Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

Comparative proteomic analysis of Pogostemon cablin leaves after continuous cropping.

A proteomic approach was used to understand the molecular mechanisms underlying obstacles to the continuous cropping of Pogostemon cablin. We examined differences in protein abundance between control (CK) and continuously cropped (TR) P. cablin leaves at different time points (90, 150, and 210 days after culture). Comparative analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) revealed 183 differentially expressed protein spots, of which 87 proteins or isoforms were identified using mass spectrometry. Among these differentially expressed proteins (DEPs), 50 proteins or isoforms showed increased abundance and 37 proteins or isoforms showed decreased abundance in the TR sample compared with the abundance in the CK sample. Bioinformatic tools were used to analyze the DEPs. These proteins were classified into 12 categories according to clusters of orthologous groups (COG) analysis, with the majority being involved in post-translational modification, protein turnover, and chaperones, followed by carbohydrate transport and metabolism, and finally, energy production and conversion. Protein-protein interactions revealed that 18 DEPs were involved in energy metabolism, 6 DEPs were involved in stress response, and 4 DEPs were involved in amino acid biosynthesis. Continuous cropping altered the expression of proteins related to energy metabolism, carbohydrate metabolism, and amino acid metabolism in P. cablin leaves. Among these processes, the most affected was energy metabolism, which may be pivotal for resistance to continuous cropping.

Molecular chaperones in protein folding and proteostasis.

Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer's disease and Parkinson's disease. Interventions in these and numerous other pathological states may spring from a detailed understanding of the pathways underlying proteome maintenance.

Characterization and expression analysis of a new small heat shock protein Hsp20.4 from Eimeria tenella.

Small heat shock proteins (sHsps) are ubiquitous and diverse molecular chaperones. Found in almost all organisms, they regulate protein refolding and protect cells from stress. Until now, no sHsp has been characterized in Eimeria tenella. In this study, the novel EtsHsp20.4 gene was cloned from E. tenella by rapid amplification of cDNA ends based on a previously identified expressed sequence tag. The full-length cDNA was 1019bp in length and contained an open reading frame of 558bp that encoded a 185-amino acid polypeptide with a calculated molecular weight of 20.4 kDa. The EtsHsp20.4 protein contained a distinct HSP20/alpha-crystallin domain that is the key determinant of their function as molecular chaperones and belongs to the HSP20 protein family. EtsHsp20.4 mRNA levels were higher in sporulated oocysts than in sporozoites or second-generation merozoites by real-time quantitative PCR, the transcription of EtsHsp20.4 was barely detectable in unsporulated oocysts. Immunolocalization with EtsHsp20.4 antibody showed that EtsHsp20.4 was mainly located on the surface of sporozoites, first-generation merozoites and second-generation merozoites. Following the development of parasites in DF-1 cells, EtsHsp20.4 protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts. Malate dehydrogenase thermal aggregation assays indicated that recombinant EtsHsp20.4 had molecular chaperone activity in vitro. These results suggested that EtsHsp20.4 might be involved in sporulation in external environments and intracellular growth of the parasite in the host.

Molecular Characterization of MaCCS, a Novel Copper Chaperone Gene Involved in Abiotic and Hormonal Stress Responses in Musa acuminata cv. Tianbaojiao.

Copper/zinc superoxide dismutases (Cu/ZnSODs) play important roles in improving banana resistance to adverse conditions, but their activities depend on the copper chaperone for superoxide dismutase (CCS) delivering copper to them. However, little is known about CCS in monocots and under stress conditions. Here, a novel CCS gene (MaCCS) was obtained from a banana using reverse transcription PCR and rapid-amplification of cDNA ends (RACE) PCR. Sequence analyses showed that MaCCS has typical CCS domains and a conserved gene structure like other plant CCSs. Alternative transcription start sites (ATSSs) and alternative polyadenylation contribute to the mRNA diversity of MaCCS. ATSSs in MaCCS resulted in one open reading frame containing two in-frame start codons to form two protein versions, which is supported by the MaCCS subcellular localization of in both cytosol and chloroplasts. Furthermore, MaCCS promoter was found to contain many cis-elements associated with abiotic and hormonal responses. Quantitative real-time PCR analysis showed that MaCCS was expressed in all tested tissues (leaves, pseudostems and roots). In addition, MaCCS expression was significantly induced by light, heat, drought, abscisic acid and indole-3-acetic acid, but inhibited by relatively high concentrations of CuSO4 and under cold treatment, which suggests that MaCCS is involved in abiotic and hormonal responses.

Emerging common molecular pathways for primary dystonia.

The dystonias are a group of hyperkinetic movement disorders whose principal cause is neuron dysfunction at 1 or more interconnected nodes of the motor system. The study of genes and proteins that cause familial dystonia provides critical information about the cellular pathways involved in this dysfunction, which disrupts the motor pathways at the systems level. In recent years study of the increasing number of DYT genes has implicated a number of cell functions that appear to be involved in the pathogenesis of dystonia. A review of the literature published in English-language publications available on PubMed relating to the genetics and cellular pathology of dystonia was performed. Numerous potential pathogenetic mechanisms have been identified. We describe those that fall into 3 emerging thematic groups: cell-cycle and transcriptional regulation in the nucleus, endoplasmic reticulum and nuclear envelope function, and control of synaptic function. © 2013 Movement Disorder Society.

Overexpression of a nascent polypeptide associated complex gene (SaßNAC) of Spartina alterniflora improves tolerance to salinity and drought in transgenic Arabidopsis.

Salinity and drought are the most important environmental constraints limiting crop growth and productivity. Here, we have characterized a gene 'SaßNAC' encoding the ß subunit of nascent polypeptide associated complex from a halophyte Spartina alterniflora and investigated its role toward abiotic stress regulation. Expression of SaßNAC was differentially regulated by abiotic stresses, including salinity, drought, cold, and ABA in leaves and roots of S. alterniflora. Constitutive over-expression of SaßNAC in Arabidopsis exhibited normal growth under non-stress conditions but enhanced tolerance to salt and drought stresses. Transgenic SaßNAC Arabidopsis retained more chlorophyll, proline, and showed improved ionic homeostasis with less damage under stress conditions compared to WT plants. This is a first report to demonstrate the involvement of ßNAC in imparting abiotic stress tolerance which might be due to protection of the newly synthesized polypeptides involved in various stress tolerance mechanisms from abiotic stress induced damage and inhibition of cell death in plant.

Identification of the tail assembly chaperone genes of T4-Like phages suggests a mechanism other than translational frameshifting for biogenesis of their encoded proteins.

Tape measure (TM) proteins are essential for the formation of long-tailed phages. TM protein assembly into tails requires the action of tail assembly chaperones (TACs). TACs (e.g. gpG and gpT of E. coli phage lambda) are usually produced in a short (TAC-N) and long form (TAC-NC) with the latter comprised of TAC-N with an additional C-terminal domain (TAC-C). TAC-NC is generally synthesized through a ribosomal frameshifting mechanism. TAC encoding genes have never been identified in the intensively studied Escherichia coli phage T4, or any related phages. Here, we have bioinformatically identified putative TAC encoding genes in diverse T4-like phage genomes. The frameshifting mechanism for producing TAC-NC appears to be conserved in several T4-like phage groups. However, the group including phage T4 itself likely employs a different strategy whereby TAC-N and TAC-NC are encoded by separate genes (26 and 51 in phage T4).

Systematic analysis of native membrane protein complexes in Escherichia coli.

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.

Dealing with misfolded proteins: examining the neuroprotective role of molecular chaperones in neurodegeneration.

Human neurodegenerative diseases arise from a wide array of genetic and environmental factors. Despite the diversity in etiology, many of these diseases are considered "conformational" in nature, characterized by the accumulation of pathological, misfolded proteins. These misfolded proteins can induce cellular stress by overloading the proteolytic machinery, ultimately resulting in the accumulation and deposition of aggregated protein species that are cytotoxic. Misfolded proteins may also form aberrant, non-physiological protein-protein interactions leading to the sequestration of other normal proteins essential for cellular functions. The progression of such disease may therefore be viewed as a failure of normal protein homeostasis, a process that involves a network of molecules regulating the synthesis, folding, translocation and clearance of proteins. Molecular chaperones are highly conserved proteins involved in the folding of nascent proteins, and the repair of proteins that have lost their typical conformations. These functions have therefore made molecular chaperones an active area of investigation within the field of conformational diseases. This review will discuss the role of molecular chaperones in neurodegenerative diseases, highlighting their functional classification, regulation, and therapeutic potential for such diseases.

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.

Stress genes and proteins in the archaea.

The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of approximately 60 kDa. These form, in various combinations depending on the species, a large structure or chaperonin complex sometimes called the thermosome. This multimolecular assembly is similar to the bacterial chaperonin complex GroEL/S, but it is made of only the large, double-ring oligomers each with eight (or nine) subunits instead of seven as in the bacterial complex. Like Hsp70(DnaK), the archaeal chaperonin subunits are remarkable for their evolution, but for a different reason. Ubiquitous among archaea, the chaperonins show a pattern of recurrent gene duplication-hetero-oligomeric chaperonin complexes appear to have evolved several times independently. The stress response and stress tolerance in the archaea involve chaperones, chaperonins, other heat shock (stress) proteins including sHsp, thermoprotectants, the proteasome, as yet incompletely understood thermoresistant features of many molecules, and formation of multicellular structures. The latter structures include single- and mixed-species (bacterial-archaeal) types. Many questions remain unanswered, and the field offers extraordinary opportunities owing to the diversity, genetic makeup, and phylogenetic position of archaea and the variety of ecosystems they inhabit. Specific aspects that deserve investigation are elucidation of the mechanism of action of the chaperonin complex at different temperatures, identification of the partners and substitutes for the Hsp70 chaperone machine, analysis of protein folding and refolding in hyperthermophiles, and determination of the molecular mechanisms involved in stress gene regulation in archaeal species that thrive under widely different conditions (temperature, pH, osmolarity, and barometric pressure). These studies are now possible with uni- and multicellular archaeal models and are relevant to various areas of basic and applied research, including exploration and conquest of ecosystems inhospitable to humans and many mammals and plants.

The protective and destructive roles played by molecular chaperones during ERAD (endoplasmic-reticulum-associated degradation).

Over one-third of all newly synthesized polypeptides in eukaryotes interact with or insert into the membrane or the lumenal space of the ER (endoplasmic reticulum), an event that is essential for the subsequent folding, post-translational modification, assembly and targeting of these proteins. Consequently, the ER houses a large number of factors that catalyse protein maturation, but, in the event that maturation is aborted or inefficient, the resulting aberrant proteins may be selected for ERAD (ER-associated degradation). Many of the factors that augment protein biogenesis in the ER and that mediate ERAD substrate selection are molecular chaperones, some of which are heat- and/or stress-inducible and are thus known as Hsps (heat-shock proteins). But, regardless of whether they are constitutively expressed or are inducible, it has been assumed that all molecular chaperones function identically. As presented in this review, this assumption may be false. Instead, a growing body of evidence suggests that a chaperone might be involved in either folding or degrading a given substrate that transits through the ER. A deeper appreciation of this fact is critical because (i) the destruction of some ERAD substrates results in specific diseases, and (ii) altered ERAD efficiency might predispose individuals to metabolic disorders. Moreover, a growing number of chaperone-modulating drugs are being developed to treat maladies that arise from the synthesis of a unique mutant protein; therefore it is critical to understand how altering the activity of a single chaperone will affect the quality control of other nascent proteins that enter the ER.

Exploring intrinsically disordered proteins in Chlamydomonas reinhardtii.

The content of intrinsically disordered protein (IDP) is related to organism complexity, evolution, and regulation. In the Plantae, despite their high complexity, experimental investigation of IDP content is lacking. We identified by mass spectrometry 682 heat-resistant proteins from the green alga, Chlamydomonas reinhardtii. Using a phosphoproteome database, we found that 331 of these proteins are targets of phosphorylation. We analyzed the flexibility propensity of the heat-resistant proteins and their specific features as well as those of predicted IDPs from the same organism. Their mean percentage of disorder was about 20%. Most of the IDPs (~70%) were addressed to other compartments than mitochondrion and chloroplast. Their amino acid composition was biased compared to other classic IDPs. Their molecular functions were diverse; the predominant ones were nucleic acid binding and unfolded protein binding and the less abundant one was catalytic activity. The most represented proteins were ribosomal proteins, proteins associated to flagella, chaperones and histones. We also found CP12, the only experimental IDP from C. reinhardtii that is referenced in disordered protein database. This is the first experimental investigation of IDPs in C. reinhardtii that also combines in silico analysis.

Studies on middle and posterior silk glands of silkworm (Bombyx mori) using two-dimensional electrophoresis and mass spectrometry.

Silk glands, present in the larval stage of the silkworm, produce threads of silky material to form the cocoon and are mainly composed of three parts: the anterior, the middle, and the posterior silk glands, each playing different roles in silk secretion. High-resolution two-dimensional polyacrylamide gel electrophoresis and computer-assisted analysis were used to investigate quantitative and qualitative differences between the middle and posterior silk glands. Silver staining revealed over 600 spots for each sample, mostly distributed from 15 to 100 kDa with pH 4-7. Computer-assisted image analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and post-source decay technology suggested that there were significant differences in spot distribution and expression between the middle and posterior silk glands. In addition, 98 spots from the posterior silk gland were excised and further investigated following trypsin digestion. The results suggested that more than 20% of the 88 proteins identified were related to heat-shock proteins and chaperones. Redox system and DNA replication proteins involved in silk protein synthesis were also detected in the posterior silk gland. Interestingly, two novel serpin proteins were identified in the middle silk gland, and to a lesser extent in the posterior gland, which were presumed to be involved in regulation of proteolytic activity and protection of silk proteins from degradation.

Protein folding in vivo: the importance of molecular chaperones.

The contribution of the two major cytosolic chaperone systems, Hsp70 and the cylindrical chaperonins, to cellular protein folding has been clarified by a number of recent papers. These studies found that, in vivo, a significant fraction of newly synthesized polypeptides transit through these chaperone systems in both prokaryotic and eukaryotic cells. The identification and characterization of the cellular substrates of chaperones will be instrumental in understanding how proteins fold in vivo.

RNA chaperones, RNA annealers and RNA helicases.

RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures. The latter is defined as "RNA chaperone activity" and is the main topic of this review. Here we define RNA chaperone activity in a stringent way and we review those proteins for which RNA chaperone activity has been clearly demonstrated. These proteins belong to quite diverse families such as hnRNPs, histone-like proteins, ribosomal proteins, cold shock domain proteins and viral nucleocapsid proteins. DExD/H-box containing RNA helicases are discussed as a special family of enzymes that restructure RNA or RNPs in an ATP-dependent manner. We further address the different mechanisms RNA chaperones might use to promote folding including the recently proposed theory of protein disorder as a key element in triggering RNA-protein interactions. Finally, we present a new website for proteins with RNA chaperone activity which compiles all the information on these proteins with the perspective to promote the understanding of their activity.

The various and varying roles of specific chaperones in type III secretion systems.

The type III secretion pathway is used by numerous Gram-negative pathogenic bacteria to deliver proteins within the membrane or the cytoplasm of eukaryotic cells with which these bacteria interact. Secretion is regulated by external signals. This requires that, before being secreted, proteins are stored in the cytoplasm where they need to be stabilised, separated from other interaction partners, and maintained in a secretion-competent state. Specialised, energy-independent chaperones play various roles in these functions by associating in the cytoplasm with proteins before their secretion. Some chaperones are also directly involved in modulating transcription in response to secretion.

Ribosome-tethered molecular chaperones: the first line of defense against protein misfolding?

Folding of many cellular proteins is facilitated by molecular chaperones. Analysis of both prokaryotic and lower eukaryotic model systems has revealed the presence of ribosome-associated molecular chaperones, thought to be the first line of defense against protein aggregation as translating polypeptides emerge from the ribosome. However, structurally unrelated chaperones have evolved to carry out these functions in different microbes. In the yeast Saccharomyces cerevisiae, an unusual complex of Hsp70 and J-type chaperones associates with ribosome-bound nascent chains, whereas in Escherichia coli the ribosome-associated peptidyl-prolyl-cis-trans isomerase, trigger factor, plays a predominant role.

Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae.

Chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. The GroE (group I) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. The central cavity is transiently closed by interaction with the co-protein, GroES. The most prominent feature specific to the group II subfamily, found in archaea and in the eukaryotic cytosol, is a long insertion in the substrate-binding region. In the archaeal complex, this forms an extended structure acting as a built-in lid, obviating the need for a GroES-like co-factor. This extension occludes a site known to bind non-native polypeptides in GroEL. The site and nature of substrate interaction are not known for the group II subfamily. The atomic structure of the thermosome, an archaeal group II chaperonin, has been determined in a fully closed form, but the entry and exit of protein substrates requires transient opening. Although an open form has been investigated by electron microscopy, conformational changes in group II chaperonins are not well characterized. Using electron cryo-microscopy and three-dimensional reconstruction, we describe three conformations of a group II chaperonin, including an asymmetric, bullet-shaped form, revealing the range of domain movements in this subfamily.