Phylogeny-dominant classification of J-proteins in Arabidopsis thaliana and Brassica oleracea.

Hsp40s or DnaJ/J-proteins are evolutionarily conserved in all organisms as co-chaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (types A-D) according to domain organization, but their phylogenetic relationships are unknown. Here, we identified 129 J-proteins in the world-wide popular vegetable Brassica oleracea, a close relative of the model plant Arabidopsis, and also revised the information of Arabidopsis J-proteins based on the latest online bioresources. According to phylogenetic analysis with domain organization and gene structure as references, the J-proteins from Arabidopsis and B. oleracea were classified into 15 main clades (I-XV) separated by a number of undefined small branches with remote relationship. Based on the number of members, they respectively belong to multigene clades, oligo-gene clades, and mono-gene clades. The J-protein genes from different clades may function together or separately to constitute a complicated regulatory network. This study provides a constructive viewpoint for J-protein classification and an informative platform for further functional dissection and resistant genes discovery related to genetic improvement of crop plants.

Predicting the types of J-proteins using clustered amino acids.

J-proteins are molecular chaperones and present in a wide variety of organisms from prokaryote to eukaryote. Based on their domain organizations, J-proteins can be classified into 4 types, that is, Type I, Type II, Type III, and Type IV. Different types of J-proteins play distinct roles in influencing cancer properties and cell death. Thus, reliably annotating the types of J-proteins is essential to better understand their molecular functions. In the present work, a support vector machine based method was developed to identify the types of J-proteins using the tripeptide composition of reduced amino acid alphabet. In the jackknife cross-validation, the maximum overall accuracy of 94% was achieved on a stringent benchmark dataset. We also analyzed the amino acid compositions by using analysis of variance and found the distinct distributions of amino acids in each family of the J-proteins. To enhance the value of the practical applications of the proposed model, an online web server was developed and can be freely accessed.

Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

BACKGROUND: In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. RESULTS: Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. CONCLUSIONS: We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP.

The heat shock protein 40 family of the malaria parasite Plasmodium falciparum.

Few diseases have had such a profound influence on human evolution and history as malaria. Despite intense efforts malaria infection continues to be a major killer. The causative agent of malaria, the unicellular eukaryote Plasmodium, displays a fascinating biology in which ubiquitous cellular concepts are modified to serve the particular needs of the malaria parasite. In this review, we explore how Plasmodium utilizes the heat shock protein 40 system, a chaperone system that ensures correct protein folding under normal and stress conditions. We highlight the peculiarities of the Plasmodium system and discuss whether any components of the system might be exploited for intervention strategies against this debilitating disease.

Identification of a consensus motif in substrates bound by a Type I Hsp40.

Protein aggregation is a hallmark of a large and diverse number of conformational diseases. Molecular chaperones of the Hsp40 family (Escherichia coli DnaJ homologs) recognize misfolded disease proteins and suppress the accumulation of toxic protein species. Type I Hsp40s are very potent at suppressing protein aggregation and facilitating the refolding of damaged proteins. Yet, the molecular mechanism for the recognition of nonnative polypeptides by Type I Hsp40s such as yeast Ydj1 is not clear. Here we computationally identify a unique motif that is selectively recognized by Ydj1p. The motif is characterized by the consensus sequence GX[LMQ]{P}X{P}{CIMPVW}, where [XY] denotes either X or Y and {XY} denotes neither X nor Y. We further verify the validity of the motif by site-directed mutagenesis and show that substrate binding by Ydj1 requires recognition of this motif. A yeast proteome screen revealed that many proteins contain more than one stretch of residues that contain the motif and are separated by varying numbers of amino acids. In light of our results, we propose a 2-site peptide-binding model and a plausible mechanism of peptide presentation by Ydj1p to the chaperones of the Hsp70 family. Based on our results, and given that Ydj1p and its human ortholog Hdj2 are functionally interchangeable, we hypothesize that our results can be extended to understanding human diseases.

The RJL family of small GTPases is an ancient eukaryotic invention probably functionally associated with the flagellar apparatus.

A patchily distributed gene family is often taken as evidence for horizontal gene transfer (HGT) events, but it may also result solely from multiple gene losses. The RJL family of uncharacterised Ras-like GTPases was previously suggested to have undergone HGT events between protists and deuterostome metazoans, owing to the apparent absence of RJL in intermediate groups (Nepomuceno-Silva, J.L., de Melo, L.D., Mendonca, S.M., Paixao, J.C., Lopes, U.G., 2004. RJLs: a new family of Ras-related GTP-binding proteins. Gene 327, 221-232). We have reanalysed the phylogenetic distribution and phylogeny of the RJL family, taking advantage of the recent expansion of sequence data available from diverse eukaryotes. We found that RJL orthologs are much more widely distributed than previously assumed. At least one representative encoding an RJL protein could be identified for each of the six major eukaryotic "supergroups" (Opisthokonta, Amoebozoa, Excavata, Archaeplastida, Chromalveolata, and Rhizaria) as well as for a species of Apusomonadida, a deep lineage that may not be specifically related to any of the recognized supergroups. Phylogenetic analyses do not support HGT of RJL genes between the major eukaryotic lineages, indicating that the RJL family was present in the last eukaryotic common ancestor and was lost several times over the course of eukaryotic evolution. Interestingly, RJL was lost from all taxa lacking flagellated cells and from a few lineages that build structurally unusual or reduced flagella, raising the intriguing possibility that RJL proteins are functionally associated with the flagellar apparatus. The RJL GTPase domain has been fused with the DnaJ domain on two separate occasions: in the Holozoa (before the split of Metazoa and choanoflagellates), giving rise to the previously known Rbj type of RJL with the DnaJ domain at the C-terminus, and independently in Alveolata resulting in novel proteins with the DnaJ domain at the N-terminus. These independent fusions suggest that RJL proteins may generally function via regulating the DnaJ-Hsp70 module.

Guidelines for the nomenclature of the human heat shock proteins.

The expanding number of members in the various human heat shock protein (HSP) families and the inconsistencies in their nomenclature have often led to confusion. Here, we propose new guidelines for the nomenclature of the human HSP families, HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), and HSPB (small HSP) as well as for the human chaperonin families HSPD/E (HSP60/HSP10) and CCT (TRiC). The nomenclature is based largely on the more consistent nomenclature assigned by the HUGO Gene Nomenclature Committee and used in the National Center of Biotechnology Information Entrez Gene database for the heat shock genes. In addition to this nomenclature, we provide a list of the human Entrez Gene IDs and the corresponding Entrez Gene IDs for the mouse orthologs.

Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones.

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.

Rapid and specific identification of 5 human pathogenic Vibrio species by multiplex polymerase chain reaction targeted to dnaJ gene.

A multiplex polymerase chain reaction (PCR) method, specifically designed for application in routine diagnostic laboratories, was developed for identifying 5 human pathogen Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, and Vibrio alginolyticus. This assay directed toward the dnaJ gene was tested on a total of 355 strains representing 13 Vibrio species and 17 non-Vibrio species. Specific PCR fragments were produced in isolates belonging to the 5 target species and were absent from all strains other than these 5 species and non-Vibrio strains, indicating a high specificity of this multiplex PCR. The multiplex PCR for the detection of Vibrio pathogens in clinical specimens was experimentally applied to spiked stool samples. Only 1 specific amplicon was observed, corresponding to the pathogen spiked into the stool sample. The detection limitation was 10(5) to 10(6) cells per milliliter stool. Our data showed that this method represented a robust tool for the specific and rapid detection of the 5 major pathogenic Vibrio species.

The Hsp40 proteins of Plasmodium falciparum and other apicomplexa: regulating chaperone power in the parasite and the host.

Extensive structural and functional remodelling of Plasmodium falciparum (malaria)-infected erythrocytes follows the export of a range of proteins of parasite origin (exportome) across the parasitophorous vacuole into the host erythrocyte. The genome of P. falciparum encodes a diverse chaperone complement including at least 43 members of the heat shock protein 40kDa (Hsp40) family, and six members of the heat shock protein 70kDa (Hsp70) family. Nearly half of the Hsp40 proteins of P. falciparum are predicted to contain a PEXEL/HT (Plasmodium export element/host targeting signal) sequence motif, and hence are likely to be part of the exportome. In this review we critically evaluate the classification, sequence similarity and clustering, and possible interactors of the P. falciparum Hsp40 chaperone machinery. In addition to the types I, II and III Hsp40 proteins all exhibiting the signature J-domain, the P. falciparum genome also encodes a number of specialized Hsp40 proteins with a J-like domain, which we have categorized as type IV Hsp40 proteins. Analysis of the potential P. falciparum Hsp40 protein interaction network revealed connections predominantly with cytoskeletal and membrane proteins, transcriptional machinery, DNA repair and replication machinery, translational machinery, the proteasome and proteolytic enzymes, and enzymes involved in cellular physiology. Comparison of the Hsp40 proteins of P. falciparum to those of other apicomplexa reveals that most of the proteins (especially the PEXEL/HT-containing proteins) are unique to P. falciparum. Furthermore, very few of the P. falciparum Hsp40 proteins have human homologs, except for those proteins implicated in fundamental biological processes. Our analysis suggests that P. falciparum has evolved an expanded and specialized Hsp40 protein machinery to enable it successfully to invade and remodel the human erythrocyte, and we propose a model in which these proteins are involved in chaperone-mediated translocation, folding, assembly and regulation of parasite and host proteins.

Phylogeny and species identification of the family Enterobacteriaceae based on dnaJ sequences.

Phylogenetic relations within the family Enterobacteriaceae were analyzed using partial dnaJ sequences of 165 strains belonging to 93 species from 27 enterobacterial genera. The dnaJ phylogeny was in relative agreement with that constructed by 16S rDNA sequences, but more monophyletic groups were obtained from the dnaJ tree than from the 16S rDNA tree. The degree of divergence of the dnaJ gene was approximately 6 times greater than that of 16S rDNA. Also, the dnaJ gene showed the most discriminatory power in comparison with tuf and atpD genes, facilitating clear differentiation of any 2 enterobacterial species by dnaJ sequence analysis. The application of dnaJ sequences to the identification was confirmed by assigning 72 clinical isolates to the correct enterobacterial species. Our data indicate that analysis of the dnaJ gene sequences can be used as a powerful marker for phylogenetic study and identification at the species level of the family Enterobacteriaceae.

Rdj2, a J protein family member, interacts with cellular prion PrP(C).

PrP(C) is a glycosylphosphatidylinositol (GPI) anchored glycoprotein of unknown function. Misfolding of normal cellular PrP(C) to the pathogenic PrP(Sc) is the hallmark of prion diseases (transmissible spongiform encephalopathies). Prion diseases are characterized by extensive neurodegeneration and early death. Understanding how PrP(C) maintains its correct conformation is a major endeavor of current inquiry. Here we demonstrate a novel interaction between PrP(C) and the J protein family member, Rdj2 (DjA2; Dj3, Dnj3, Cpr3, and Hirip4). The importance of the J protein family in the cellular folding machinery has been recognized for many years. The PrP(C)/Rdj2 association was direct and concentration-dependent. Other J proteins such as CSPalpha and auxilin did not associate with PrP(C) in the absence of ATP, demonstrating the specificity of the PrP(C)/J protein interaction. These findings suggest that the J protein family serves as a 'folding catalyst' for PrP(C) and implicates Rdj2 as a factor in the protection against prion diseases.