DnaJ Protein Research Articles

Global analysis of expression profile of members of DnaJ gene families involved in capsaicinoids synthesis in pepper (Capsicum annuum L)

BackgroundThe DnaJ proteins play critical roles in plant development and stress responses. Recently, seventy-six DnaJ genes were identified through a comprehensive bioinformatics analysis in the pepper genome. However, there were no reports on understanding of phylogenetic relationships and diverse expression profile of pepper DnaJ genes to date. Herein, we performed the systemic analysis of the phylogenetic relationships and expression profile of pepper DnaJ genes in different tissues and in response to both abiotic stress and plant hormones.ResultsPhylogenetic analysis showed that all the pepper DnaJ genes were grouped into 7 sub-families (sub-family I, II, III, IV, V, VI and VII) according to sequence homology. The expression of pepper DnaJs in different tissues revealed that about 38% (29/76) of pepper DnaJs were expressed in at least one tissue. The results demonstrate the potentially critical role of DnaJs in pepper growth and development. In addition, to gain insight into the expression difference of pepper DnaJ genes in placenta between pungent and non-pungent, their expression patterns were also analyzed using RNA-seq data and qRT-PCR. Comparison analysis revealed that eight genes presented distinct expression profiles in pungent and non-pungent pepper. The CaDnaJs co-expressed with genes involved in capsaicinoids synthesis during placenta development. What is more, our study exposed the fact that these eight DnaJ genes were probably regulated by stress (heat, drought and salt), and were also regulated by plant hormones (ABA, GA3, MeJA and SA).ConclusionsIn summary, these results showed that some DnaJ genes expressed in placenta may be involved in plant response to abiotic stress during biosynthesis of compounds related with pungency. The study provides wide insights to the expression profiles of pepper DanJ genes and contributes to our knowledge about the function of DnaJ genes in pepper.

Co-chaperones DNAJA1 and DNAJB6 are critical for regulation of polyglutamine aggregation

Huntington’s disease (HD) is caused by CAG repeat expansion in the huntingtin gene. The expanded polyglutamine (polyQ) repeat of the encoded protein leads to protein misfolding and aggregation, resulting in increased neuronal cell death. DNAJ co-chaperones play a crucial role in transferring misfolded/unfolded proteins to HSP70 chaperones, which play an essential role for protein folding. Here, we investigated the effect of knock out (KO) of three individual DNAJ genes in HEK293 cells expressing polyglutamine74exon1 huntingtin (polyQ74htt). Flourescence microscopy analysis revealed that KO of DNAJB6 resulted in a 5-fold increase in polyQ74htt aggregation and that DNAJA1 KO resulted in a 4-fold decrease of polyQ74htt aggregation. KO of DNAJB1 did not change the propensity of polyQ74htt to aggregate in cells. These findings where confirmed both by fluorescence microscopy analysis and filter trap assay (FTA). DNAJB6 KO cells displayed an increased rate of cell death as assessed by trypan blue exclusion and propidium iodide (PI) uptake assays. These results demonstrate that the DNAJ proteins DNAJA1 and DNAJB6 can modulate polyQ aggregation in opposite manners, and thus that fine-tuning the cellular levels of DNAJ proteins is critical for suppression of polyQ aggregation and cell survival.

Exploring Protein‐Protein Interactions for MS2‐L Lysis Activity

Bacteriophage like MS2 are viruses that infect and replicate in host cell bacteria. MS2 is a small single‐stranded RNA bacteriophage that infects Escherichia coli. MS2 has a lysis protein L that hypothetically requires a host protein for cell lysis. Expression of L alone in E. coli is sufficient for cell lysis, although the mechanism remains unknown. Interestingly, our data suggests that N‐terminally tagged L is functional for lysis, although with slower kinetics than an un‐tagged version. As a control experiment for exploring protein‐protein interactions, we are overexpressing the chaperone protein DnaJ to confirm its known interaction with L. 1 To accomplish this we are using the bi‐cistronic expression vector pET‐Duet‐1 to co‐express DnaJ and L. pET‐Duet provides epitope tags to permit confirmation of expression and interaction via immunoblotting and pull‐down experiments, respectively. It is known that DnaJ and L interact, however, L does not require DnaJ for lysis. 1 Using Photo‐leucine‐crosslinking, a process that incorporates a leucine analog reactive to UV light into expressed proteins, we hope to covalently capture other E. coli proteins that are necessary for lysis. Overexpressed DnaJ will serve as a positive control to confirm covalent cross‐linking with a known interaction partner.

Expression profiling of Cordyceps DnaJ protein family in Tolypocladium guangdongense during developmental and temperature stress processes.

DnaJ is an important molecular chaperone, with significant roles in growth, development, and stress resistance. Studies on the DnaJ gene family in macro-fungi such as Cordyceps spp. s.l. is scare. In this study, 22, 20, and 24 putative DnaJ genes were identified in Tolypocladium guangdongense, Ophiocordyceps sinensis, and C. militaris, respectively. They were classified into four groups based on the presence of the J, zinc finger, and C-terminal domains. We mainly studied the T. guangdongense DnaJ genes being located in the endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus. Phylogenetic analysis revealed gene duplications during the evolutionary process. Multiple cis-elements and transcription factor binding sites were observed in the promoter, suggesting their involvement in the response to multiple stresses. qRT-PCR analysis showed that 63.63% and 45.45% of T. guangdongense DnaJ genes were differentially expressed under cold and heat stress, respectively, indicating their involvement in the response to temperature stress. Many T. guangdongense DnaJ genes in the primordium and fruiting body exhibited differential expression, in comparison to those in the mycelium, suggesting a regulatory role in its growth and development process. These findings will facilitate further functional analysis, and provide information on the classification and conservative functions of DnaJ proteins in macro-fungi.

The role of the VosA-repressed dnjA gene in development and metabolism in Aspergillus species.

The DnaJ family of proteins (or J-proteins) are molecular chaperones that govern protein folding, degradation, and translocation in many organisms. Although J-proteins play key roles in eukaryotic and prokaryotic biology, the role of J-proteins in Aspergillus species is currently unknown. In this study, we characterized the dnjA gene, which encodes a putative DnaJ protein, in two Aspergillus species: Aspergillus nidulans and Aspergillus flavus. Expression of the dnjA gene is inhibited by the velvet regulator VosA, which plays a pivotal role in spore survival and metabolism in Aspergillus. The deletion of dnjA decreased the number of asexual spores (conidia), produced abnormal conidiophores, and reduced sexual fruiting bodies (cleistothecia) or sclerotia. In addition, the absence of dnjA caused increased sterigmatocystin or aflatoxin production in A. nidulans and A. flavus, respectively. These results suggest that DnjA plays a conserved role in asexual and sexual development and mycotoxin production in Aspergillus species. However, DnjA also plays a species-specific role; AniDnjA but not AflDnjA, affects conidial viability, trehalose contents, and thermal tolerance of conidia. In plant virulence assay, the infection ability of the ΔAfldnjA mutant decreased in the kernels, suggesting that DnjA plays a crucial role in the pathogenicity of A. flavus. Taken together, these results demonstrate that DnjA is multifunctional in Aspergillus species; it is involved in diverse biological processes, including fungal differentiation and secondary metabolism.

A DnaJ protein that interacts with soybean mosaic virus coat protein serves as a key susceptibility factor for viral infection

Soybean mosaic virus (SMV)-disease is one of the most serious and widespread diseases in soybean (Glycine max). In the present study, a DnaJ protein in soybean designated GmCPIP (SMV coat protein-interacting protein) was screened by the QIS-Seq (quantitative interactor screening with next-generation sequencing) method, and the interaction between SMV CP and GmCPIP was confirmed by the yeast two-hybrid (Y2H) system and bimolecular fluorescence complementation (BiFC) assay. Subcellular localization analysis indicated that both proteins are localized in the cytoplasm, cytomembrane and nucleus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that infection with SMV-SC4 temporarily increased the transcription of GmCPIP. Virus-induced gene silencing (VIGS) down-regulated the GmCPIP gene by 82%, and the accumulation of SMV was decreased by 88.6% in GmCPIP-silenced plants inoculated with SMV-SC4. The interaction of GmCPIP with SMV CP seems to contribute to SMV infection in soybean.

Growth hormone activates X-box binding protein 1 in a sexually dimorphic manner through the extracellular signal-regulated protein kinase and CCAAT/enhancer-binding protein β pathway in rat liver.

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein β (C/EBPβ) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPβ pathway.

Identification of GCC-box and TCC-box motifs in the promoters of differentially expressed genes in rice (Oryza sativa L.): Experimental and computational approaches.

The transcription factor selectively binds with the cis-regulatory elements of the promoter and regulates the differential expression of genes. In this study, we aimed to identify and validate the presence of GCC-box and TCC-box motifs in the promoters of upregulated differentially expressed genes (UR-DEGs) and downregulated differentially expressed genes (DR-DEGs) under anoxia using molecular beacon probe (MBP) based real-time PCR. The GCC-box motif was detected in UR-DEGs (DnaJ and 60S ribosomal protein L7 genes), whereas, the TCC-box was detected in DR-DEGs (DnaK and CPuORF11 genes). In addition, the mechanism of interaction of AP2/EREBP family transcription factor (LOC_Os03g22170) with GCC-box promoter motif present in DnaJ gene (LOC_Os06g09560) and 60S ribosomal protein L7 gene (LOC_Os08g42920); and TCC-box promoter motif of DnaK gene (LOC_Os02g48110) and CPuORF11 gene (LOC_Os02g01240) were explored using molecular dynamics (MD) simulations analysis including binding free energy calculations, principal component analyses, and free energy landscapes. The binding free energy analysis revealed that AP2/EREBP model residues such as Arg68, Arg72, Arg83, Lys87, and Arg90 were commonly involved in the formation of hydrogen bonds with GCC and TCC-box promoter motifs, suggesting that these residues are critical for strong interaction. The movement of the entire protein bound to DNA was restricted, confirming the stability of the complex. This study provides comprehensive binding information and a more detailed view of the dynamic interaction between proteins and DNA.

Mutation of Mediator subunit CDK8 counteracts the stunted growth and salicylic acid hyperaccumulation phenotypes of an Arabidopsis MED5 mutant.

The Mediator complex functions as a hub for transcriptional regulation. MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semidominant mutation (ref4-3) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism. By contrast, loss of MED5 results in increased concentrations of phenylpropanoids. We used a reverse genetic approach to identify suppressors of ref4-3 and found that ref4-3 requires CDK8, a kinase module subunit of Mediator, to repress plant growth. The genetic interaction between MED5 and CDK8 was further characterized using mRNA-sequencing (RNA-seq) and metabolite analysis. Growth inhibition and suppression of phenylpropanoid metabolism can be genetically separated in ref4-3 by elimination of CDK8 kinase activity; however, the stunted growth of ref4-3 is not dependent on the phosphorylation event introduced by the G383S mutation. In addition, rather than perturbation of lignin biosynthesis, misregulation of DJC66, a gene encoding a DNAJ protein, is involved in the dwarfism of the med5 mutants. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants.

Phenylalanine hydroxylase variants interact with the co-chaperone DNAJC12.

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co-chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild-type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu1/1 mouse model, homozygous for the V106A-Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin-tagged PAH. This form represented a major fraction of PAH in the Enu1/1 but was also present in liver of wild-type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin-dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.

Cover Image: DNA methylation and gene expression

Genome‐wide DNA methylation patterns in plants are dynamically regulated by the DNA methylation and demethylation pathways. In this issue, Xiao et al. (110–119) and Zhao et al. (120–139) published two studies back to back to show that a protein complex composed of Su(var)3–9 homologs (SUVHs) and SUVH‐interacting DNAJ (SDJ) proteins can recognize DNA methylation in the promoter of demethylase ROS1 and is required for activating the expression of ROS1 and other promoter‐methylated genes in Arabidopsis, thus dynamically regulate DNA methylation.

Novel DnaJ Protein Facilitates Thermotolerance of Transgenic Tomatoes.

DnaJ proteins, which are molecular chaperones that are widely present in plants, can respond to various environmental stresses. At present, the function of DnaJ proteins was studied in many plant species, but only a few studies were conducted in tomato. Here, we examined the functions of a novel tomato (Solanum lycopersicum) DnaJ protein (SlDnaJ20) in heat tolerance using sense and antisense transgenic tomatoes. Transient conversion assays of Arabidopsis protoplasts showed that SlDnaJ20 was targeted to chloroplasts. Expression analysis showed that SlDnaJ20 expression was induced by chilling, NaCl, polyethylene glycol, and H2O2, especially via heat stress. Under heat stress, sense plants showed higher fresh weights, chlorophyll content, fluorescence (Fv/Fm), and D1 protein levels, and a lower accumulation of reactive oxygen species (ROS) than antisense plants. These results suggest that SlDnaJ20 overexpression can reduce the photoinhibition of photosystem II (PSII) by relieving ROS accumulation. Moreover, higher expression levels of HsfA1 and HsfB1 were observed under heat stress in sense plants, indicating that SlDnaJ20 overexpression contributes to HSF expression. The yeast two-hybrid system proved that SlDnaJ20 can interact with the chloroplast heat-shock protein 70. Our results indicate that SlDnaJ20 overexpression enhances the thermotolerance of transgenic tomatoes, whereas suppression of SlDnaJ20 increases the heat sensitivity of transgenic tomatoes.

DnaJ molecules as potential effectors in Meloidogyne arenaria. An unexplored group of proteins in plant parasitic nematodes

ABSTRACTPlant pathogenic organisms secrete proteins called effectors that recognize, infect and promote disease within host cells. Bacteria, like Pseudomona syringae, use effectors with DnaJ function to disrupt plant defenses. DnaJ proteins (also called Hsp40) are a group of co-chaperone molecules, which assist in the folding of proteins. Despite the described role of DnaJs as effectors in several groups of pathogens, this group of proteins has never been correlated with the infection process in plant parasitic nematodes. In this study, we analyze the importance of DnaJ for plant parasitic nematodes. To do that, we compare the number of DnaJ proteins in nematodes with different lifestyles. Then, we predict the secreted DnaJ proteins in order to detect effector candidates. We found that Meloidogyne species have more secreted DnaJs than the rest of the nematodes analyzed in the study. Particularly, M. arenaria possess the highest proportion of secreted DnaJ sequences in comparison to total DnaJ proteins. Furthermore, we found in this species at least five sequences with a putative nuclear localization signal, three of them with a serine rich region with an unknown function. Then, we chose one of these sequences (MG599854) to perform an expression analysis. We found that MG599854 is over-expressed from 3 days post inoculation onwards in tomato plants. Moreover, MG599854 seems to be enough to produce cell death in Nicotiana benthamiana under transient expression conditions. In concordance with our results, we propose that DnaJ proteins are a potential source of effector proteins in plant parasitic nematodes.

The DnaJ Gene Family in Shiitake Culinary-Medicinal Mushroom, Lentinus edodes (Agaricomycetes): Comprehensive Identification, Characterization, and Expression Profiles under Different Conditions.

The DnaJ proteins, also called heat-shock protein 40 based on their molecular weight, play significant roles in organism growth and development and resistance to abiotic and biotic stresses. However, studies on the DnaJ gene family in Lentinus edodes (= Lentinula edodes) are less well known. In this study, 29 putative L. edode DnaJ genes (LeDnaJ01 to LeDnaJ29) were identified using bioinformatics analysis and were classified into four groups according to the presence of the J protein and zinc finger as well as C-terminal domain. Multiple cis elements related to the phytohormone and stresses were found in the promotor region of the LeDnaJ genes. In addition, qRT-PCR analysis revealed that 79.31% of LeDnaJ genes were induced by cadmium, 55.17% were induced by Trichoderma atroviride, and 37.93% were induced by heat stress, indicating that the LeDnaJ proteins might participate in the response of L. edodes to the multiple stresses. Meanwhile, qRT-PCR analysis also revealed that all LeDnaJs are expressed in at least one development stage, indicating that they could be involved in the process of L. edodes growth and development and the response to the abiotic and biotic stresses. Taken together, these results advance the functional analysis of DnaJ genes in Basidiomycetes.

Gene expression analysis reveals diversified responsiveness to salt stress in rice genotypes

Salt stress is a major factor affecting rice (Oryza sativa) growth and productivity, limiting its distribution globally. Rice production is primarily gets affected due to its vulnerability to salinity stress at seedling stage, as well as reproductive stage leading to reduction in yield. We report the analysis of 12 diverse rice genotypes collected from different parts of coastal places of northern Andra Pradesh and southern Orissa in India for salt tolerance and categorized their tolerance levels on the basis of reduction in growth and transcript expression pattern. The analysis identified four contrasting salt tolerant genotypes and other eight genotypes exhibited moderate tolerance. Two tolerant and two sensitive genotypes were biochemically explored and used further for gene expression analysis. Proline content showed significant difference between salt tolerant and sensitive genotypes suggesting high tolerance level in tolerant CR1014 and Kudrat-5. The gene-expression analysis of selected CR1014 (salt-tolerant) and Kudrat-5 (salt-sensitive) genotypes differed at transcript expression levels for antioxidant specific A2YPX2/plant peroxidase—POX (19.03-fold) and SODCP/superoxide dismutase (SOD) copper/zinc binding (3.77-fold) in shoots of Kudrat-5 and CATA1/catalase (2.49-fold) and SODCP/SOD copper/zinc binding (4.83-fold) in CR1014 in stress conditions). The salt-stress responsive-specific transcripts HAK17/K+ potassium transporter, A2XMP7/heat shock protein DnaJ, cysteine-rich domain and Q10D68/serine hydroxymethyltransferase in CR1014 after treatment showed increased expression. Interestingly, the transcripts specific for methionine biosynthesis mainly A2Z9C9/Cys/Met metabolism, pyridoxal phosphate-dependent enzyme and B8AF89/spermine synthase induced their expression in Kudrat-5 under salinity stress, where the similar trend was not reflected in salt-tolerant genotype. Thus, the present study highlights on transcript expression pattern for salt-stress responsive candidate genes in two-contrasting rice genotypes selected from the pool of farmer’s cultivated varieties along the coastal belts of India.

The heat shock protein 40 LeDnaJ regulates stress resistance and indole-3-acetic acid biosynthesis in Lentinula edodes

DnaJ proteins, termed heat shock proteins based on their molecular weight, function as molecular chaperones that play critical roles in regulating organism growth and development as well as adaptation to the environment. However, little has been reported on their gene function in higher basidiomycetes. Here, the heat shock protein 40 (LeDnaJ) gene was cloned and characterized from Lentinula edodes. RNA interference was used to explore the function of LeDnaJ in response to heat stress and Trichoderma atroviride. Integration of the target gene into the L. edodes genome was confirmed by Southern blot analysis, and the silence efficiency of LeDnaJ was analyzed by qRT-PCR. The results revealed that LeDnaJ silence caused defects in mycelial growth and resistance to heat stress and T. atroviride, but increased the mycelial density compared with the wild type (WT) strain S606. Additionally, the IAA content showed a more than 10-fold increase in the WT after heat stress, but an about two-fold increase in the two LeDnaJ RNAi transfortants (LeDnaJ-i-6 and LeDnaJ-i-8). Previous study has shown that enhanced IAA (indole-3-acetic acid) content enhanced the thermotolerance of the heat-sensitive strain YS3357. In this study, it was documented that IAA amendments could partly restore the resistance to T. atroviride and thermotolerance of the two LeDnaJ RNAi transformants. Overall, LeDnaJ is nvolved in fungal growth, T. atroviride resistance, and thermotolerance by regulating the IAA biosynthesis in L. edodes.

Abstract 2901: Identifying synthetic lethal interactions in castration-resistant prostate cancer using HSP40 and HSP70 inhibitors

Abstract Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of alternative nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. These alternative receptors include ARv7, a ligand binding domain (LBD)-deficient, constitutively active splice variant that correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy. As a nuclear hormone receptor, AR exists in a dynamic cycle with molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 which foster proper folding and remodeling of the LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that LBD-deficient ARv7 functions independently of HSP90, we have observed that loss of the LBD does not abrogate HSP40/HSP70 binding, as ARv7 interacts with HSP40/HSP70 in CRPC cells. As such, targeting either HSP40 (C86) or HSP70 (JG98/JG231) inhibits stability and function of AR/ARv7. However, this is complicated by the fact that over 40 isoforms of HSP40 (DnaJ proteins) and 13 HSP70 paralogs exist in humans. Therefore, we sought to identify the requirements for components of the HSP40/HSP70 network in mediating intracellular stability of AR/ARv7, and determine which chaperones orchestrate sensitivity to HSP40/HSP70 inhibitors. Using a doxycycline-inducible lentiviral proteostasis shRNA library, we identified genes that appear to be required for proliferation of 22Rv1 CRPC cells (expressing endogenous AR and ARv7) distinct from those found in ARv7-deficient LNCaP prostate cancer cells. These include genes from several chaperone families including DnaJA3 (HSP40), HSPA2 (HSP70-2), and HSPD1 (HSP60), which may be unique regulators of ARv7 stability. Synthetic lethal screens using C86 and JG231 were also performed to determine which genes may mediate sensitivity to these chaperone inhibitors. Knockdown of HSP40 isoforms (DnaJA1, DnaJA2), HSP70 paralog HSPA4, or stress factors (ATF6, HSF1) in combination with C86 lead to growth impairment in 22Rv1 cells. DnaJA1, DnaJA2, and DnaJA3 were all found in the cytosol and nucleus of 22Rv1 cells, and in a complex with AR/ARv7 after a biotin-C86 IP, suggesting they be required for AR/ARv7 stability, dimerization, and/or nuclear translocation. Other genes from chaperone families HSP40 (DnaJC7, DnaJC8, DnaJC16), HSP70 (HSPA5, HSPA8, HSPA9), and HSP90/HSP90 co-chaperones (HSP90AB1, Cdc37, PTGES3 [p23]) were found to be important mediators of sensitivity to JG231, revealing targets for possible combination therapies. Taken together, these data help identify HSP40/HSP70 subnetworks regulating AR/ARv7 and those mediating sensitivity to chaperone inhibitors to aid in identifying new drug targets for efficacy in CRPC. Citation Format: Michael A. Moses, Genesis Rivera-Marquez, Isabelle Taylor, Hao Shao, Jason Gestwicki, Jane Trepel, Len Neckers. Identifying synthetic lethal interactions in castration-resistant prostate cancer using HSP40 and HSP70 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2901.

Cloning and constructing transformation vector carrying IbOr from the sweetpotato (Impomea batatas L.) involved in accumulation of carotenoids

The Orange gene (Or gene) is known as a gene encoding the protein containing a cysteine-rich Zinc finger domain. The Or protein is an important component of the structure that involes in accumulation of carotenoids and enhancing the resistant ability of plant to enviromental stresses. In an attempt to improve the nutritional quality and resistance to adverse environmental conditions of maize, in this article, we present the results on cloning IbOr gene from the sweetpotato cultivar Hoang Long and constructing transformation vectors carrying cloned IbOr gene under control of Ubiquitin (Ubi) or Globulin 1 (Glo1) promoter. The cloned IbOr gene was 942 bp in size and had similarity level of 100% comparing to the IbOr gene that was deposited in GenBank with Accession number HQ828087.1. The IbOr gene from sweedpotato Hoang Long was registered in GenBank under Accession number KX792094.1. The deduced protein consisted of 313 amino acids having Zin finger domain of DnaJ and HSP40 proteins. We have also constructed two transformation vectors pCambia2300/Ubiquitin/IbOr/Nos and pCambia2300/Glo1/IbOr/Nos. These vectors were transformed successfully into Agrobacterium tumefaciens strain C58. The presence of the target gene (IbOr) and promoter (Ubi or Glo1) in the A. tumefaciens strains were confirmed by colony-PCR amplification with respective specific primer pairs and digested by suitable restriction enzymes. These vectors will provide important materials for further gene transfer research for the expression of IbOr gene and production of transgenic maize with ability to accumulate high carotenoids.

Construction of yeast two-hybrid library of Salvia miltiorrhiza and screening of SmJAZ8 interaction protein

The study is aimed to construct high quality Salvia miltiorrhiza cDNA library and obtain the SmJAZ8 gene of S. miltiorrhiza by yeast two-hybrid system. In this study， full-length cDNA was synthesized from roots， stems， leaves， flowers and hairy roots of S. miltiorrhiza. The full-length cDNA library was synthesized by SMART method and constructed with DSN homogenization technique. The results showed that the library capacity was 1.45×10⁶， the recombination rate was 100%， and the average size of the insert was 500-2 000 bp. The recombinant vector of pDEST-pGADT7-SmJAZ8 was constructed and transformed into Y2HGold strain. The interaction protein was screened by yeast two-hybrid system. The DnaJ protein and UBQ protein were screened by yeast two-hybrid system. This study has successfully constructed a full-length cDNA library of S. miltiorrhiza， and laid the foundation for the follow-up study on functional gene screening and gene function of S. miltiorrhiza.

Mechanistic insights into acephalic spermatozoa syndrome–associated mutations in the human SUN5 gene

Acephalic spermatozoa syndrome has been reported for many decades; it is characterized by very few intact spermatozoa and tailless sperm heads in the semen and causes severe male infertility. The only gene in which mutations have been found to be associated with this syndrome encodes Sad1 and UNC84 domain-containing 5 (SUN5), a testis-specific nuclear envelope protein. The functional role of SUN5 has been well-studied in mouse models, but the molecular basis for the pathogenic effects of mutations in the human SUN5 gene remains elusive. Here, we report a new SUN5 mutation (c.475C→T; p.Arg159*), and explore the pathogenic effects of all known SUN5 mutations on acephalic spermatozoa syndrome. Using an artificial splicing system, we found that the intronic mutation affects the splicing of SUN5 mRNA, yielding a premature stop codon that results in a truncated SUN5 protein. We also found that SUN5 interacts with the coupling apparatus protein DnaJ heat shock protein family (Hsp40) member B13 (DNAJB13) during spermatogenesis, and the substitutions in the SUN5 SUN domain impair its interaction with DNAJB13. Furthermore, we observed that many SUN5 mutations affect the secondary structure of the protein and influence its folding and cellular localization. In summary, our findings indicate an interaction of SUN5 with DNAJB13 during spermatogenesis, provide mechanistic insights into the functional role of this interaction in sperm head-tail integration, and elucidate the molecular etiology of acephalic spermatozoa syndrome-associated SUN5 mutations.