Designing a High Throughput Refolding Array Using a Combination of the GroEL Chaperonin and Osmolytes

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL–GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin–substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a ‘folding array’ to identify superior folding conditions. The stable chaperonin–substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (~mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.     

Rescue of Folding Defects in ABC Transporters Using Pharmacological Chaperones

The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (ΔF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in ΔF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of ΔF508 CFTR as well as other CFTR processing mutants in the presence of 1 μM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.     

The therapeutic potential of chemical chaperones in protein folding diseases

Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.     

固定化分子伴侣GroE促进变性溶菌酶复性的研究

利用重组大肠杆菌表达制备了分子伴侣GroE(GroEL和GroES),研究了GroE以及GroEL辅助变性溶菌酶复性的作用。结果表明,不仅游离GroEL单独作用可使溶菌酶复性收率达到90%以上,而且固定化GroEL亦可有效地促进蛋白质复性,最佳复性温度为37℃,最佳pH值范围为6～8,复性酶的活性收率在85%以上。另外,固定化GroEL可反复回收利用,表明固定化GroEL有可能在实际生物下游过程中得到应用。     

Strategies for Correcting the AF508 CFTR Protein-Folding Defect

Many human diseases arise as a result of mutations within genes encoding essential proteins. In many cases, the mutations are not so severe as to render the protein biologically inactive. Rather, the mutations oftentimes result in only subtle protein-folding abnormalities. In the case of the CFTR protein, a mutation leading to the loss of a single amino acid is responsible for the diseased state in the majority of individuals with cystic fibrosis. Here the newly synthesized mutant CFTR protein, missing a phenylalanine residue at position 508 (F508 CFTR), is unable to transit from the endoplasmic reticulum to the plasma membrane, where it functions as a regulator of chloride transport. All of the available evidence indicate that the newly synthesized F508 CFTR protein adopts a slightly altered conformation and therefore is retained at the level of the endoplasmic reticulum, ostensibly by the actions of the cellular quality control system. Because the mutant protein is capable of functioning as a chloride channel, developing ways to elicit its release out of the ER and to the plasma membrane has important clinical implications. Herein, we discuss our recent studies showing that the protein folding defect associated with the F508 CFTR mutation, as well as a number of other temperature-sensitive mutations, can be overcome by strategies designed to influence protein folding inside the cell. Specifically we show that a number of low-molecular-weight compounds, all of which are known to stabilize proteins in their native conformation, are effective in rescuing the folding and/or processing defects associated with different mutations that oftentimes lead to human disease.     

Chaperone mediated solubilization of 69-kDa recombinant maltodextrin glucosidase in Escherichia coli

Aims:  To investigate the factors affecting expression and solubilization of Escherichia coli maltodextrin glucosidase in E. coli .
Methods and Results:  Expression level and solubilization of the recombinant E. coli maltodextrin glucosidase was studied in E. coli at different temperatures, in presence of overexpressed GroEL, GroES and externally supplemented glycerol. Aggregation of maltodextrin glucosidase in the cytoplasm was partially prevented by the co-expression of GroEL and GroES, and using externally supplemented glycerol or lowering the culture temperature. Co-expression of GroEL and GroES or simultaneous presence of overexpressed GroEL, GroES and externally supplemented glycerol together resulted significant increase of the activity of maltodextrin glucosidase. The growth rate of E. coli was inhibited by the formation of inclusion bodies whereas the presence of overexpressed GroEL, GroES alone or together with glycerol enhanced the growth rate of E. coli substantially.
Conclusions:  The results indicated that lowering the temperature, use of GroEL, GroES and glycerol could be few controlling factors for the solubilization of recombinant aggregation-prone maltodextrin glucosidase in E. coli.
Significance and Impact of the Study:  Our study could help in developing the strategy for enhancing the production of soluble industrial enzymes and finding the therapeutic agents against protein misfolding diseases.     

Quality control of the proteins associated with neurodegenerative diseases

Most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease and other polyglutamine diseases are associated with degeneration and death of specific neuronal populations due to misfolding or aggregation of certain proteins. These aggregates often contain ubiquitin that is the signal for proteolysis by the ubiquitin-proteasome system, and chaperone proteins that are involved in the assistance of protein folding. Here we review the role of protein quality control systems in the pathogenesis of neurodegenerative diseases, and aim to learn more from the cooperation between molecular chaperones and ubiquitin-proteasome system responding to cellular protein aggregates, in order to find molecular targets for therapeutic intervention.     

Proteomics: a strategy to understand the novel targets in protein misfolding and cancer therapy

Proteins carry out important functions as they fold themselves. Protein misfolding occurs during different biochemical processes and may lead to the development of diseases such as cancer, which is characterized by genetic instability. The cancer microenvironment exposes malignant cells to a variety of stressful conditions that may further promote protein misfolding. Tumor development and progression often arises from mutations that interfere with the appropriate function of tumor-suppressor proteins and oncogenes. These may be due to alteration of catalytic activity of the protein, loss of binding sites for effector proteins or alterations of the native folded protein conformation. Src family kinases, p53, mTOR and C-terminus of HSC70 interacting protein (CHIPs) are some examples associated with protein misfolding and tumorigenesis. Molecular chaperones, such as heat-shock protein (HSP)70 and HSP90, assist protein folding and recognize target misfolded proteins for degradation. It is likely that this misfolding in cancer is linked by common principles, and may, therefore, present an exciting possibility to identify common targets for therapeutic intervention. Here we aim to review a number of examples that show how alterations in the folding of tumor-suppressor proteins or oncogenes lead to tumorigenesis. The possibility of targeting the targets to repair or degrade protein misfolding in cancer therapy is discussed.     

Effects of hesperidin,a flavanone glycoside interaction on the conformation,stability, and aggregation of lysozyme: multispectroscopic and molecular dynamic simulation studies?

Hesperidin (HESP), a flavanone glycoside, shows high antioxidant properties and posses ability to go through the blood–brain barrier. Therefore, it could be a potential drug molecule against aggregation based diseases such as Alzheimer’s, Parkinson’s, and systemic amyloidoses. In this work, we investigated the potential of HESP to interact with hen egg-white lysozyme (HEWL) monomer and prevent its aggregation. The HESP–HEWL binding studies were performed using a fluorescence quenching technique, molecular docking and molecular dynamics simulations. We found a strong interaction of HESP with the lysozyme monomer (K_a, ~ 5 × 10⁴ M^?1) mainly through hydrogen bonding, water bridges, and hydrophobic interactions. We showed that HESP molecule spanned the highly aggregation prone region (amino acid residues 48-101) of HEWL and prevented its fibrillar aggregation. Further, we found that HESP binding completely inhibited amorphous aggregation of the protein induced by disulfide-reducing agent tries-(2-carboxyethyl) phosphine. Conformational and stability studies as followed by various tertiary and secondary structure probes revealed that HESP binding only marginally affected the lysozyme monomer conformation and increased both stability and reversibility of the protein against thermal denaturation. Future studies should investigate detail effects of HESP on solvent dynamics, structure, and toxicity of various aggregates. The answers to these questions will not only target the basic sciences, but also have application in biomedical and biotechnological sciences.     

Protein folding and chaperonins

The folding of polypeptide chains in cells, following either translation or translocation through membranes, must take place under conditions of extremely high protein concentrations. In addition, folding into a correct structure must occur in the presence of other rapidly folding species, and at temperatures known to destabilize aggregation-prone folding intermediates. To facilitate folding in vivo, molecular chaperones have evolved that stabilize protein folding intermediates, thus partitioning them towards a pathway leading to the native state rather than forming inactive aggregated structures.     

Protein Folding, Misfolding, and Aggregation. Formation of Inclusion Bodies and Aggresomes

In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.