Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins.

Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the ß-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.

Autophagy, protein aggregation and hyperthermia: a mini-review.

PURPOSE: We aim to explore the role of macroautophagy in cellular responses to hyperthermia. Protein damage incurred during hyperthermia can either lead to cell death or may be repaired by polypeptide quality control pathways including: (1) the deterrence of protein unfolding by molecular chaperones and (2) proteolysis of the denatured proteins within the proteasome. A third pathway of protein quality control is triggered by formation of protein aggregates in the heat shocked cell. This is the macroautophagy pathway in which protein aggregates are transported to specialised organelles called autolysosomes capable of degrading the aggregates. The consequences for cell viability of triggering this pathway are complex and may involve cell death, although under many circumstances, including exposure of cells to hyperthermia, autophagy leads to enhanced cell survival. We have discussed mechanisms by which cells detect protein aggregates and recruit them into the macroautophagy pathway as well as the potential role of inhibiting this process in hyperthermia. CONCLUSIONS: Directed macroautophagy, with its key role in protein quality control, seems an attractive target for a therapy such as hyperthermia that functions principally through denaturing the proteome. However, much work is needed to decode the mechanisms of thermal stress-mediated macroautophagy and their role in survival/death of cancer cells before recommendations can be made on targeting this pathway in combination with hyperthermia.

Proteomics displays cytoskeletal proteins and chaperones involvement in Hedyotis corymbosa-induced photokilling in skin cancer cells.

Photodynamic therapy was found to be an effective therapy for local malignant tumors. This study demonstrated that 80 µg/ml Hedyotis corymbosa extracts with 0.8 J/cm(2) fluence dose caused M21 skin cancer cell death. Photoactivated H. corymbosa-induced M21 cell death is a typical apoptosis that is accompanied by nuclear condensation, externalization of phosphatidylserine and the changes in protein expression of apoptosis-related proteins, such as Bcl-2 and caspase family members. This study applied 2D electrophoresis to analyse the proteins involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We found 12 proteins to be markedly changed. According to the results of protein sequence analysis of these altered protein spots, we identified that the expression of cytoskeletal proteins and chaperones were involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We further demonstrated that photoactivated H. corymbosa caused a significant effect on the cytoskeleton distribution and mitochondrial activity in M21 cells. Based on the above findings, this study characterized the effects and mechanisms of the photoactivated H. corymbosa-induced apoptosis in M21 skin cancer cells.

The metal chelating and chaperoning effects of clioquinol: insights from yeast studies.

Clioquinol (CQ), a once popular antibiotic, was used to inhibit the growth of microorganisms. Recently, CQ and its analog PBT2 have shown encouraging effects in the animal and clinical trials for Alzheimer's disease (AD). However, the mechanism by which this class of molecules works remains controversial. In this work, we used the yeast Saccharomyces cerevisiae as a model to study how CQ affects molecular and cellular functions and particularly, copper, iron, and zinc homeostasis. We observed a CQ-induced inhibition of yeast growth, which could be slightly relieved by supplementation of copper or iron. Microarray results indicated that yeast cells treated with CQ sense a general deficiency in metals, despite elevated total cellular contents of copper and iron. Consistent with this, reduced activities of some metal-sensitive enzymes were observed. Intriguingly, CQ can increase the SOD1 activity, likely through Ccs1's accessibility to CQ-bound copper ions. Further studies revealed that CQ sequestrates copper and iron at the cellular membrane, likely the plasma membrane, resulting overall metal accumulation but cytosolic metal depletion. CQ's effects on metal-sensitive metalloenzymes were also verified in mammalian cell line SH-SY5Y. Together, our results revealed that CQ can regulate metal homeostasis by binding metal ions, resulting the cell sensing a state of deficiency of bioavailable metal ions while simultaneously increasing available metals to SOD1 (via Ccs1) and possibly some other metalloproteins that can access CQ-bound metals. We hope this regulation of metal homeostasis may be helpful in explaining the therapeutic effects of CQ used in disease treatment.

Mini-alphaB-crystallin: a functional element of alphaB-crystallin with chaperone-like activity.

Alpha-crystallin is a member of the family of small heat-shock proteins (sHSP) and is composed of two subunits, alphaA-crystallin and alphaB-crystallin, which exhibit molecular chaperone-like properties. In a previous study, we found that residues 70-88 in alphaA-crystallin can function like a molecular chaperone by preventing the aggregation and precipitation of denaturing substrate proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we show that the complementary sequence in alphaB-crystallin, residues 73-92 (DRFSVNLDVKHFSPEELKVK), is the functional chaperone site of alphaB-crystallin. Like the mini-alphaA-crystallin chaperone, the mini-alphaB-crystallin chaperone interacts with 1,1'-bi(4-anilino) naphthalene-5,5'-disulphonic acid (bis-ANS) and also possesses significant beta-sheet and random coil structure. Deletion of four residues (DRFS) from the N-terminus or deletion of C-terminus LKVK residues from the 73-92 peptide abolishes the chaperone-like activity against denaturing alcohol dehydrogenase. However, removal of DRFS or HFSPEELKVK is necessary to completely abolish the antiaggregation property of the peptide in insulin reduction assay. Substitution of Asp at a site corresponding to D80 in alphaB-crystallin with d-Asp or beta-Asp results in a significant loss of chaperone-like activity. Kynurenine modification of His in the peptide abolishes the antiaggregation property of the mini-chaperone. These data suggest that the 73-92 region in alphaB-crystallin is one of the substrate binding sites during chaperone activity.

The DnaJ-related factor Mrj interacts with nuclear factor of activated T cells c3 and mediates transcriptional repression through class II histone deacetylase recruitment.

The calcium-regulated protein phosphatase calcineurin (PP2B) functions as a regulator of gene expression in diverse tissues through the dephosphorylation and activation of a family of transcription factors known as nuclear factor of activated T cells (NFAT). Here we show that NFATc3, in addition to being calcium responsive, is regulated through an indirect recruitment of class II histone deacetylases (HDACs). Specifically, yeast two-hybrid screening with the rel homology domain of NFATc3 identified the chaperone mammalian relative of DnaJ (Mrj) as a specific interacting factor. Mrj and NFATc3 were shown to directly associate with one another in mammalian cells and in vitro. Mrj served as a potent inhibitor of NFAT transcriptional activity within the nucleus through a mechanism involving histone deacetylase recruitment in conjunction with heat shock stimulation. Indeed, Mrj was determined to interact with class II histone deacetylases, each of which translocated to the nucleus following heat shock stimulation. Mrj also decreased NFATc3 occupancy of the tumor necrosis factor-alpha promoter in cardiomyocytes in an HDAC-dependent manner, and Mrj blocked calcineurin-induced cardiomyocyte hypertrophic growth. Conversely, small-interfering-RNA-mediated reduction of Mrj augmented NFAT transcriptional activity and spontaneously induced cardiac myocyte growth. Collectively, our results define a novel response pathway whereby NFATc3 is negatively regulated by class II histone deacetylases through the DnaJ (heat shock protein-40) superfamily member Mrj.

Protracted lithium treatment protects against the ER stress elicited by thapsigargin in rat PC12 cells: roles of intracellular calcium, GRP78 and Bcl-2.

We investigated the cytoprotective effects of lithium, the mood-stabilizer, on thapsigargin-induced stress on the endoplasmic reticulum (ER) in rat PC12 cells. Protracted lithium pretreatment of PC12 cells elicited cytoprotection against thapsigargin-induced cytotoxicity. Lithium protection was concurrent with inhibition of thapsigargin-induced intracellular calcium increase and with elevated expression of the molecular chaperone GRP78. Moreover, lithium pretreatment upregulated the antiapoptotic protein Bcl-2, and blocked Bcl-2 downregulation elicited by thapsigargin. Prior to the induction of GRP78, lithium treatment alone increased the expression of c-Fos whose induction by ER stress is necessary for GRP78 induction. Curcumin, an inhibitor of transcription factor AP-1, blocked lithium cytoprotection against thapsigargin cytotoxicity. Thus, the induction of GRP78 and Bcl-2, and activation of AP-1 likely contribute to lithium-induced protection against cytotoxicity resulting from ER stress. Additionally, thapsigargin-induced cytotoxicity was suppressed by pretreatment with another mood-stabilizer, valproate, indicating that cytoprotection against ER stress is a common action of mood-stabilizing drugs.

Deep brain stimulation in the treatment of dyskinesia and dystonia.

Deep brain stimulation (DBS) has become a mainstay of treatment for patients with movement disorders. This modality is directed at modulating pathological activity within basal ganglia output structures by stimulating some of their nuclei, such as the subthalamic nucleus (STN) and the globus pallidus internus (GPi), without making permanent lesions. With the accumulation of experience, indications for the use of DBS have become clearer and the effectiveness and limitations of this form of therapy in different clinical conditions have been better appreciated. In this review the authors discuss the efficacy of DBS in the treatment of dystonia and levodopa-induced dyskinesias. The use of DBS of the STN and GPi is very effective for the treatment of movement disorders induced by levodopa. The relative benefits of using the GPi as opposed to the STN as a target are still being investigated. Bilateral GPi stimulation is gaining importance in the therapeutic armamentarium for the treatment of dystonia. The DYT1 forms of generalized dystonia and cervical dystonias respond to DBS better than secondary dystonia does. Discrimination between the diverse forms of dystonia and a better understanding of the pathophysiological features of this condition will serve as a platform for improved outcomes.

Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator.

The Sm-like protein Hfq is involved in post-transcriptional regulation by small, noncoding RNAs in Escherichia coli that act by base pairing. Hfq stabilises the small RNAs and mediates their interaction with the target mRNA by an as yet unknown mechanism. We show here a novel chaperoning use of Hfq in the regulation by small RNAs. We analysed in vitro and in vivo the role of Hfq in the interaction between the small RNA RyhB and its sodB (iron superoxide dismutase) mRNA target. Hfq bound strongly to sodB mRNA and altered the structure of the mRNA, partially opening a loop. This gives access to a sequence complementary to RyhB and encompassing the translation initiation codon. RyhB binding blocked the translation initiation codon of sodB and triggered the degradation of both RyhB and sodB mRNA. Thus, Hfq is a critical chaperone in vivo and in vitro, changing the folding of the target mRNA to make it subject to the small RNA regulator.

Targeting aggregation in the development of therapeutics for the treatment of Huntington's disease and other polyglutamine repeat diseases.

Huntington's disease (HD) is one of a number of familial polyglutamine (polyQ) repeat diseases. These neurodegenerative disorders are caused by expression of otherwise unrelated proteins that contain an expansion of a polyQ tract, rendering them toxic to specific subsets of vulnerable neurons. These expanded repeats have an inherent propensity to aggregate; insoluble neuronal nuclear and cytoplasmic polyQ aggregates or inclusions are hallmarks of the disorders [1,2]. In HD, inclusions in diseased brains often precede onset of symptoms, and have been proposed to be involved in pathogenicity [3-5]. Various strategies to block the process of aggregation have been developed in an effort to create drugs that decrease neurotoxicity. A discussion of the effect of antibodies, caspase inhibitors, chemical inhibitors, heat-shock proteins, suppressor peptides and transglutaminase inhibitors upon aggregation and disease is presented.

Pectin methylesterases: cell wall enzymes with important roles in plant physiology.

Pectin methylesterases catalyse the demethylesterification of cell wall polygalacturonans. In dicot plants, these ubiquitous cell wall enzymes are involved in important developmental processes including cellular adhesion and stem elongation. Here, I highlight recent studies that challenge the accepted views of the mechanism and function of pectin methylesterases, including the co-secretion of pectins and pectin methylesterases into the apoplasm, new action patterns of mature pectin methylesterases and a possible function of the pro regions of pectin methylesterases as intramolecular chaperones.

The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies.

Multiple molecular chaperones, including Hsp90 and p23, interact with members of the intracellular receptor (IR) family. To investigate p23 function, we compared the effects of three p23 proteins on IR activities, yeast p23 (sba1p) and the two human p23 homologs, p23 and tsp23. We found that Sba1p was indistinguishable from human p23 in assays of seven IR activities in both animal cells and in yeast; in contrast, certain effects of tsp23 were specific to that homolog. Transcriptional activation by two IRs was increased by expression of any of the p23 species, whereas activation by five other IRs was decreased by Sba1p or p23, and unaffected by tsp23. p23 was expressed in all tissues examined except striated and cardiac muscle, whereas tsp23 accumulated in a complementary pattern; hence, p23 proteins might contribute to tissue-specific differences in IR activities. Unlike Hsp90, which acts on IR aporeceptors to stimulate ligand potency (i.e., hormone-binding affinity), p23 proteins acted on IR holoreceptors to alter ligand efficiencies (i.e., transcriptional activation activity). Moreover, the p23 effects developed slowly, requiring prolonged exposure to hormone. In vitro, p23 interacted preferentially with hormone-receptor-response element ternary complexes, and stimulated receptor-DNA dissociation. The dissociation was reversed by addition of a fragment of the GRIP1 coactivator, suggesting that the two reactions may be in competition in vivo. Our findings suggest that p23 functions at one or more late steps in IR-mediated signal transduction, perhaps including receptor recycling and/or reversal of the response.

The role of calnexin in NK-target cell interaction.

In this study, a relationship between target cell sensitivity to natural killing and target cell expression of the molecular chaperone++ calnexin was assessed. The NK-resistant cell line NKR was originally derived from the NK-sensitive, human T-cell line CEM and does not synthesize calnexin protein or mRNA. The cell lines CEM, NKR and 1B9 (NKR transfected with a calnexin cDNA) were compared in a number of assays. All the lines but CEM were resistant to NK in conventional 4 h cytotoxicity assay, but were highly sensitive to IL-2 activated NK. Incubation of NK cells with CEM but not with the other two lines led to increased expression of the NK cell activation marker CD69. Treatment of effector cells with PGE2 and TGF-beta resulted in an inhibition of NK activity and CD69 expression. The calnexin transfected clone 1B9 clone had intermediate ability to block cytotoxicity in cold target inhibition assay compared to CEM and NKR. Expression of the adhesion molecules CD44 and LFA-1alpha was significantly higher on both calnexin positive cell lines compared to NKR. These data suggest that calnexin controls the expression of some, but not all, target structures that are necessary for binding and activation of NK cells.

Molecular chaperones in the etiology and therapy of cancer.

Molecular chaperones are ubiquitous, well-conserved proteins that account for 2-5 % of all cellular proteins in most cells. The present review summarizes our current knowledge about their involvement in the etiology and therapy of cancer with special emphasis on the expression of chaperones in malignant cells, their role in folding of (proto)oncogene products, cell cycle regulation, cell differentiation and apoptosis, development of metastasis, and their participation in the recognition of malignant cells. We also overview the importance of chaperones in hyperthermia, drug resistance, and recent approaches in chaperone-immunotherapy.

Natural defenses and autoprotection: naturotherapy, an old concept of healing in a new perspective.

Recent molecular-biological and molecular-genetic research has shown that important cellular-based autoprotective mechanisms are mediated by heat-shock proteins (HSPs) or stress-response proteins, also called 'chaperones'. This can happen because cells react to extracellular stimuli by activating signal transduction pathways which result in activating the genetic program. Molecular biologists and cardiologists are tempted to evaluate these phenomena in respect to their potential meaning for a better understanding of the complex notions of health and disease. When molecular geneticists or cardiologists talk about autoprotective or natural defense mechanisms, and physicians talk about salutogenesis, they all mean something very specific. The phenomenon seen here belongs to the body's own defense mechanisms which make it capable of reacting to harmful influences and allow it to stabilize a structure and/or function of the body for a certain period. Here we see a connecting link to the historically grounded term self-healing forces, which has challenged medical doctors in the different historical periods of medical science. They tried to explain these effects based on the current model of the organism. Their understanding of this phenomenon played a role in defining the concept of health and disease. Thus, it seems very fitting to look back into history, since the phenomena discussed here as well as the insights into autoprotective mechanisms will continue to influence medical understanding of health and disease.