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Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide. The etiological agent responsible for this disease is "Candidatus Liberibacter asiaticus" (CLas), a phloem-restricted bacterium transmitted by psyllid vectors. To date, effective practical strategies for curing Citrus HLB remain elusive. Additionally, no susceptibility genes associated with HLB have been identified in Citrus species, thereby complicating the application of gene-editing techniques such as CRISPR-Cas9 to enhance resistance to HLB. The co-chaperone DnaJ plays a crucial role in protein folding and the regulation of various physiological activities, and it is also associated with multiple pathological processes. DnaJ has been extensively studied in many species, including Arabidopsis, rice, and wheat. However, there is limited information available regarding the DnaJ gene family in citrus. In this study, we conducted a comprehensive genome-wide analysis of the DnaJ family genes in various Citrus species. The Citrus genome was identified to contain 86 DnaJ genes, which were unevenly distributed across nine chromosomes. Phylogenetic analysis indicated that these genes could be classified into six distinct groups. Furthermore, transcriptomic analysis revealed that nine DnaJ genes exhibited significantly higher induction in HLB-infected samples relative to non-HLB-infected Citrus. Cis-acting elements within the promoters of DnaJ genes were also examined, revealing the presence of hormone and defense/stress responsiveness elements (TC-rich) distributed on the ClDJC24 gene. The results were validated using quantitative real-time PCR (qRT-PCR). Additionally, the silencing of ClDJC24 suggested that this gene negatively regulates disease resistance in Citrus. Our study provided useful clues for further functional characterization and constructed a theoretical foundation for disease-resistant breeding in Citrus. [ABSTRACT FROM AUTHOR]

DnaJ, or heat shock protein 40 (HSP40), is a conserved protein that functions as a molecular chaperone with a strong cytoprotective effect, participating in plant signal transduction, growth, development, and the response to heat stress and other adverse environmental conditions. Although functional characteristics of DnaJ/HSP40 have been described in several plant species, the underlying mechanisms are not completely understood. In this study, 99 putative maize (Zea mays L.) DnaJ genes (ZmDnaJ01–99) randomly distributed on 10 chromosomes were identified in the whole genome using bioinformatics approaches and classified into three groups according to protein structure. The conservation of the gene structure was closely related to the evolutionary tree. Collinearity analysis of DnaJ family genes among species revealed a close evolutionary relationship between maize and sorghum. Transposon analysis revealed helitrons inserted in 49 ZmDnaJs. Multiple stress-related cis-elements were found in the promoter regions of ZmDnaJ genes, suggesting a role in gene expression regulation in response to abiotic stress. The co-expression network screened 12 ZmDnaJ genes that could be induced by stress (drought, high temperature, and salt) and regulated by abscisic acid. ZmDnaJ96 overexpression increased antioxidant enzyme activity and reduced the damage caused by drought and high-temperature stress in Arabidopsis chloroplasts. Virus-induced silencing of ZmDnaJ96 reduced drought and heat tolerance in maize by reducing antioxidant enzyme activity. These results provide genetic resources for maize stress resistance molecular breeding, enhance knowledge of DnaJ gene function in maize, and offer a solid foundation for further in-depth study of the relationship between maize DnaJ/HSP40 members. [ABSTRACT FROM AUTHOR]

Abstract Background The aqueous environment directs the protein folding process towards the generation of micelle-type structures, which results in the exposure of hydrophilic residues on the surface (polarity) and the concentration of hydrophobic residues in the center (hydrophobic core). Obtaining a structure without a hydrophobic core requires a different type of external force field than those generated by a water. The examples are membrane proteins, where the distribution of hydrophobicity is opposite to that of water-soluble proteins. Apart from these two extreme examples, the process of protein folding can be directed by chaperones, resulting in a structure devoid of a hydrophobic core. Results The current work presents such example: DnaJ Hsp40 in complex with alkaline phosphatase PhoA-U (PDB ID—6PSI)—the client molecule. The availability of WT form of the folding protein—alkaline phosphatase (PDB ID—1EW8) enables a comparative analysis of the structures: at the stage of interaction with the chaperone and the final, folded structure of this biologically active protein. The fuzzy oil drop model in its modified FOD-M version was used in this analysis, taking into account the influence of an external force field, in this case coming from a chaperone. Conclusions The FOD-M model identifies the external force field introduced by chaperon influencing the folding proces. The identified specific external force field can be applied in Ab Initio protein structure prediction as the environmental conditioning the folding proces.

Plant DnaJA proteins act as molecular chaperones in response to environmental stressors. The purpose of this study was to characterize the function and regulatory mechanisms of DnaJA genes in soybean. Gene expression profiles in various soybean tissues at various stages of development indicated that GmDnaJAs function in the coordination of stress and plant hormone responses. GmDnaJA6 was identified as a candidate regulator of saline and alkaline stress resistance and GmDnaJA6 overexpression lines showed increased soybean saline and alkaline tolerance. DnaJ interacted with Hsp70, and GmHsp70 increased the saline and alkaline tolerance of plants with chimeric soybean hairy roots.

The brown planthopper (BPH) Nilaparvata lugens (Stål) is a main pest on rice. It secretes saliva to regulate plant defense responses, when penetrating rice plant and sucking phloem sap through its stylet. However, the molecular mechanisms of BPH salivary proteins regulating plant defense responses remain poorly understood. A N. lugens DNAJ protein (NlDNAJB9) gene was highly expressed in salivary glands, and the knock down of NlDNAJB9 significantly enhanced honeydew excretion and fecundity of the BPH. NlDNAJB9 could induce plant cell death, and the overexpression of NlDNAJB9 gene in Nicotiana benthamiana induced calcium signaling, mitogen-activated protein kinase (MAPK) cascades, reactive oxygen species (ROS) accumulation, jasmonic acid (JA) hormone signaling and callose deposition. The results from different NlDNAJB9 deletion mutants indicated that the nuclear localization of NlDNAJB9 was not necessary to induce cell death. The DNAJ domain was the key region to induce cell death, and the overexpression of DNAJ domain in N. benthamiana significantly inhibited insect feeding and pathogenic infection. NlDNAJB9 might interact indirectly with NlHSC70-3 to regulate plant defense responses. NlDNAJB9 and its orthologs were highly conserved in three planthopper species, and could induce ROS burst and cell death in plants. Our study provides new insights into the molecular mechanisms of insect-plant interactions. [ABSTRACT FROM AUTHOR]

Background: The aqueous environment directs the protein folding process towards the generation of micelle-type structures, which results in the exposure of hydrophilic residues on the surface (polarity) and the concentration of hydrophobic residues in the center (hydrophobic core). Obtaining a structure without a hydrophobic core requires a different type of external force field than those generated by a water. The examples are membrane proteins, where the distribution of hydrophobicity is opposite to that of water-soluble proteins. Apart from these two extreme examples, the process of protein folding can be directed by chaperones, resulting in a structure devoid of a hydrophobic core. Results: The current work presents such example: DnaJ Hsp40 in complex with alkaline phosphatase PhoA-U (PDB ID—6PSI)—the client molecule. The availability of WT form of the folding protein—alkaline phosphatase (PDB ID—1EW8) enables a comparative analysis of the structures: at the stage of interaction with the chaperone and the final, folded structure of this biologically active protein. The fuzzy oil drop model in its modified FOD-M version was used in this analysis, taking into account the influence of an external force field, in this case coming from a chaperone. Conclusions: The FOD-M model identifies the external force field introduced by chaperon influencing the folding proces. The identified specific external force field can be applied in Ab Initio protein structure prediction as the environmental conditioning the folding proces. [ABSTRACT FROM AUTHOR]

The present study reports the recognition and characterization of the gene encoding the co-chaperone DnaJ in the halophilic strain Mesobacillus persicus B48. The new extracted gene was sequenced and cloned in E. coli, followed by protein purification using a C-terminal His-tag. The stability and function of the recombinant DnaJ protein under salt and pH stress conditions were evaluated. SDS-PAGE revealed a band on nearly 40-kDa region. The homology model structure of new DnaJ demonstrated 56% similarity to the same protein from Streptococcus pneumonia. Fluorescence spectra indicated several hydrophobic residues located on the protein surface, which is consistent with the misfolded polypeptide recognition function of DnaJ. Spectroscopic results showed 56% higher carbonic anhydrase activity in the presence of the recombinant DnaJ homolog compared to its absence. In addition, salt resistance experiments showed that the survival of recombinant E. coli+DnaJ was 2.1 times more than control cells in 0.5 M NaCl. Furthermore, the number of recombinant E. coli BL21+DnaJ colonies was 7.7 times that of the control colonies in pH 8.5. Based on the results, DnaJ from the M. persicus can potentially be employed for improving the functional features of enzymes and other proteins in various applications. [ABSTRACT FROM AUTHOR]

The role of the structural genes groESL and dnaJ and the regulatory gene hrcA, which are involved in the synthesis of heat shock proteins, in biosurfactant production by R. pyridinivorans 5Ap was determined. CIRCE binding sites for the regulatory protein encoded by hrcA were detected in the promoter regions of groESL, groEL2, and fmdB. The expression of groESL and groEL2 during the late exponential phase in the medium with hexadecane at 42°C was higher than at 28°C (4.4 and 5.3 times, respectively). At the same time, no changes in hrcA and fmdB expression were observed at two different temperature modes (28 and 42°C). In the absence of the negative regulator HrcA, groESL expression increased 14.4 and 3.5 times, that of groEL2, 9.6 and 2.7 times, and that of fmdB, 1.82 and 2.52 times at 28 and 42°C, respectively. Products of dnaJ and hrcA genes were required for trehalolipid synthesis at different temperature modes, with their role increasing at higher temperature (synthesis of trehalolipids by the mutant with impaired dnaJ gene decreased 1.8 and 2.5 times compared to 1.5 and 6.6 times, for the mutant with impaired hrcA at 28 and 42°C, respectively). At the same time, emulsifying activity of all mutant variants did not change at 28°C and decreased 1.4 and 1.9 times at 42°C for the mutants with impaired groESL and hrcA genes, respectively. Our results indicated the complex chemical nature of the biosurfactants produce by R. pyridinivorans 5Ap (emulsifiers, including trehalolipids and compounds of other chemical composition). The Gro chaperones and the regulatory protein HrcA play the key roles in the synthesis of these compounds at different temperatures, while the dnaJ product is required only for trehalolipid synthesis. [ABSTRACT FROM AUTHOR]

Methamphetamine (MA) is spread worldwide and is a highly addictive psychostimulant that can induce neurodegeneration and cognitive disorder, which lacks effective treatments. We and other researchers have found that the crucial member of Hsp70 chaperone machinery, DnaJ, is liable to be co-aggregated with aberrant proteins, which has been confirmed a risk factor to promote neurodegeneration. In the current study, we demonstrated that tailing with a hyper-acidic fusion partner, tua2, human DnaJB1 could resist the formation of toxic mutant Tau aggregates both in prokaryote and eukaryote models. We found that aberrant Tau aggregates could deplete the antioxidant enzyme pool and disturb Hsp70 molecular chaperone system by co-aggregating with the principal members of these systems. Stability-enhanced DnaJB1-tua2 could stop the chain reaction of Tau aggregates as well as maintain redox balance and protein homeostasis. With an MA-induced cognitive disorder mouse model, we found that the cognitive disorder of MA mice was rescued and the overactivated inflammatory response was relieved by the expression of DnaJB1-tua2 in the hippocampus. Furthermore, the Tau neurofibrillary tangles and apoptotic neurons were diminished with the escorting of DnaJB1-tua2. These findings demonstrate that delivering DnaJB1-tua2 in hippocampus may have a therapeutic potential in the treatment of MA-induced cognitive disorder.

Abstract Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of carbon fixation performed by photosynthetic organisms. Form I of this enzyme found in plants and cyanobacteria is composed of eight large (RbcL) and eight small (RbcS) subunits. To form a functional enzyme, Rubisco subunits need to be properly folded, with the assistance of cellular chaperone machinery, and consecutively assembled in a strictly orchestrated manner, with the help of multiple auxiliary factors. In recent years, multiple Rubisco assembly chaperones and their function in enzyme biogenesis have been extensively characterized. Little is known about the potential specialized factors involved in Rubisco subunits folding at the pre-chaperonin stage, yet this knowledge is greatly needed for the fast and efficient testing of new Rubisco variants. Synechococcus sp. PCC 6803 Rubisco shows limited solubility and a lack of assembly in the Escherichia coli expression system. In this study, we aim to identify which additional chaperones are necessary and sufficient in sustaining the heterologous assembly of native Rubisco. Our findings prove that upon the introduction of Synechocystis DnaK2 to the E. coli system, RbcL is produced in soluble form. The addition of specific DnaJ (Sll1384) enhances this effect. We explain these combined effects based on binding constancies, measured for particular partners in vitro, as well as our analysis of the putative tertiary structure of the proteins. Our results have potential implications for Rubisco engineering.

Reduced crop productivity results from altered plant physiological processes caused by dysfunctional proteins due to environmental stressors. In this study, a novel DnaJ Type-I encoding gene, VaDJI having a zinc finger motif in its C-terminal domain was found to be induced early upon treatment with heat stress (within 5 min) in a heat tolerant genotype of Vigna aconitifolia RMO-40. VaDJI is induced by multiple stresses. In tobacco, ectopic expression of VaDJI reduced ABA sensitivity during seed germination and the early stages of seedling growth of transgenic tobacco plants. Concomitantly, it also improved the ability of transgenic tobacco plants to withstand drought stress by modulating the photosynthetic efficiency, with the transgenic plants having higher Fv/Fm ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H2O2 and lower MDA levels and higher chlorophyll content during drought stress, which attenuated cell damage and reduced oxidative damage. An analysis using the qRT-PCR study demonstrated that VaDJI overexpression is associated with the expression of some ROS-detoxification-related genes and stress-marker genes that are often induced during drought stress responses. These findings suggest a hypothesis whereby VaDJI positively influences drought stress tolerance and ABA signalling in transgenic tobacco, and suggests that it is a potential gene for genetic improvement of drought and heat stress tolerance in crop plants. [ABSTRACT FROM AUTHOR]

Background: Endosomally produced by eukaryotic cells, exosomes are microvesicles involved in cell-to-cell communication. Exosomes have shown a wide range of therapeutic potential as a drug or vaccine delivery system, and they are useful as biomarkers in several disease processes. Another biological function described is pathogen dissemination through host-derived molecules released during infection, thus modulating the immune response in the host. Results: This work characterizes the exosomal fraction recovered from serum of Piscirickttesia salmonis-challenged Salmo salar specimens and from the corresponding non-challenged controls. Exosomes presented a spherical morphology and particle size distribution within 50–125 nm, showing similar parameters in both groups. The mass spectrometry analysis of exosomes isolated at 14 and 21 d post-challenge showed the presence of peptides corresponding to the three proteins of Hsp70/DnaK chaperone system (DnaK, DnaJ, and GrpE). BLAST search of these peptides showed the specificity to P. salmonis. Data are available via ProteomeXchange with identifier PXD023594. Conclusions: The chaperones were found with >95% identity in the core genome when aligned to 73 genomes of P. salmonis. The proteins also showed a high degree of similarity with other microorganisms, where this system has proven to be vital for their survival under stress conditions.The presence of these three proteins in exosomes isolated from challenged fish sera calls for further study into their potential role in bacterium pathogenicity.How to cite: Muñoz C, Carmona M, Luna O, et al. Serum-isolated exosomes from Piscirickettsia salmonis-infected Salmo salar specimens enclose bacterial DnaK, DnaJ and GrpE chaperones. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.07.003.

Reduced crop productivity results from altered plant physiological processes caused by dysfunctional proteins due to environmental stressors. In this study, a novel DnaJ Type-I encoding gene, VaDJI having a zinc finger motif in its C-terminal domain was found to be induced early upon treatment with heat stress (within 5 min) in a heat tolerant genotype of Vigna aconitifolia RMO-40. VaDJI is induced by multiple stresses. In tobacco, ectopic expression of VaDJI reduced ABA sensitivity during seed germination and the early stages of seedling growth of transgenic tobacco plants. Concomitantly, it also improved the ability of transgenic tobacco plants to withstand drought stress by modulating the photosynthetic efficiency, with the transgenic plants having higher Fv/Fm ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H2O2 and lower MDA levels and higher chlorophyll content during drought stress, which attenuated cell damage and reduced oxidative damage. An analysis using the qRT-PCR study demonstrated that VaDJI overexpression is associated with the expression of some ROS-detoxification-related genes and stress-marker genes that are often induced during drought stress responses. These findings suggest a hypothesis whereby VaDJI positively influences drought stress tolerance and ABA signalling in transgenic tobacco, and suggests that it is a potential gene for genetic improvement of drought and heat stress tolerance in crop plants.

DnaJs are the common molecular chaperone proteins with strong structural and functional diversity. In recent years, only several DnaJ family members have been found to be able to regulate leaf color, and it remains to be explored whether there are other potential members that also regulate this character. Here, we identified 88 putative DnaJ proteins from Catalpa bungei, and classified them into four types according to their domain. Gene-structure analysis revealed that each member of CbuDnaJ family had same or similar exon-intron structure. Chromosome mapping and collinearity analysis showed that tandem and fragment duplication occurred in the process of evolution. Promoter analyses suggested that CbuDnaJs might be involved in a variety of biological processes. The expression levels of DnaJ family members in different color leaves of Maiyuanjinqiu were respectively extracted from the differential transcriptome. Among these, CbuDnaJ49 was the largest differentially expressed gene between the green and yellow sectors. Ectopic overexpression of CbuDnaJ49 in tobacco showed that the positive transgenic seedlings exhibited albino leaves, and the contents of chlorophyll and carotenoid were significantly reduced compared with those of wild type. The results suggested that CbuDnaJ49 played an important role in regulating leaf color. This study not only identified a novel gene of DnaJ family members regulating leaf color, but also provided new germplasm for landscaping. [ABSTRACT FROM AUTHOR]

DnaJ (Orf40), a late-expressed factor of Bombyx mori nucleopolyhedrovirus (BmNPV), is essential for the budding of virions and influences the transfer of the nucleocapsid from the nucleus to the cytoplasm. Previous studies showed that the knockdown of dnaj could prevent the nucleocapsid from exiting the nucleus, but the underlying regulatory mechanism remains unknown. In our previous acetylomic analysis of BmN cells infected with wild-type BmNPV, we found that a lysine residue (K17) was acetylated 36h post-infection, and the acetylation level of this site was upregulated about 3.5-fold. Here, we found that deacetylation of DnaJ K17 significantly inhibited viral proliferation without affecting viral DNA replication. Furthermore, deacetylation of DnaJ K17 affected the interaction with two nucleocapsid-associated proteins, Ac66 and VP80, which in turn affected the production of nucleocapsids, as well as their transport within the nucleus along F-actin fibers, leading to a decrease in the export of nucleocapsids from the nucleus. The reduced amount of nucleocapsids in the cytoplasm ultimately led to a decrease in the production of budded virions and consequently inhibited viral proliferation. In conclusion, acetylation of DnaJ affects nucleocapsid production and transport, thereby influencing viral proliferation. • Acetylation of DnaJ promotes viral proliferation. • Acetylation of DnaJ facilitates nucleocapsid generation and transport. • Acetylation of DnaJ affects its ability to interact with Ac66, VP80 and Actin. [ABSTRACT FROM AUTHOR]

DnaJ proteins are key molecular chaperones that act as a part of the stress response to stabilize plant proteins, thereby maintaining protein homeostasis under stressful conditions. Herein we used transgenic plants to explore the role of the tomato (Solanum lycopersicum) SlDnaJ20 chloroplast DnaJ protein in to the resistance of these proteins to cold. When chilled, transgenic plants exhibited superior cold resistance, with reduced growth inhibition and cellular damage and increased fresh mass and chlorophyll content relative to control. These transgenic plants further exhibited increased Fv/Fm, P700 oxidation, φRo, and δRo relative to control plants under chilling conditions. Under these same cold conditions, these transgenic plants also exhibited higher levels of core proteins in the photosystem I (PSI) and II (PSII) complexes (PsaA and PsaB; D1 and D2) relative to control wild-type plants. Together these results suggested that the overexpression of SlDnaJ20 is sufficient to maintain PSI and PSII complex stability and to alleviate associated photoinhibition of these complexes, thereby increasing transgenic plant resistance to cold stress.

Background: Endosomally produced by eukaryotic cells, exosomes are microvesicles involved in cell-tocell communication. Exosomes have shown a wide range of therapeutic potential as a drug or vaccine delivery system, and they are useful as biomarkers in several disease processes. Another biological function described is pathogen dissemination through host-derived molecules released during infection, thus modulating the immune response in the host. Results: This work characterizes the exosomal fraction recovered from serum of Piscirickttesia salmonischallenged Salmo salar specimens and from the corresponding non-challenged controls. Exosomes presented a spherical morphology and particle size distribution within 50-125 nm, showing similar parameters in both groups. The mass spectrometry analysis of exosomes isolated at 14 and 21 d post-challenge showed the presence of peptides corresponding to the three proteins of Hsp70/DnaK chaperone system (DnaK, DnaJ, and GrpE). BLAST search of these peptides showed the specificity to P. salmonis. Data are available via ProteomeXchange with identifier PXD023594. Conclusions: The chaperones were found with >95% identity in the core genome when aligned to 73 genomes of P. salmonis. The proteins also showed a high degree of similarity with other microorganisms, where this system has proven to be vital for their survival under stress conditions. The presence of these three proteins in exosomes isolated from challenged fish sera calls for further study into their potential role in bacterium pathogenicity. [ABSTRACT FROM AUTHOR]

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates. [Display omitted] • Trigger factor binds collapsed folding intermediates and outcompetes DnaJ • DnaJ recruits DnaK to both partially and extensively folded intermediates • Chaperone binding is disfavored close to the ribosome surface • Chaperone binding does not reverse cotranslational folding Several unrelated classes of molecular chaperones guide the folding of nascent polypeptides as they emerge from the ribosome. Using in vitro reconstitution, Roeselová et al. show that different chaperones recognize distinct features of nascent proteins. As a result, the chaperone network persistently engages nascent chains without antagonizing cotranslational folding. [ABSTRACT FROM AUTHOR]

Soybean [Glycine max (L.) Merri.] is an important oilseed and food crop. In recent years, environmental degradation has accelerated soil alkalization, salinization, and water deficit, which have seriously threatened the soybean quality and yield. Chaperone DNAJ proteins play important roles in plant response to a number of abiotic and biotic stresses. Here, we investigated the function of a soybean DNAJ gene, GmDNAJC7, in plant tolerance to abiotic stresses. GmDNAJC7 gene expression was induced by alkaline-salt, salt, and drought treatments in soybean roots, suggesting its possible role in soybean response to these stresses. GmDNAJC7 overexpression improved the alkaline-salt tolerance of soybean composite plants, which showed a higher SPAD (Soil and Plant Analysis Development) value for chlorophyll content and leaf relative water content than the control plants after NaHCO3 treatment. Moreover, the GmDNAJC7 overexpressing Arabidopsis had a higher germination rate and average root length than the wild type and dnajc7 mutant, under NaHCO3, NaCl, and mannitol stresses, indicating that the ectopic expression of the GmDNAJC7 gene enhanced the alkaline-salt, salt, and drought tolerance in Arabidopsis. These findings suggest that GmDNAJC7 is involved in the alkaline-salt, salt, and drought tolerance in Arabidopsis and soybean. This study provides new insights into the role of DNAJ proteins in plant tolerance to abiotic stress. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many neurodegenerative diseases, including Alzheimer's, originate from the conversion of proteins into pathogenic conformations. The microtubule-associated protein tau converts into β-sheet-rich amyloid conformations, which underlie pathology in over 25 related tauopathies. Structural studies of tau amyloid fibrils isolated from human tauopathy tissues have revealed that tau adopts diverse structural polymorphs, each linked to a different disease. Molecular chaperones play central roles in regulating tau function and amyloid assembly in disease. New data supports the model that chaperones selectively recognize different conformations of tau to limit the accumulation of proteotoxic species. The challenge now is to understand how chaperones influence disease processes across different tauopathies, which will help guide the development of novel conformation-specific diagnostic and therapeutic strategies. Tau adopts distinct structures in disease and its assembly is mediated by soluble pathogenic seeds. J-domain proteins (JDPs) alone recognize different structural states of tau and, in conjunction with Hsp70, drive disaggregation of tau fibrils and refolding. Hsp90 binds to partially folded tau intermediates using exposed hydrophobic motifs in the repeat domains. Small heat shock proteins (sHSPs) use weak/transient interactions to stabilize amyloid motifs on tau to control aggregation. [ABSTRACT FROM AUTHOR]

As their name indicates, polyomaviruses (PyVs) can induce tumors. Mouse PyV, hamster PyV and raccoon PyV have been shown to cause tumors in their natural host. During the last 30 years, 15 PyVs have been isolated from humans. From these, Merkel cell PyV is classified as a Group 2A carcinogenic pathogen (probably carcinogenic to humans), whereas BKPyV and JCPyV are class 2B (possibly carcinogenic to humans) by the International Agency for Research on Cancer. Although the other PyVs recently detected in humans (referred to here as novel HPyV; nHPyV) share many common features with PyVs, including the viral oncoproteins large tumor antigen and small tumor antigen, as their role in cancer is questioned. This review discusses whether the nHPyVs may play a role in cancer based on predicted and experimentally proven functions of their early proteins in oncogenic processes. The functional domains that mediate the oncogenic properties of early proteins of known PyVs, that can cause cancer in their natural host or animal models, have been well characterized and we examined whether these functional domains are conserved in the early proteins of the nHPyVs and presented experimental evidence that these conserved domains are functional. Furthermore, we reviewed the literature describing the detection of nHPyV in human tumors. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are secreted vesicles of diverse size and cargo that are implicated in the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. Mutant huntingtin, the disease-causing entity in Huntington's disease, has an expanded polyglutamine track at the N terminus that causes the protein to misfold and form toxic intracellular aggregates. In Huntington's disease, mutant huntingtin aggregates are transferred between cells by several routes. We have previously identified a cellular pathway that is responsible for the export of mutant huntingtin via extracellular vesicles. Identifying the EV sub-populations that carry misfolded huntingtin cargo is critical to understanding disease progression. In this work we expressed a form of polyglutamine expanded huntingtin (GFP-tagged 72Qhuntingtinexon1) in cells to assess the EVs involved in cellular export. We demonstrate that the molecular chaperone, cysteine string protein (CSPα; DnaJC5), facilitates export of disease-causing-polyglutamine-expanded huntingtin cargo in 180–240 nm vesicles as well as larger 10–30 μm vesicles. [ABSTRACT FROM AUTHOR]

Introduction: To prevent pneumococcal infections, especially meningitis and bacteremia, and to overcome the serotype-dependent limitation of polysaccharide-based vaccines, the development of conserved protein-based vaccines is essential. This study aimed at investigate the in-silico analysis and epitope mapping of pneumococcal DnaJ for the first time, and to design the multi-epitope based vaccines with different categories by focusing on induction of both humoral and cellular immunities. Methods: We predicted B- and T-cell epitopes, IL-4, IL-17, IL-10, and IFN-γ inducer epitopes of DnaJ using Immunoinformatics tools. The immunogenicity and conservation score of the predicted epitopes among pneumococcal prevalent clinical serotypes, the immune simulation of DnaJ administration in mammals and potential regions involved in DnaJTLRs interactions were analyzed. Finally, we proposed three classes of multi-epitope DnaJ-based vaccine candidates. Results: This protein had 24 and 15 predicted linear Bcell and helper T-cell epitopes, respectively, with a conservation score of 86-100% among prevalent clinical pneumococcal serotypes. DnaJ also had many IL-4 and IFN-γ inducing epitopes and was considered an IL-10 and IL-17 inducer protein. The immune simulation showed induction of both humoral and cellular immunity against DnaJ. The residues at positions 274, 280, 292, 297, 300, 316-319, 333, 336-340, 358, 363-366, and 372 were predicted to be involved in DnaJ-TLR2 and DnaJ-TLR4 interactions. Three classes of proposed DnaJ-based constructs were based on only B-cell epitopes, only helper T-cell epitopes, and multi-epitopes of B- and T-cell and IL-17 epitopes. Conclusion: The results showed that although DnaJ has been reported to play an important role in cellular immunity, our results indicated the high potential of DnaJ to stimulate mucosal, humoral, and cellular immunity. [ABSTRACT FROM AUTHOR]

Hsp70 proteins and their Hsp40 co‐chaperones are essential components of cellular chaperone networks in both prokaryotes and eukaryotes. Here, we performed a genetic analysis to define the protein domains required for the key functions of the major Hsp40/DnaJ protein Sll0897 of the cyanobacterium Synechocystis sp. PCC6803. The expression of the N‐terminally located J‐ and G/F‐domains is essential and sufficient for the proteins’ fundamental in vivo functions, whereas the presence of the full‐length protein, containing the C‐terminal substrate‐binding domains, is crucial under stress conditions.

As their name indicates, polyomaviruses (PyVs) can induce tumors. Mouse PyV, hamster PyV and raccoon PyV have been shown to cause tumors in their natural host. During the last 30 years, 15 PyVs have been isolated from humans. From these, Merkel cell PyV is classified as a Group 2A carcinogenic pathogen (probably carcinogenic to humans), whereas BKPyV and JCPyV are class 2B (possibly carcinogenic to humans) by the International Agency for Research on Cancer. Although the other PyVs recently detected in humans (referred to here as novel HPyV; nHPyV) share many common features with PyVs, including the viral oncoproteins large tumor antigen and small tumor antigen, as their role in cancer is questioned. This review discusses whether the nHPyVs may play a role in cancer based on predicted and experimentally proven functions of their early proteins in oncogenic processes. The functional domains that mediate the oncogenic properties of early proteins of known PyVs, that can cause cancer in their natural host or animal models, have been well characterized and we examined whether these functional domains are conserved in the early proteins of the nHPyVs and presented experimental evidence that these conserved domains are functional. Furthermore, we reviewed the literature describing the detection of nHPyV in human tumors.

Extracellular vesicles (EVs) are secreted vesicles of diverse size and cargo that are implicated in the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. Mutant huntingtin, the disease-causing entity in Huntington’s disease, has an expanded polyglutamine track at the N terminus that causes the protein to misfold and form toxic intracellular aggregates. In Huntington’s disease, mutant huntingtin aggregates are transferred between cells by several routes. We have previously identified a cellular pathway that is responsible for the export of mutant huntingtin via extracellular vesicles. Identifying the EV sub-populations that carry misfolded huntingtin cargo is critical to understanding disease progression. In this work we expressed a form of polyglutamine expanded huntingtin (GFP-tagged 72Qhuntingtinexon1) in cells to assess the EVs involved in cellular export. We demonstrate that the molecular chaperone, cysteine string protein (CSPα; DnaJC5), facilitates export of disease-causing-polyglutamine-expanded huntingtin cargo in 180–240 nm vesicles as well as larger 10–30 μm vesicles.

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Proteostasis is defined by the orchestrated control of anabolic and catabolic protein pathways. Disruption of proteostasis results in cell stress and adaptation to proteostasis imbalance is mediated by adaptive pathways such as the Heat Shock Response (including heat-shock proteins) or the unfolded protein response (UPR). The BCR-ABL1 kinase (Philadelphia chromosome) is the hallmark of chronic myeloid leukemia (CML) and defines a historically poor subset in acute lymphoblastic leukemia (Ph(+) ALL). We previously demonstrated the importance of the UPR and particularly of the IRE1/XBP1 signaling axis in Ph(+) ALL, while others demonstrated the therapeutic relevance of HSP70 in ALL. In this regard, HSP70 is regulated by smaller HSP40 s, whose function is so far poorly characterized. RESULTS: Herein, we characterize the expression of HSP40 s in Ph(+) ALL and CML. We show that these genes are not regulated in a pan-class manner and identify a homologous gene pair, namely Auxilin-1 (DNAJC6) and Auxilin-2 (GAK) with a unique expression profile. Overexpression of Auxilin-2, the ubiquitously expressed homologue of Auxilin-1 correlated with superior clinical outcome in ALL and was tightly linked to both IRE1 RNase and BCR-ABL1 kinase activities. CONCLUSIONS: Our findings suggest that HSP40 gens are uniquely regulated and provide a rationale for further studies between BCR-ABL1/IRE1-based therapies in combination with HSP40 inhibitors, thus opening potentially novel therapeutic avenues.

Seagrass meadows are one of the most important marine resources that grow along the coast. They provide habitat and a food source for animals. They also protect the coast, fix sediment and purify seawater. In the current period of global climate change, anomalies in coastal water temperatures are increasing. A sudden increase in water temperature owing to a heat wave can have a profound effect on seagrass. Zostera japonica is a type of intertidal seagrasses, which is exposed to the air at low tide. High temperatures in the summer often lead to a decline in seagrass meadows. DnaJ proteins, also known as J proteins, are a family of conserved chaperone proteins. They are designated as J proteins because they contain a highly conserved J domain. They function as chaperones of heat shock proteins in organisms. In this study, the role of DnaJ protein (ZjDjB1) of Z. japonica under heat stress was studied. ZjDjB1 was localized to the cytoplasm and nucleus. The overexpression of ZjDjB1 in Arabidopsis thaliana results in an increase in thermotolerance and a decrease in the accumulation of reactive oxygen species and also a reduction in membrane damage. ZjDjB1 may achieve this goal by maintaining a low activity of proteolytic enzymes. [ABSTRACT FROM AUTHOR]

Introduction: To prevent pneumococcal infections, especially meningitis and bacteremia, and to overcome the serotype-dependent limitation of polysaccharide-based vaccines, the development of conserved protein-based vaccines is essential. This study aimed at investigate the in-silico analysis and epitope mapping of pneumococcal DnaJ for the first time, and to design the multi-epitope based vaccines with different categories by focusing on induction of both humoral and cellular immunities. Methods: We predicted B- and T-cell epitopes, IL-4, IL-17, IL-10, and IFN-γ inducer epitopes of DnaJ using Immunoinformatics tools. The immunogenicity and conservation score of the predicted epitopes among pneumococcal prevalent clinical serotypes, the immune simulation of DnaJ administration in mammals and potential regions involved in DnaJTLRs interactions were analyzed. Finally, we proposed three classes of multi-epitope DnaJ-based vaccine candidates. Results: This protein had 24 and 15 predicted linear Bcell and helper T-cell epitopes, respectively, with a conservation score of 86-100% among prevalent clinical pneumococcal serotypes. DnaJ also had many IL-4 and IFN-γ inducing epitopes and was considered an IL-10 and IL-17 inducer protein. The immune simulation showed induction of both humoral and cellular immunity against DnaJ. The residues at positions 274, 280, 292, 297, 300, 316-319, 333, 336-340, 358, 363-366, and 372 were predicted to be involved in DnaJ-TLR2 and DnaJ-TLR4 interactions. Three classes of proposed DnaJ-based constructs were based on only B-cell epitopes, only helper T-cell epitopes, and multi-epitopes of B- and T-cell and IL-17 epitopes. Conclusion: The results showed that although DnaJ has been reported to play an important role in cellular immunity, our results indicated the high potential of DnaJ to stimulate mucosal, humoral, and cellular immunity. [ABSTRACT FROM AUTHOR]

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70–Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease.

Soybean [Glycine max (L.) Merri.] is an important oilseed and food crop. In recent years, environmental degradation has accelerated soil alkalization, salinization, and water deficit, which have seriously threatened the soybean quality and yield. Chaperone DNAJ proteins play important roles in plant response to a number of abiotic and biotic stresses. Here, we investigated the function of a soybean DNAJ gene, GmDNAJC7, in plant tolerance to abiotic stresses. GmDNAJC7 gene expression was induced by alkaline-salt, salt, and drought treatments in soybean roots, suggesting its possible role in soybean response to these stresses. GmDNAJC7 overexpression improved the alkaline-salt tolerance of soybean composite plants, which showed a higher SPAD (Soil and Plant Analysis Development) value for chlorophyll content and leaf relative water content than the control plants after NaHCO3 treatment. Moreover, the GmDNAJC7 overexpressing Arabidopsis had a higher germination rate and average root length than the wild type and dnajc7 mutant, under NaHCO3, NaCl, and mannitol stresses, indicating that the ectopic expression of the GmDNAJC7 gene enhanced the alkaline-salt, salt, and drought tolerance in Arabidopsis. These findings suggest that GmDNAJC7 is involved in the alkaline-salt, salt, and drought tolerance in Arabidopsis and soybean. This study provides new insights into the role of DNAJ proteins in plant tolerance to abiotic stress.

SUMMARYHeat shock protein 90 (Hsp90) participates in proteostasis by facilitating protein folding, activation, disaggregation, prevention of aggregation, degradation, and protection against degradation of various cellular proteins. It is highly conserved from bacteria to humans. In bacteria, protein remodeling by Hsp90 involves collaboration with the Hsp70 molecular chaperone and Hsp70 cochaperones. In eukaryotes, protein folding by Hsp90 is more complex and involves collaboration with many Hsp90 cochaperones as well as Hsp70 and Hsp70 cochaperones. This review focuses primarily on bacterial Hsp90 and highlights similarities and differences between bacterial and eukaryotic Hsp90. Seminal research findings that elucidate the structure and the mechanisms of protein folding, disaggregation, and reactivation promoted by Hsp90 are discussed. Understanding the mechanisms of bacterial Hsp90 will provide fundamental insight into the more complex eukaryotic chaperone systems., Competing Interests: The authors declare no conflict of interest.

Bacteria encode a wide range of antiphage systems and a subset of these proteins are homologous to components of the human innate immune system. Mammalian nucleotide-binding and leucine-rich repeat containing proteins (NLRs) and bacterial NLR-related proteins use a central NACHT domain to link infection detection with initiation of an antimicrobial response. Bacterial NACHT proteins provide defense against both DNA and RNA phages. Here we determine the mechanism of RNA phage detection by the bacterial NLR-related protein bNACHT25 in E. coli . bNACHT25 was specifically activated by Emesvirus ssRNA phages and analysis of MS2 phage suppressor mutants that evaded detection revealed Coat Protein (CP) was sufficient for activation. bNACHT25 and CP did not physically interact. Instead, we found bNACHT25 requires the host chaperone DnaJ to detect CP. Our data suggest that bNACHT25 detects a wide range of phages by guarding a host cell process rather than binding a specific phage-derived molecule., Competing Interests: Declaration of interests The authors declare no competing interests.

Salix is a dioecious plant. Research on the molecular regulation mechanism of male and female inflorescence differentiation and development is necessary to analyze sex differentiation in the willow and the underlying mechanisms of unisexual flower development. However, at present, there are no reference genes suitable for stable expression in the process of willow inflorescence development. In this study, Salix suchowensis was used as the research material, nine candidate reference genes (α-TUB1, α-TUB2, ACT, H2A, DnaJ, CDC2, GAPDH, TIP41, β-TUB) were selected, and qRT-PCR technology was used to detect the expression of each candidate reference gene in female and male flowers at different developmental stages and using five algorithms (geNorm, Normfinder, Delta Ct, BestKeeper, and RefFinder) to comprehensively evaluate the stability of candidate reference genes. The results showed that ACT and DnaJ were stably expressed in all samples and could be used as reference genes. In addition, the reliability of the screening results was further verified via an expression pattern analysis of the CFS gene that encodes flower specific transcription factor in different samples. The stable reference genes selected in this study provide the basis for future research on the expression analysis of functional genes related to the development of male and female flowers of S. suchowensis.

Hsp70 proteins and their Hsp40 co‐chaperones are essential components of cellular chaperone networks in both prokaryotes and eukaryotes. Here, we performed a genetic analysis to define the protein domains required for the key functions of the major Hsp40/DnaJ protein Sll0897 of the cyanobacterium Synechocystis sp. PCC6803. The expression of the N‐terminally located J‐ and G/F‐domains is essential and sufficient for the proteins' fundamental in vivo functions, whereas the presence of the full‐length protein, containing the C‐terminal substrate‐binding domains, is crucial under stress conditions. [ABSTRACT FROM AUTHOR]

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.

Main conclusion: Despite AtJ3 and AtJ2 sharing a high protein-sequence identity and both being substrates of protein farnesyltransferase (PFT), AtJ3 but not AtJ2 mediates in Arabidopsis the heat-dependent phenotypes derived from farnesylation modification. Arabidopsis HEAT-INTOERANT 5 (HIT5)/ENHANCED RESPONSE TO ABA 1 (ERA1) encodes the β-subunit of the protein farnesyltransferase (PFT), and the hit5/era1 mutant is better able to tolerate heat-shock stress than the wild type. Given that Arabidopsis AtJ2 (J2) and AtJ3 (J3) are heat-shock protein 40 (HSP40) homologs, sharing 90% protein-sequence identity, and each contains a CaaX box for farnesylation; atj2 (j2) and atj3 (j3) mutants were subjected to heat-shock treatment. Results showed that j3 but not j2 manifested the heat-shock tolerant phenotype. In addition, transgenic j3 plants that expressed a CaaX- abolishing J3C417S construct maintained the same capacity to tolerate heat shock as j3. The basal transcript levels of HEAT-SHOCK PROTEIN 101 (HSP101) in hit5/era1 and j3 were higher than those in the wild type. Although the capacities of j3/hsp101 and hit5/hsp101 double mutants to tolerate heat-shock stress declined compared to those of j3 and hit5/era1, they were still greater than that of the wild type. These results show that a lack of farnesylated J3 contributes to the heat-dependent phenotypes of hit5/era1, in part by the modulation of HSP101 activity, and also indicates that (a) mediator(s) other than J3 is (are) involved in the PFT-regulated heat-stress response. In addition, because HSP40s are known to function in dimer formation, bimolecular fluorescence complementation experiments were performed, and results show that J3 could dimerize regardless of farnesylation. In sum, in this study, a specific PFT substrate was identified, and its roles in the farnesylation-regulated heat-stress responses were clarified, which could be of use in future agricultural applications. [ABSTRACT FROM AUTHOR]

The DNAJC protein family is a subclass of heat shock proteins that has attracted recent attention due to the identification of mutations that are linked with parkinsonism, a feature of Parkinson's disease and other neurological disorders. In this review, we discuss the current genetic and functional evidence of the association of these DNAJC proteins with disease and how mutations in these proteins may contribute to disease pathogenesis. Although DNAJC6 (Auxilin), DNAJC12, and DNAJC5 (CSPα) exhibit strong genetic association with disease, DNAJC26 (GAK), DNAJC13 (RME‐8), and DNAJC10 (Erdj5) require additional evidence to definitively link reported variants to parkinsonism. Remarkably, multiple DNAJC proteins (Auxilin, GAK, RME‐8, CSPα) functionally converge on pathways of synaptic trafficking and clathrin dynamics, highlighting an important role of those pathways in the pathogenesis of parkinsonism. Further research is required to define the mechanisms through which these mutations contribute to disease etiology. [ABSTRACT FROM AUTHOR]

Members of the Hsp90 and Hsp70 families of molecular chaperones are imp\ortant for the maintenance of protein homeostasis and cellular recovery following environmental stresses, such as heat and oxidative stress. Moreover, the two chaperones can collaborate in protein remodeling and activation. In higher eukaryotes, Hsp90 and Hsp70 form a functionally active complex with Hop (Hsp90–Hsp70 organizing protein) acting as a bridge between the two chaperones. In bacteria, which do not contain a Hop homolog, Hsp90 and Hsp70, DnaK, directly interact during protein remodeling. Although yeast possesses a Hop-like protein, Sti1, Hsp90, and Hsp70 can directly interact in yeast in the absence of Sti1. Previous studies showed that residues in the middle domain of Escherichia coli Hsp90 are important for interaction with the J-protein binding region of DnaK. The results did not distinguish between the possibility that (i) these sites were involved in direct interaction and (ii) the residues in these sites participate in conformational changes which are transduced to other sites on Hsp90 and DnaK that are involved in the direct interaction. Here we show by crosslinking experiments that the direct interaction is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, J-protein promotes the Hsp70–Hsp90 interaction in the presence of ATP, likely by converting Hsp70 into the ADP-bound conformation. The identification of the protein–protein interaction site is anticipated to lead to a better understanding of the collaboration between the two chaperones in protein remodeling. Unlabelled Image • Hsp90 interacts with Hsp70 in bacteria and yeast. • Specific crosslinking is used to define the sites of direct Hsp90–Hsp70 interaction. • Residues in the Hsp90 M-domain directly contact the Hsp70 J-protein binding site. • The J-protein promotes the Hsp90–Hsp70 interaction in the presence of ATP. • The Hsp90–Hsp70 regions of direct interaction are conserved in E. coli and yeast. [ABSTRACT FROM AUTHOR]

Δlon mutant of Escherichia coli becomes hypersensitive to DNA damaging agents and over‐produce capsule due to stabilization of the Lon substrates, namely, SulA and RcsA, respectively. These phenotypes were earlier found to be suppressed in Δlon ssrA::cat/pUC4 K and Δlon faa (DnaJ, G232D) strains, called as "Alp" strains. We observed that a plasmid carrying an E. coli chromosomal fragment harboring few genes, a heat shock gene htpY and a portion of dnaK capable of encoding truncated N‐terminal ATPase domain (244 aa) could suppress lon mutant phenotypes. Deletion of htpY did not affect the efficiency of suppression. Clones expressing DnaK′ (244 aa) peptide alone could suppress both Δlon phenotypes in copy number dependent manner. Inactivation of clpQ did not affect the MMSR phenotype of Δlon strain carrying dnaK′ clones indicating that ClpYQ protease does not degrade SulA. We hypothesize that the high levels of defective DnaK′‐DnaJ chaperone complex formed in these strains might lead to aggregation of SulA and RcsA and, thereby the suppression of Δlon phenotypes. Systematic deletion analysis of dnaK′ revealed that, ∼220 aa N‐terminal DnaK peptide is required for suppression of cps‐lac over‐expression and ∼169 aa peptide is enough for the suppression of MMSS phenotype of Δlon mutant. [ABSTRACT FROM AUTHOR]

The aberrant accumulation of tau protein is implicated as a pathogenic factor in many neurodegenerative diseases. Tau seeding may underlie its predictable spread in these diseases. Molecular chaperones can modulate tau pathology, but their effects have mainly been studied in isolation. This study employed a semi-high throughput assay to identify molecular chaperones influencing tau seeding using Tau RD P301S FRET Biosensor cells, which express a portion of tau containing the frontotemporal dementia-related P301S tau mutation fused to a FRET biosensor. Approximately fifty chaperones from five major families were screened using live cell imaging to monitor FRET-positive tau seeding. Among the tested chaperones, five exhibited significant effects on tau in the primary screen. Notably, three of these were from the DnaJ family. In subsequent studies, overexpression of DnaJA2, DnaJB1, and DnaJB6b resulted in significant reductions in tau levels. Knockdown experiments by shRNA revealed an inverse correlation between DnaJB1 and DnaJB6b with tau levels. DnaJB6b overexpression, specifically, reduced total tau levels in a cellular model with a pre-existing pool of tau, partially through enhanced proteasomal degradation. Further, DnaJB6b interacted with tau complexes. These findings highlight the potent chaperone activity within the DnaJ family, particularly DnaJB6b, towards tau. • Molecular chaperones across five distinct families were assessed to determine their effect on tau seeding. • Hsp90α, FKBP19, DnaJA2, DnaJB1, and DnaJB6b significantly reduced tau seeding. • DnaJA2, DnaJB1, and DnaJB6b reduce intracellular tau levels. • DnaJB1 and DnaJB6b knockdown increases tau levels. • DnaJB6b forms complexes with tau. [ABSTRACT FROM AUTHOR]

As a member of the damage-associated molecular patterns, heat shock proteins (HSPs) are widely recognized for their role in initiating innate immune responses. These highly conserved proteins are expressed ubiquitously in both prokaryotes and eukaryotes. In this study, our aim was to investigate how DnaJ, a HSP40 homolog derived from Pseudomonas aeruginosa (P. aeruginosa), influences the regulation of IL-8 expression in macrophages. Treatment with DnaJ served as a stimulus, inducing a more robust expression of IL-8 compared to other HSP homologs, including DnaK, GroEL, and HtpG. This effect was achieved through the activation of the NF-κB signaling pathway. Interestingly, DnaJ treatment also significantly increased the expression of microRNA-146a (miR-146a), which appears to play a role in modulating the expression of innate defense genes. As a consequence, pre-treatment with DnaJ led to a reduction in the extent of IL-8 induction in response to P. aeruginosa treatment. Notably, this reduction was counteracted by transfection of a miR-146a inhibitor, highlighting the involvement of miR-146a in P. aeruginosa -mediated induction of IL-8 expression. Therefore, this study uncovers the role of DnaJ in triggering the expression of miR-146a, which, in turn, modulates the excessive expression of IL-8 induced by P. aeruginosa infection. • DnaJ, HSP40 homolog, is sufficient to promote the expression of miR-146a. • DnaJ pre-treatment reduces IL-8 induced by P. aeruginosa treatment. • MiR-146a modulates the excessive IL-8 expression triggered by P. aeruginosa. [ABSTRACT FROM AUTHOR]

DnaJ, also known as Hsp40, plays important roles in maintaining the normal physiological state of an organism under stress conditions by mediating essential processes, such as protein synthesis, degradation, folding and metabolism. However, the exact functions of most DnaJ members are not fully understood in insects. Here, we identified three genes, AccDnaJA1, AccDnaJB12, and AccDnaJC8, in Apis cerana cerana and explored their connection with the environmental stress response. Quantitative real-time PCR results showed that the mRNA levels of AccDnaJA1, AccDnaJB12, and AccDnaJC8 were all induced under cold, UV, H2O2 and different pesticides treatment. The expression patterns of AccDnaJB12 and AccDnaJC8 were upregulated by CdCl2 and HgCl2 stress, while the transcriptional levels of AccDnaJA1 were downregulated by CdCl2 and HgCl2 stress. Western blot findings further indicated that AccDnaJB12 protein levels were increased by some stress conditions. Knockdown of each of these three genes downregulated the transcriptional patterns of several stress response-related genes at different levels. Functional analysis further demonstrated that the resistance of A. cerana cerana to lambda-cyhalothrin stress was reduced with knockdown of AccDnaJA1, AccDnaJB12, or AccDnaJC8, indicating that these three genes may be involved in the tolerance to this pesticide. Taken together, these findings indicate that AccDnaJA1, AccDnaJB12, and AccDnaJC8 may play pivotal roles in the stress response by facilitating honeybee survival under some adverse circumstances. To our knowledge, this is the first report that reveals the roles of DnaJ family proteins under different adverse circumstances in A. cerana cerana.