and combinatorial array of PTMs that modulate Hsp90 chaperone function, a phenomenon termed the "chaperone code."

The endoplasmic reticulum HSP40 co-chaperone ERdj3/DNAJB11 assembles and functions as a tetramer.

ERdj3/DNAJB11 is an endoplasmic reticulum (ER)-targeted HSP40 co-chaperone that performs multifaceted functions involved in coordinating ER and extracellular proteostasis. Here, we show that ERdj3 assembles into a native tetramer that is distinct from the dimeric structure observed for other HSP40 co-chaperones. An electron microscopy structural model of full-length ERdj3 shows that these tetramers are arranged as a dimer of dimers formed by distinct inter-subunit interactions involving ERdj3 domain II and domain III Targeted deletion of residues 175-190 within domain II renders ERdj3 a stable dimer that is folded and efficiently secreted from mammalian cells. This dimeric ERdj3 shows impaired substrate binding both in the ER and extracellular environments and reduced interactions with the ER HSP70 chaperone BiP. Furthermore, we show that overexpression of dimeric ERdj3 exacerbates ER stress-dependent reductions in the secretion of a destabilized, aggregation-prone protein and increases its accumulation as soluble oligomers in extracellular environments. These results reveal ERdj3 tetramerization as an important structural framework for ERdj3 functions involved in coordinating ER and extracellular proteostasis in the presence and absence of ER stress.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect.

The ClC-2 channel plays a critical role in maintaining ion homeostasis in the brain and the testis. Loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the white matter disease leukodystrophy. Clcn2-deficient mice display neuronal myelin vacuolation and testicular degeneration. Leukodystrophy-causing ClC-2 mutant channels are associated with anomalous proteostasis manifesting enhanced endoplasmic reticulum (ER)-associated degradation. The molecular nature of the ER quality control system for ClC-2 protein remains elusive. In mouse testicular tissues and Leydig cells, we demonstrated that endogenous ClC-2 co-existed in the same protein complex with the molecular chaperones heat shock protein 90ß (Hsp90ß) and heat shock cognate protein (Hsc70), as well as the associated co-chaperones Hsp70/Hsp90 organizing protein (HOP), activator of Hsp90 ATPase homolog 1 (Aha1), and FK506-binding protein 8 (FKBP8). Further biochemical analyses revealed that the Hsp90ß-Hsc70 chaperone/co-chaperone system promoted mouse and human ClC-2 protein biogenesis. FKBP8 additionally facilitated membrane trafficking of ClC-2 channels. Interestingly, treatment with the Hsp90-targeting small molecule 17-allylamino-17-demethoxygeldanamycin (17-AAG) substantially boosted ClC-2 protein expression. Also, 17-AAG effectively increased both total and cell surface protein levels of leukodystrophy-causing loss-of-function ClC-2 mutant channels. Our findings highlight the therapeutic potential of 17-AAG in correcting anomalous ClC-2 proteostasis associated with leukodystrophy.

Hsp90 and Hsp70 chaperones: Collaborators in protein remodeling.

Heat shock proteins 90 (Hsp90) and 70 (Hsp70) are two families of highly conserved ATP-dependent molecular chaperones that fold and remodel proteins. Both are important components of the cellular machinery involved in protein homeostasis and participate in nearly every cellular process. Although Hsp90 and Hsp70 each carry out some chaperone activities independently, they collaborate in other cellular remodeling reactions. In eukaryotes, both Hsp90 and Hsp70 function with numerous Hsp90 and Hsp70 co-chaperones. In contrast, bacterial Hsp90 and Hsp70 are less complex; Hsp90 acts independently of co-chaperones, and Hsp70 uses two co-chaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70, with an emphasis on bacterial chaperones. We describe the structure and conformational dynamics of these chaperones and their interactions with each other 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 the groundwork for understanding the more complex eukaryotic Hsp90 system and its modulation by Hsp90 co-chaperones.

Multifaceted roles of HEAT SHOCK PROTEIN 90 molecular chaperones in plant development.

HEAT SHOCK PROTEINS 90 (HSP90s) are molecular chaperones that mediate correct folding and stability of many client proteins. These chaperones act as master molecular hubs involved in multiple aspects of cellular and developmental signalling in diverse organisms. Moreover, environmental and genetic perturbations affect both HSP90s and their clients, leading to alterations of molecular networks determining respectively plant phenotypes and genotypes and contributing to a broad phenotypic plasticity. Although HSP90 interaction networks affecting the genetic basis of phenotypic variation and diversity have been thoroughly studied in animals, such studies are just starting to emerge in plants. Here, we summarize current knowledge and discuss HSP90 network functions in plant development and cellular homeostasis.

Peptides in proteins.

During evolution C-terminal peptide extensions were added to proteins on the gene level. These convey additional functions such as interaction with partner proteins or oligomerisation. IgM antibodies and molecular chaperones are two prominent examples discussed.

Anticancer properties and mechanism of action of the quassinoid ailanthone.

Ailanthone (AIT) is a quassinoid natural product isolated from the worldwide-distributed plant Ailanthus altissima. The drug displays multiple pharmacological properties, in particular significant antitumor effects against a variety of cancer cell lines in vitro. Potent in vivo activities have been evidenced in mice bearing hepatocellular carcinoma, nonsmall cell lung cancer and castration-resistant prostate cancer. This review focusses on the mechanism of action of AIT, notably to highlight the capacity of the drug to activate DNA damage responses, to inhibit the Hsp90 co-chaperone p23 and to modulate the expression of several microRNA. The interconnexion between these effects is discussed. The unique capacity of AIT to downregulate oncogenic miR-21 and to upregulate the tumor suppressor miRNAs miR-126, miR-148a, miR-195, and miR-449a is presented. AIT exploits several microRNAs to exert its anticancer effects in distinct tumor types. AIT is one of the rare antitumor natural products that binds to and strongly inhibits cochaperone p23, opening interesting perspectives to treat cancers. However, the toxicity profile of the molecule may limit its development as an anticancer drug, unless it can be properly formulated to prevent AIT-induced gastro-intestinal damages in particular. The antitumor properties of AIT and analogs are underlined, with the aim to encourage further pharmacological studies with this underexplored natural product and related quassinoids. HIGHLIGHTS: Ailanthone (AIT) is an anticancer quassinoid isolated from Ailanthus altissima It inhibits proliferation and induces cell death of many cancer cell types The drug activates DNA damage response and targets p23 cochaperone Up or downregulation of several microRNA by AIT contributes to the anticancer activity Analogs or specific formulations must be developed to prevent the toxicity of AIT.