High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size.

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.

Chaperone-like protein DAY plays critical roles in photomorphogenesis.

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.

Two J domains ensure high cochaperone activity of DnaJ, Escherichia coli heat shock protein 40.

Heat shock protein 70 (Hsp70) chaperone systems consist of Hsp70, Hsp40 and a nucleotide-exchange factor and function to help unfolded proteins achieve their native conformations. Typical Hsp40s assume a homodimeric structure and have both chaperone and cochaperone activity. The dimeric structure is critical for chaperone function, whereas the relationship between the dimeric structure and cochaperone function is hardly known. Here, we examined whether two intact protomers are required for cochaperone activity of Hsp40 using an Escherichia coli Hsp70 chaperone system consisting of DnaK, DnaJ and GrpE. The expression systems were generated and two heterodimeric DnaJs that included a mutated protomer lacking cochaperone activity were purified. Normal chaperone activity was demonstrated by assessing aggregation prevention activity using urea-denatured luciferase. The heterodimeric DnaJs were investigated for cochaperone activity by measuring DnaK ATPase activity and the heat-denatured glucose-6-phosphate dehydrogenase refolding activity of the DnaK chaperone system, and they showed reduced cochaperone activity. These results indicate that two intact protomers are required for high cochaperone activity of DnaJ, suggesting that one homodimeric DnaJ molecule promotes the simultaneous binding of multiple DnaK molecules to one substrate molecule, and that this binding mode is required for the efficient folding of denatured proteins.

The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x.

Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

[Stress proteins in the cells of Porphyra purpurea (Rhodophyta) thallus].

Heat shock proteins have been revealed for the first time by the methods of Western blotting using alkaline phosphatase and ECL in the cells of Porphyra purpurea from Kattegat area of the Baltic Sea in normal and experimental stress conditions. It was demonstrated with application of monoclonal anti-Hsp70 antibodies that a slight band about 70 kDa is present constitutively at the film; additionally the polypeptide of about 40 kDa ("Hsp40") has been detected. After heat shock at 28 degrees C during 1 hr significant "expenditure" of Hsp70 was observed, as well as the pronounced induction of "Hsp40"; the induction was expressed especially strongly in 24 hr after the stress application.

[Effects of Schistosoma japonicum heat-shock protein 40 on macrophage activation].

OBJECTIVE: To clone and express heat-shock protein 40 gene of Schistosoma japonicum (SjHSP40) and analyze its effect on macrophage activation. METHODS: The fragment of gene encoding SjHSP40 was amplified by PCR. The gene was sub-cloned into the prokaryotic expression vector pGEX-6P-1. The recombinant plasmid pGEX-6P-1-SjHSP40 was transformed into E. coli BL21 (DE3) and induced with IPTG. The recombinant protein was purified with Glutathione-Sepharose 4B resin and analyzed by SDS-PAGE and Western-blot. The fusion protein of GST-SjHSP40 was loaded to the macrophage cell-line RAW264.7 for 48 h. Following that, the surface molecules of the macrophages were analyzed by flow cytometry. RESULTS: DNA sequencing showed that the recombinant plasmid, pGEX-6P-1-SjHSP40, was successfully constructed. The fusion protein of GST-SjHSP40 was induced, purified and specifically recognized by anti-GST antibody. Compared to GST and medium control groups, this fusion protein significantly induced the expression of co-stimulatory molecules (CD40, CD80, and CD86) and MHC-II on the surface of the macrophages. CONCLUSIONS: SjHSP40 significantly up-regulates the expression of co-stimulatory molecules and MHC-II on the surface of the macrophages. These data indicate that SjHSP40 may initiate macrophage activation.

Purification, crystallization and preliminary X-ray diffraction analysis of the Hsp40 protein CPIP1 from Nicotiana tabacum.

Chaperones promote many different molecular processes, including the folding, targeting and degradation of proteins. The best-studied chaperone system consists of the Hsp70s and their co-chaperones the Hsp40s. Chaperone function can be hijacked by viruses in plants. Potato virus Y interacts via its coat protein with an Hsp40 from Nicotiana tabacum, referred to as NtCPIP1, in order to regulate replication. To understand the molecular determinants of this mechanism, different variants of NtCPIP1 were expressed, purified and crystallized. While crystals of wild-type NtCPIP1 diffracted to 8.0 Å resolution, the deletion mutant NtCPIP1-Δ(1:127) crystallized in space group P2(1)2(1)2 and diffracted to 2.4 Å resolution.

Reactivation of protein aggregates by mortalin and Tid1--the human mitochondrial Hsp70 chaperone system.

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.

The crystal structure of the human co-chaperone P58(IPK).

P58(IPK) is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58(IPK), expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58(IPK), and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58(IPK) to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58(IPK) monomer to have a very elongated overall shape. In addition to the conserved J domain, P58(IPK) contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58(IPK) from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding.

[Cloning, prokaryotic expression of novel swine gene P58IPK and its polyclonal antibody preparation].

AIM: To clone a novel swine gene P58(IPK)[58-kDa(inhibitor of protein kinase) protein] and prepare its polyclonal antibody for further research of influenza and host interaction. METHODS: The swine P58(IPK); gene was first identified in silico through homology searching in the swine EST database. Then this gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA of the gene contained the complete open reading frame(ORF) of 1 518 bp, and encoded 505 amino acid residues (Accession No.HQ287801). The gene was first analyzed using bioinformatics methods. Then P58(IPK) was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET-P58(IPK). The fusion protein his-P58(IPK) was expressed in E.coli BL21 and purified using a his-tag protein purification column. Subsequently rabbits were immunized with the purified protein. RESULTS: Specific polyclonal antibody against the fusion protein his-P58(IPK) was obtained. The activity of the antibody was determined through double-immunodiffusion test. The titer of the antibody was 1:20 000 as shown by ELISA. specifically recognized the protein P58(IPK) by Western blot and immunofluorescence assay. CONCLUSION: The novel swine gene P58(IPK) has been successfully cloned and its polyclonal antibody has been prepared.

Purification, crystallization and preliminary X-ray crystallographic analysis of the human heat-shock protein 40 Hdj1 and its C-terminal peptide-binding domain.

Hsp40 is a co-chaperone of Hsp70 that correctly folds polypeptides that exist in non-native forms. The C-terminal peptide-binding domain (CTD) of the human Hsp40 Hdj1 has been purified and crystallized. In the presence of the C-terminal octapeptide of human Hsp70, four types of crystals, types I-B, II, III and IV, were grown and diffracted to 1.85, 2.51, 2.10 and 2.80 Šresolution, respectively. In the absence of the octapeptide, type I-A crystals of the CTD were grown that diffracted to 2.05 Šresolution. The full-length Hdj1 was also purified and crystallized (type V crystals); the crystal diffracted to 3.90 Šresolution.

Overproduction, purification and characterisation of Tbj1, a novel Type III Hsp40 from Trypanosoma brucei, the African sleeping sickness parasite.

The heat shock protein 40 (Hsp40) family of proteins act as co-chaperones of the heat shock protein 70 (Hsp70) chaperone family, and together they play a vital role in the maintenance of cellular homeostasis. The Type III class of Hsp40s are diverse in terms of both sequence identity and function and have not been extensively characterised. The Trypanosoma brucei parasite is the causative agent of Human African Trypanosomiasis, and possesses an unusually large Hsp40 complement, consisting mostly of Type III Hsp40s. A novel T. brucei Type III Hsp40, Tbj1, was heterologously expressed, purified, and found to exist as a compact monomer in solution. Using polyclonal antibodies to the full-length recombinant protein, Tbj1 was found by Western analysis to be expressed in the T. brucei bloodstream-form. Tbj1 was found to be able to assist two different Hsp70 proteins in the suppression of protein aggregation in vitro, despite being unable to stimulate their ATPase activity. This indicated that while Tbj1 did not possess independent chaperone activity, it potentially functioned as a novel co-chaperone of Hsp70 in T. brucei.

Preliminary X-ray crystallographic studies of mouse UPR responsive protein P58(IPK) TPR fragment.

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), which can promote protein folding and misfolded protein degradation and attenuate protein translation and protein translocation into the ER. P58(IPK) has been proposed to function as a molecular chaperone to maintain protein-folding homeostasis in the ER under normal and stressed conditions. P58(IPK) contains nine TPR motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the ER, a P58(IPK) TPR fragment without the C-terminal J-domain was crystallized. The crystals diffract to 2.5 A resolution using a synchrotron X-ray source. The crystals belong to space group P2(1), with unit-cell parameters a = 83.53, b = 92.75, c = 84.32 A, alpha = 90.00, beta = 119.36, gamma = 90.00 degrees. There are two P58(IPK) molecules in the asymmetric unit, which corresponds to a solvent content of approximately 60%. Structure determination by MAD methods is under way.

TAT-Hsp40 inhibits oxidative stress-mediated cytotoxicity via the inhibition of Hsp70 ubiquitination.

Heat shock protein 40 (Hsp40) functions as a co-chaperone of mammalian Heat shock protein 70 (Hsp70) and facilitates the ATPase activity of Hsp70, and also promotes the cellular protein folding and renaturation of misfolded proteins. In an effort to assess the effects of Hsp40, we generated TAT-fused Hsp40 (TAT-Hsp40). The cells were transduced with TAT-Hsp40 and exposed to H(2)O(2). We demonstrated that the TAT-Hsp40-transduced cells were more resistant to cellular cytotoxicity and cell death. In particular, the degradation of Hsp70 was significantly reduced in TAT-Hsp40-containing cells as a consequence of reduced ubiquitin-proteasome activity after oxidative injury. These data support the notion that Hsp40 may confer resistance to oxidative stress via the prevention of proteasome activity.

Induction of tobacco genes in response to oligochitosan.

Oligochitosan has a variety of biological activities. To understand its mechanism, DDRT-PCR, reverse Northern blot and quantitative relative RT-PCR were used to identify and isolate genes whose transcription were altered in cultured Nicotiana tabacum (var. Samsun NN) plants that were treated with oligochitosan. Three genes whose mRNA levels significantly changed in response to oligochitosan were isolated and identified. One gene is up-regulated, and two genes are down-regulated. These genes encode a DNAJ heat shock N-terminal domain-containing protein, a histone H1 gene and a hypothetical protein, whose function is unknown. The results suggest that the usefulness of mRNA differential display technique for the detection of plant metabolic pathways affected by oligochitosan.

Purification and assay of the chaperone-dependent ubiquitin ligase of the carboxyl terminus of Hsc70-interacting protein.

It is notable that both chaperone and ubiquitin-proteasome systems are required for the removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. The carboxyl terminus of Hsc70-interacting protein (CHIP), originally identified as a cochaperone of Hsc70, has both a TPR motif and a U-box domain. The TPR motif associates with Hsp70 and Hsp90, whereas the U-box domain executes ubiquitin ligase activity. Thus, CHIP is an ideal molecule, acting as a protein quality control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. This chapter describes methods of analyzing chaperone-dependent ubiquitin ligase activity of CHIP using firefly luciferase as a model substrate.

[Prokaryotic expression of DnaJ-homologous chaperon PBP and preparation of rabbit antibody against PBP].

AIM: To express DnaJ-homologous chaperon peripherin-binding protein(PBP) gene in E.coli and prepare the rabbit antibody against PBP. METHODS: The PBP cDNA was amplified from the human fetal brain tissue by RT-PCR. After confirmed by DNA sequencing, the PBP-cDNA was cloned into expression vector pET28a and then the PBP gene was expressed in E.coli under the IPTG induction. The expressed protein was purified through Ni-NTA affinity chromatography column. The rabbit antibody against PBP was prepared by immunizing two New Zealand white rabbits using the purified PBP as immunogen. The titer and specificity of the antisera were determined by Western blot. RESULTS: The 720 bp PBP gene was amplified, cloned, and expressed in E.coli. The expressed product existed in the bacterial inclusion body and the supernatant of the bacteria lysate. The purified PBP reached electrophoretic purity. The rabbit antibody against PBP was prepared and its titer was about 1:1,600. Western blot analysis showed that the antibody could bind to the expressed PBP protein specifically. CONCLUSION: The PBP protein was expressed in E.coli and rabbit antibody against PBP was prepared successfully, which lays the foundation for further study on the structure and biological function of PBP.