Chaperone-based procedure to increase yields of soluble recombinant proteins produced in <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins.     

Overproduced <Emphasis Type="Italic">Brucella abortus</Emphasis> PdhS-mCherry forms soluble aggregates in <Emphasis Type="Italic">Escherichia coli</Emphasis>, partially associating with mobile foci of IbpA-YFP

Background  

When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.     

The small heat shock protein (sHSP) genes in the silkworm, Bombyx mori, and comparative analysis with other insect sHSP genes

Background  

Small heat shock proteins (sHSPs) are products of heat shock response and of other stress responses, and ubiquitous in all three domains of life, archaea, bacteria, and eukarya. They mainly function as molecular chaperones to protect proteins from being denatured in extreme conditions. Study on insect sHSPs could provide some insights into evolution of insects that have adapted to diverse niches in the world.     

Puromycin-based vectors promote a ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70 and Proteasomes

Background  

Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation.     

Effects of small <Emphasis Type="Italic">Hsp</Emphasis>genes on developmental stability and microenvironmental canalization

Background  

Progression of development has to be insulated from the damaging impacts of environmental and genetic perturbations to produce highly predictable phenotypes. Molecular chaperones, such as the heat shock proteins (HSPs), are known to buffer various environmental stresses, and are deeply involved in protein homeostasis. These characteristics of HSPs imply that they might affect developmental buffering and canalization.     

Hook2 contributes to aggresome formation

Background  

Aggresomes are pericentrosomal accumulations of misfolded proteins, chaperones and proteasomes. Their positioning near the centrosome, like that of other organelles, requires active, microtubule-dependent transport. Linker proteins that can associate with the motor protein dynein, organelles, and microtubules are thought to contribute to the active maintenance of the juxtanuclear localization of many membrane bound organelles and aggresomes. Hook proteins have been proposed to serve as adaptors for the association of cargos with dynein for transport on microtubules. Hook2 was shown to localize to the centrosome, bind centriolin, and contribute to centrosomal function.     

Stage-specific expression of the mitochondrial co-chaperonin of Leishmania donovani,CPN10

Background  

Leishmania spp., in the course of their parasitic life cycle, encounter two vastly different environments: the gut of sandflies and the phagosomes of mammalian macrophages. During transmission into a mammal, the parasites are exposed to increased ambient temperature as well as to different carbon sources. Molecular chaperones or heat shock proteins are implicated in the necessary adaptations which involve the ordered differentiation from the flagellated, extracellular promastigote to the intracellular amastigote stage.     

Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

Background  

Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators.     

The <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

Background  

Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study.     

Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana

Background

Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures.

Methods

Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation.

Results

We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence.

General significance

The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.     

Transient increase of ATP as a response to temperature up-shift in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift.     

Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.)

Background

Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs.

Results

We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP⁺ reductase was fused to either the N- or C-terminal end of glutathione S-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide.

Conclusions

AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.     

Proteomic analysis of acidic chaperones, and stress proteins in extreme halophile Halobacterium NRC-1: a comparative proteomic approach to study heat shock response

Background  

Halobacterium sp. NRC-1 is an extremely halophilic archaeon and has adapted to optimal growth under conditions of extremely high salinity. Its proteome is highly acidic with a median pI of 4.9, a unique characteristic which helps the organism to adapt high saline environment. In the natural growth environment, Halobacterium NRC-1 encounters a number of stressful conditions including high temperature and intense solar radiation, oxidative and cold stress. Heat shock proteins and chaperones play indispensable roles in an organism's survival under many stress conditions. The aim of this study was to develop an improved method of 2-D gel electrophoresis with enhanced resolution of the acidic proteome, and to identify proteins with diverse cellular functions using in-gel digestion and LC-MS/MS and MALDI-TOF approach.     

The crystal structure of the putative peptide-binding fragment from the human Hsp40 protein Hdj1

Background  

The mechanism by which Hsp40 and other molecular chaperones recognize and interact with non-native polypeptides is a fundamental question. How Hsp40 co-operates with Hsp70 to facilitate protein folding is not well understood. To investigate the mechanisms, we determined the crystal structure of the putative peptide-binding fragment of Hdj1, a human member of the type II Hsp40 family.     

Myofiber stress-response in myositis: parallel investigations on patients and experimental animal models of muscle regeneration and systemic inflammation

Introduction  

The endoplasmic reticulum (ER) stress-response, evoked in mice by the overexpression of class I major histocompatibility complex antigen (MHC-I), was proposed as a major mechanism responsible for skeletal muscle damage and dysfunction in autoimmune myositis. The present study was undertaken to characterize in more detail the ER stress-response occurring in myofibers of patients with inflammatory myopathies, focusing on the expression and distribution of Grp94, calreticulin and Grp75, three ER chaperones involved in immunomodulation.     

The small heat-shock proteins IbpA and IbpB reduce the stress load of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> and delay degradation of inclusion bodies

Background  

The permanently impaired protein folding during recombinant protein production resembles the stress encountered at extreme temperatures, under which condition the putative holding chaperones, IbpA/IbpB, play an important role. We evaluated the impact of ibpAB deletion or overexpression on stress responses and the inclusion body metabolism during production of yeast α-glucosidase in Escherichia coli.     

Defective Protein Folding and Aggregation as the Basis of Neurodegenerative Diseases: The Darker Aspect of Proteins

Abstract  

The ability of a polypeptide to fold into a unique, functional, and three-dimensional structure depends on the intrinsic properties of the amino acid sequence, function of the molecular chaperones, proteins, and enzymes. Every polypeptide has a finite tendency to misfold and this forms the darker side of the protein world. Partially folded and misfolded proteins that escape the cellular quality control mechanism have the high tendency to form inter-molecular hydrogen bonding between the same protein molecules resulting in aggregation. This review summarizes the underlying and universal mechanism of protein folding. It also deals with the factors responsible for protein misfolding and aggregation. This article describes some of the consequences of such behavior particularly in the context of neurodegenerative conformational diseases such as Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis and other non-neurodegenerative conformational diseases such as cancer and cystic fibrosis etc. This will encourage a more proactive approach to the early diagnosis of conformational diseases and nutritional counseling for patients.     

Quality control of inclusion bodies in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Bacterial inclusion bodies (IBs) are key intermediates for protein production. Their quality affects the refolding yield and further purification. Recent functional and structural studies have revealed that IBs are not dead-end aggregates but undergo dynamic changes, including aggregation, refunctionalization of the protein and proteolysis. Both, aggregation of the folding intermediates and turnover of IBs are influenced by the cellular situation and a number of well-studied chaperones and proteases are included. IBs mostly contain only minor impurities and are relatively homogenous.