Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli.

Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E. coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.

Determining arsenic stress tolerance genes in rice (Oryza sativa L.) via genomic insights and QTL mapping with double haploid lines.

Arsenic, a hazardous heavy metal with potent carcinogenic properties, significantly affects key rice-producing regions worldwide. In this study, we present a quantitative trait locus (QTL) mapping investigation designed to identify candidate genes responsible for conferring tolerance to arsenic toxicity in rice (Oryza sativa L.) during the seedling stage. This study identified 17 QTLs on different chromosomes, including qCHC-1 and qCHC-3 on chromosome 1 and 3 related to chlorophyll content and qRFW-12 on chromosome 12 related to root fresh weight. Gene expression analysis revealed eight candidate genes exhibited significant upregulation in the resistant lines, OsGRL1, OsDjB1, OsZIP2, OsMATE12, OsTRX29, OsMADS33, OsABCG29, and OsENODL24. These genes display sequence alignment and phylogenetic tree similarities with other species and engaging in protein-protein interactions with significant proteins. Advanced gene-editing techniques such as CRISPR-Cas9 to precisely target and modify the candidate genes responsible for arsenic tolerance will be explore. This approach may expedite the development of arsenic-resistant rice cultivars, which are essential for ensuring food security in regions affected by arsenic-contaminated soil and water.

Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

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.

Phage detection by a bacterial NLR-related protein is mediated by DnaJ.

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.

Comparative genomic analysis reveals expansion of the DnaJ gene family in Lagerstroemia indica and its members response to salt stress.

DnaJs/Hsp40s/JPDs are obligate co-chaperones of heat shock proteins (Hsp70), performing crucial biological functions within organisms. A comparative genome analysis of four genomes (Vitis vinifera, Eucalyptus grandis, Lagerstroemia indica, and Punica granatum) revealed that the DnaJ gene family in L. indica has undergone expansion, although not to the extent observed in P. granatum. Inter-genome collinearity analysis of four plants indicates that members belonging to Class A and B are more conserved during evolution. In L. indica, the expanded members primarily belong to Class-C. Tissue expression patterns and the biochemical characterization of LiDnaJs further suggested that DnaJs may be involved in numerous biological processes in L. indica. Transcriptome and qPCR analyses of salt stressed leaves identified at least ten LiDnaJs that responded to salt stress. In summary, we have elucidated the expansion mechanism of the LiDnaJs, which is attributed to a recent whole-genome triplication. This research laid the foundation for functional analysis of LiDnaJs and provides gene resources for breeding salt-tolerant varieties of L. indica.

Hsp90, a team player in protein quality control and the stress response in bacteria.

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.

Case report: Mutations in DNAJC30 causing autosomal recessive Leber hereditary optic neuropathy are common amongst Eastern European individuals.

Background: Leber Hereditary Optic Neuropathy (LHON) is the most common inherited mitochondrial disease characterized by bilateral, painless, subacute visual loss with a peak age of onset in the second to third decade. Historically, LHON was thought to be exclusively maternally inherited due to mutations in mitochondrial DNA (mtDNA); however, recent studies have identified an autosomal recessive form of LHON (arLHON) caused by point mutations in the nuclear gene, DNAJC30. Case Presentations: In this study, we report the cases of three Eastern European individuals presenting with bilateral painless visual loss, one of whom was also exhibiting motor symptoms. After a several-year-long diagnostic journey, all three patients were found to carry the homozygous c.152A>G (p.Tyr51Cys) mutation in DNAJC30. This has been identified as the most common arLHON pathogenic variant and has been shown to exhibit a significant founder effect amongst Eastern European individuals. Conclusion: This finding adds to the growing cohort of patients with arLHON and demonstrates the importance of DNAJC30 screening in patients with molecularly undiagnosed LHON, particularly in Eastern European individuals. It is of heightened translational significance as patients diagnosed with arLHON exhibit a better prognosis and response to therapeutic treatment with the co-enzyme Q10 analog idebenone.

Ab initio protein structure prediction: the necessary presence of external force field as it is delivered by Hsp40 chaperone.

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.

The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-ß peptide fibrils and inhibits secondary nucleation.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD ß-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first ß-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-ß (Aß42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aß, the CTD constructs instead bind to Aß42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two ß-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

DnaJs are enriched in tau regulators.

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.

DnaJ-induced miRNA-146a negatively regulates the expression of IL-8 in macrophages.

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.

MsDjB4, a HSP40 Chaperone in Alfalfa (Medicago sativa L.), Improves Alfalfa Hairy Root Tolerance to Aluminum Stress.

The toxicity of aluminum (Al) in acidic soils poses a significant limitation to crop productivity. In this study, we found a notable increase in DnaJ (HSP40) expression in the roots of Al-tolerant alfalfa (WL-525HQ), which we named MsDjB4. Transient conversion assays of tobacco leaf epidermal cells showed that MsDjB4 was targeted to the membrane system including Endoplasmic Reticulum (ER), Golgi, and plasma membrane. We overexpressed (MsDjB4-OE) and suppressed (MsDjB4-RNAi) MsDjB4 in alfalfa hairy roots and found that MsDjB4-OE lines exhibited significantly better tolerance to Al stress compared to wild-type and RNAi hairy roots. Specifically, MsDjB4-OE lines had longer root length, more lateral roots, and lower Al content compared to wild-type and RNAi lines. Furthermore, MsDjB4-OE lines showed lower levels of lipid peroxidation and ROS, as well as higher activity of antioxidant enzymes SOD, CAT, and POD compared to wild-type and RNAi lines under Al stress. Moreover, MsDjB4-OE lines had higher soluble protein content compared to wild-type and RNAi lines after Al treatment. These findings provide evidence that MsDjB4 contributes to the improved tolerance of alfalfa to Al stress by facilitating protein synthesis and enhancing antioxidant capacity.

Complementation Assays for Co-chaperone Function.

The development of mutant microorganisms lacking J domain proteins (JDPs; formerly called Hsp40s) has enabled the development of complementation assays for testing the co-chaperone function of JDPs. In these assays, an exogenously expressed novel JDP is tested for its ability to functionally substitute for a non-expressed or nonfunctional endogenous JDP(s) by reversing a stress phenotype. For example, the in vivo functionality of prokaryotic JDPs can be tested on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant strain of the bacterium Escherichia coli (OD259). Similarly, the in vivo functionality of eukaryotic JDPs can be assessed in a thermosensitive ydj1 mutant strain of the yeast Saccharomyces cerevisiae (JJ160). Here we outline the use of these thermosensitive microorganisms in complementation assays to functionally characterize a JDP from the bacterium, Agrobacterium tumefaciens (AgtDnaJ), and a JDP from the trypanosomal parasite, Trypanosoma cruzi (TcJ2).

Human Endogenous Retrovirus-H-Derived miR-4454 Inhibits the Expression of DNAJB4 and SASH1 in Non-Muscle-Invasive Bladder Cancer.

Although most human endogenous retroviruses (HERVs) have been silenced and lost their ability to translocate because of accumulated mutations during evolution, they still play important roles in human biology. Several studies have demonstrated that HERVs play pathological roles in numerous human diseases, especially cancer. A few studies have revealed that long non-coding RNAs that are transcribed from HERV sequences affect cancer progression. However, there is no study on microRNAs derived from HERVs related to cancer. In this study, we identified 29 microRNAs (miRNAs) derived from HERV sequences in the human genome. In particular, we discovered that miR-4454, which is HERV-H-derived miRNA, was upregulated in non-muscle-invasive bladder cancer (NMIBC) cells. To figure out the effects of upregulated miR-4454 in NMIBC, genes whose expression was downregulated in NMIBC, as well as tumor suppressor genes, were selected as putative target genes of miR-4454. The dual-luciferase assay was used to determine the negative relationship between miR-4454 and its target genes, DNAJB4 and SASH1, and they were confirmed to be promising target genes of miR-4454. Taken together, this study suggests that the upregulation of miR-4454 derived from HERV-H in NMIBC reduces the expression of the tumor suppressor genes, DNAJB4 and SASH1, to promote NMIBC progression.

Gene cloning and characterization of a novel recombinant 40-kDa heat shock protein from Mesobacillus persicus B48.

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.

DnaJ, a heat shock protein 40 family member, is essential for the survival and virulence of plant pathogenic Pseudomonas cichorii JBC1.

Bacterial plant pathogens must cope with various environmental conditions and defenses from their hosts for colonization and infection. Heat shock proteins (HSPs) play critical roles in a variety of cellular processes, such as the maintenance of cellular homeostasis in response to environmental stress. However, the significance of HSP40 family protein DnaJ in virulence of plant pathogenic bacteria has not yet been explored. To elucidate the function of DnaJ in Pseudomonas cichorii JBC1 (PcJBC1) virulence, we generated dnaJ-deficient (JBC1ΔdnaJ) mutant using CRISPR-CAS9. The disease severity by JBC1ΔdnaJ was significantly reduced compared with wild-type (WT) and dnaJ-complemented (JBC1ΔdnaJ + pdnaJ) strain. The defect of DnaJ suppressed siderophore production, extracellular DNA (eDNA) release, biofilm formation, and swarming motility and made the strain sensitive to stresses such as heat and H2O2. The supplementation of eDNA recovered the amount of biofilm formation by JBC1ΔdnaJ. Our results indicate that DnaJ is a key player in the survival and colonization of bacterial plant pathogens on plant surfaces as well as bacterial responses to abiotic and biotic stresses, which are determinative to cause disease. These findings can broaden our understanding of plant and bacterial pathogen interactions.

J-domain Proteins form Binary Complexes with Hsp90 and Ternary Complexes with Hsp90 and Hsp70.

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.

Ectopic expression of DnaJ type-I protein homolog of Vigna aconitifolia (VaDJI) confers ABA insensitivity and multiple stress tolerance in transgenic tobacco plants.

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.

Verification of the Interaction Target Protein of the Effector ApCE22 of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis.

The study of interaction proteins of the pathogen A. phaeospermum effector protein is an important means to analyze the disease-resistance mechanism of Bambusa pervariabilis × Dendrocalamopsis grandis shoot blight. To obtain the proteins interacting with the effector ApCE22 of A. phaeospermum, 27 proteins interacting with the effector ApCE22 were initially identified via a yeast two-hybrid assay, of which four interaction proteins were obtained after one-to-one validation. The B2 protein and the chaperone protein DnaJ chloroplast protein were then verified to interact with the ApCE22 effector protein by bimolecular fluorescence complementation and GST pull-down methods. Advanced structure prediction showed that the B2 protein contained the DCD functional domain related to plant development and cell death, and the DnaJ protein contained the DnaJ domain related to stress resistance. The results showed that both the B2 protein and DnaJ protein in B. pervariabilis × D. grandis were the target interaction proteins of the ApCE22 effector of A. phaeospermum and related to the stress resistance of the host B. pervariabilis × D. grandis. The successful identification of the pathogen effector interaction target protein in B. pervariabilis × D. grandis plays an important role in the mechanism of pathogen-host interaction, thus providing a theoretical basis for the control of B. pervariabilis × D. grandis shoot blight.

The stability and function of human cochaperone Hsp40/DNAJA1 are affected by zinc removal and partially restored by copper.

The imbalance in metal homeostasis can be associated with several human diseases, and exposure to increasing concentrations of metals promotes cell stress and toxicity. Therefore, understanding the cytotoxic effect of metal imbalance is important to unravel the biochemical mechanism of homeostasis and the action of potential protective proteins against metal toxicity. Several studies, including gene deletion in yeast, provide evidence indicating the possible indirect involvement of cochaperones from the Hsp40/DNAJA family in metal homeostasis, possibly through modulating the activity of Hsp 70.This work first investigated the effect of zinc and copper on the conformation and function of the human Hsp40 cochaperone DNAJA1, a zinc-binding protein. DNAJA1 was capable to complement the phenotype of a yeast strain deleted of the ydj1 gene, which was more sensitive to the presence of zinc and copper than the wild-type strain. To gain further insight about the role of the DNAJA family in metal binding, the recombinant human DNAJA1 protein was studied. Zinc removal from DNAJA1 affected both its stability and ability to act as a chaperone, i.e., to protect other proteins from aggregation. The reintroduction of zinc restored the native properties of DNAJA1 and, surprisingly, the addition of copper partially restored the native properties.