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).

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.

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.

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.

Development of a phenotypic high-content assay to identify pharmacoperone drugs for the treatment of primary hyperoxaluria type 1 by high-throughput screening.

Primary hyperoxaluria is a severe disease for which the best current therapy is dialysis or organ transplantation. These are risky, inconvenient, and costly procedures. In some patients, pyridoxine treatment can delay the need for these surgical procedures. The underlying cause of particular forms of this disease is the misrouting of a specific enzyme, alanine:glyoxylate aminotransferase (AGT), to the mitochondria instead of the peroxisomes. Pharmacoperones are small molecules that can rescue misfolded proteins and redirect them to their correct location, thereby restoring their function and potentially curing disease. In the present study, we miniaturized a cell-based assay to identify pharmacoperone drugs present in large chemical libraries to selectively correct AGT misrouting. This assay employs AGT-170, a mutant form of AGT that predominantly resides in the mitochondria, which we monitor for its relocation to the peroxisomes through automated image acquisition and analysis. Over the course of a pilot screen of 1,280 test compounds, we achieved an average Z'-factor of 0.72±0.02, demonstrating the suitability of this assay for HTS.

A primer on peroxiredoxin biochemistry.

Peroxiredoxins were not recognized as a family of enzymes until the 1990s but are now known to be the dominant peroxidases in most organisms. Here, the history and fundamental properties of peroxiredoxins are briefly reviewed, with a special focus on describing how an exquisitely tunable balance between fully folded and locally unfolded conformations plays a large role in peroxiredoxin catalytic properties.

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.

Evolution of chloroplast J proteins.

Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance.

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.

Chaperone-usher pathways: diversity and pilus assembly mechanism.

Up to eight different types of secretion systems, and several more subtypes, have been described in Gram-negative bacteria. Here, we focus on the diversity and assembly mechanism of one of the best-studied secretion systems, the widespread chaperone-usher pathway known to assemble and secrete adhesive surface structures, called pili or fimbriae, which play essential roles in targeting bacterial pathogens to the host.

Dissection of the dimerization modes in the DJ-1 superfamily.

The DJ-1 superfamily (DJ-1/ThiJ/PfpI superfamily) is distributed across all three kingdoms of life. These proteins are involved in a highly diverse range of cellular functions, including chaperone and protease activity. DJ-1 proteins usually form dimers or hexamers in vivo and show at least four different binding orientations via distinct interface patches. Abnormal oligomerization of human DJ-1 is related to neurodegenerative disorders including Parkinson's disease, suggesting important functional roles of quaternary structures. However, the quaternary structures of the DJ-1 superfamily have not been extensively studied. Here, we focus on the diverse oligomerization modes among the DJ-1 superfamily proteins and investigate the functional roles of quaternary structures both computationally and experimentally. The oligomerization modes are classified into 4 types (DJ-1, YhbO, Hsp, and YDR types) depending on the distinct interface patches (I-IV) upon dimerization. A unique, rotated interface via patch I is reported, which may potentially be related to higher order oligomerization. In general, the groups based on sequence similarity are consistent with the quaternary structural classes, but their biochemical functions cannot be directly inferred using sequence information alone. The observed phyletic pattern suggests the dynamic nature of quaternary structures in the course of evolution. The amino acid residues at the interfaces tend to show lower mutation rates than those of non-interfacial surfaces.

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.

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.

Expression profiles and physiological roles of two types of prefoldins from the hyperthermophilic archaeon Thermococcus kodakaraensis.

The hyperthermophilic archaeon Thermococcus kodakaraensis possesses four prefoldin genes encoding two alpha subunits (pfdA and pfdC) and two beta subunits (pfdB and pfdD) of prefoldins on the genome. pfdC and pfdD are unique genes whose orthologues are not found in Pyrococcus spp., whereas pfdA and pfdB are commonly found in both Thermococcus and Pyrococcus spp. The pfdA and pfdB are located at different loci, and pfdC and pfdD were tandemly arranged on the genome. Immunoprecipitation experiments using specific antisera, anti-PfdB and anti-PfdD, revealed that PfdB and PfdD make a complex only with PfdA and PfdC, respectively. Both PfdA/PfdB and PfdC/PfdD complexes obtained as recombinant forms showed inhibitory activity against the thermal aggregation of citrate synthase. Immunoblot experiments indicated that the PfdA/PfdB complex was expressed at all examined temperatures; however, the PfdC/PfdD complex was specifically expressed under heat-stress conditions at 93 degrees C. Transcriptional analyses showed that pfdA and pfdB were transcribed at equal levels at all examined temperatures but pfdC and pfdD were transcribed at higher levels at 93 degrees C. Furthermore, pfdA and pfdB were transcribed individually, but pfdD was cotranscribed with pfdC. A typical Pyrococcus heat-shock regulator (Phr) recognition sequence was identified at the upstream region of pfdC. The transcriptional level of pfdCD was measured in a phr disruptant, showing that the pfdCD transcript in the phr disruptant was drastically increased in comparison with that of the wild type. However, the pfdCD level was also elevated at higher temperature, indicating that heat induction of PfdC/PfdD is mainly achieved by Phr derepression but that a certain degree of induction is not under Phr control. The pfdB and pfdD disruptants were then constructed, and the growth profiles were compared. At 85 degrees C cultivation, no significant difference was observed between the wild type and the pfdD disruptant; however, less growth was observed in the pfdB disruptant. At 93 degrees C, the pfdD disruptant grew less than the wild type, and the pfdB disruptant grew the least. The results suggest that the PfdA/PfdB complex plays a crucial role at all growth temperatures and the PfdC/PfdD complex contributes to survival in a high-temperature environment.

Tipping the delicate balance: defining how proteasome maturation affects the degradation of a substrate for autophagy and endoplasmic reticulum associated degradation (ERAD).

An increasing body of data links endoplasmic reticulum (ER) function to autophagy. Not surprisingly, then, some aberrant proteins in the ER can be destroyed either via ER associated degradation (ERAD), which is proteasome-mediated, or via autophagy. One such substrate is the "Z" variant of the alpha-1 protease inhibitor (A1Pi), variably known as A1Pi-Z or AT-Z ("anti-trypsin, Z variant"). The wild type protein is primarily synthesized in the liver and is secreted. In contrast, AT-Z, like other ERAD substrates, is retro-translocated from the ER and delivered to the proteasome. However, AT-Z can form high molecular weight polymers that are degraded via autophagy, and cells that accumulate AT-Z polymers ultimately succumb, which leads to liver disease. Therefore, identifying genes that have an impact AT-Z turnover represents an active area of research. To this end, a yeast expression system for AT-Z has proven valuable. For example, a recent study using this system indicates that the activity of a proteasome assembly chaperone (PAC) is critical for maximal AT-Z turnover, which suggests a new role for PACs. Because PACs are conserved, it will be critical to analyze whether these dedicated chaperones are implicated in other diseases associated with ERAD and autophagy.

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.