Factors affecting protein recovery during Hsp40 affinity profiling.

The identification and quantification of misfolded proteins from complex mixtures is important for biological characterization and disease diagnosis, but remains a major bioanalytical challenge. We have developed Hsp40 Affinity Profiling as a bioanalytical approach to profile protein stability in response to cellular stress. In this assay, we ectopically introduce the Hsp40 FlagDNAJB8H31Q into cells and use quantitative proteomics to determine how protein affinity for DNAJB8 changes in the presence of cellular stress, without regard for native clients. Herein, we evaluate potential approaches to improve the performance of this bioanalytical assay. We find that although intracellular crosslinking increases recovery of protein interactors, this is not enough to overcome the relative drop in DNAJB8 recovery. While the J-domain promotes Hsp70 association, it does not affect the yield of protein association with DNAJB8 under basal conditions. By contrast, crosslinking and J-domain ablation both substantially increase relative protein interactor recovery with the structurally distinct Class B Hsp40 DNAJB1 but are completely compensated by poorer yield of DNAJB1 itself. Cellular thermal stress promotes increased affinity between DNAJB8H31Q and interacting proteins, as expected for interactions driven by recognition of misfolded proteins. DNAJB8WT does not demonstrate such a property, suggesting that under stress misfolded proteins are handed off to Hsp70. Hence, we find that DNAJB8H31Q is still our most effective recognition element for the recovery of destabilized client proteins following cellular stress.

The heat shock protein DNAJB8 inhibits pseudorabies virus replication by autophagy.

Pseudorabies virus (PRV) effectively utilizes numerous host proteins and pathways to establish a successful infection. Consequently, it becomes imperative to investigate novel host factors implicated in viral infections to gain a deeper understanding of PRV pathogenesis. In this study, we reveal that the host heat shock protein, DNAJB8, functions as a negative regulator in PRV replication. Our findings indicated that both mRNA and protein levels of DNAJB8 were downregulated in cells infected with PRV. Further analysis demonstrated that overexpressing DNAJB8 suppressed PRV replication, whereas its knockdown enhanced viral replication. From a mechanistic perspective, DNAJB8 promoted cellular autophagy, subsequently impeding viral replication. Additionally, we discovered that the transcription factor SOX30 regulated DNAJB8 expression, thereby influencing viral replication. Collectively, these findings enhance our comprehension of the roles played by DNAJB8 and SOX30 in viral replication, broadening our knowledge of virus-host interactions.

DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity.

J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8 that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.

DNAJB8 facilitates autophagic-lysosomal degradation of viral Vif protein and restricts HIV-1 virion infectivity by rescuing APOBEC3G expression in host cells.

HSP40/DNAJ family of proteins is the most diverse chaperone family, comprising about 49 isoforms in humans. Several reports have demonstrated the functional role of a few of these isoforms in the pathogenesis of various viruses, including HIV-1. Our earlier study has shown that several isoforms of HSP40 get significantly modulated at the mRNA level during HIV-1 infection in T cells. To explore the biological role of these significantly modulated isoforms, we analyzed their effect on HIV-1 gene expression and virus production using knockdown and overexpression studies. Among these isoforms, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 seem to positively regulate virus replication, whereas DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate virus replication. Further investigation on the infectivity of the progeny virion demonstrated that only DNAJB8 negatively regulates the progeny virion infectivity. It was further identified that DNAJB8 protein is involved in the downregulation of Vif protein, required for the infectivity of HIV-1 virions. DNAJB8 seems to direct Vif protein for autophagic-lysosomal degradation, leading to rescue of the cellular restriction factor APOBEC3G from Vif-mediated proteasomal degradation, resulting in enhanced packaging of APOBEC3G in budding virions and release of less infective progeny virion particles. Finally, our results also indicate that during the early stage of HIV-1 infection, enhanced expression of DNAJB8 promotes the production of less infective progeny virions, but at the later stage or at the peak of infection, reduced expression of DNJAB8 protein allows the HIV-1 to replicate and produce more infective progeny virion particles.

Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry.

The quantitative multiplexing capacity of isobaric tandem mass tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t-test and Pearson's R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8H31Q followed by stringent washing, and reversible association to 14-3-3ζ with gentle washing. Correlation-derived t-statistics performed at least as well as Student's t-statistics for each sample and with substantial improvement in performance for experiments with high bait-level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase the robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.

Dnajb8, a target gene of SOX30, is dispensable for male fertility in mice.

BACKGROUND: The DNAJ family of molecular chaperones maintains protein homeostasis in mitotic and postmeiotic cells, especially germ cells. Recently, we found that the transcription factor SOX30 initiates transcription of Dnajb8 during late meiosis and spermiogenesis in mouse testes. METHODS: We used the CRISPR/Cas9 system to generate Dnajb8 mutant mice and analyze the phenotype of the Dnajb8 mutants. RESULTS: Although Dnajb8 is an evolutionarily conserved gene, it is not essential for spermatogenesis and male fertility. We provide this phenotypic information, which could prevent duplicative work by other groups.

Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.

Identification of antigenic peptides from novel renal cancer stem-like cell antigen, DNAJB8.

OBJECTIVES: To identify antigenic peptides of cancer stem-like cells (CSCs) antigen, DNAJB8, and establish a mouse CSCs-targeting immunotherapy model. MATERIALS AND METHODS: To induce DNAJB8-specific immune reaction, we stimulated human CD8+ lymphocytes with antigen-presenting cells pulsed with a cocktail of three candidate HLA-A*24:02 restricted peptides and assessed peptide specific human cytotoxic T lymphocytes (CTLs) induction. One of the antigenic peptides showed identical amino acid sequence as corresponding mouse DNAJB8. We evaluated CTL induction with the peptide immunization in mouse model. RESULTS: We confirmed peptide-specific interferon-γ secretions and cytotoxic activities of induced human CTLs. In vivo immunization with the peptide to mice, peptide-specific CTL response could be observed in mouse CD8+ T cells. Furthermore, immunization with the peptide showed significant anti-tumor effects compared with negative controls. CONCLUSION: DNAJB8-derived peptide is a novel candidate for CSCs-targeting immunotherapy, and mouse models can be used to evaluate CSCs-targeting immunotherapy.

Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells.

The aim of the present study was to establish cancer stem-like cell/cancer-initiating cell (CSC/CIC)-targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8-specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8-derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC-targeting immunotherapy.