Genome-wide RNAi screen and in vivo protein aggregation reporters identify degradation of damaged proteins as an essential hypertonic stress response

The damaging effects of hypertonic stress on cellular proteins are poorly defined, and almost nothing is known about the pathways that detect and repair hypertonicity-induced protein damage. To begin addressing these problems, we screened approximately 19,000 Caenorhabditis elegans genes by RNA interference (RNAi) feeding and identified 40 that are essential for survival during acute hypertonic stress. Half (20 of 40) of these genes encode proteins that function to detect, transport, and degrade damaged proteins, including components of the ubiquitin-proteasome system, endosomal sorting complexes, and lysosomes. High-molecular-weight ubiquitin conjugates increase during hypertonic stress, suggesting a global change in the ubiquitinylation state of endogenous proteins. Using a polyglutamine-containing fluorescent reporter, we demonstrate that cell shrinkage induces rapid protein aggregation in vivo and that many of the genes that are essential for survival during hypertonic stress function to prevent accumulation of aggregated proteins. High levels of urea, a strong protein denaturant, do not cause aggregation, suggesting that factors such as macromolecular crowding also contribute to protein aggregate formation during cell shrinkage. Acclimation of C. elegans to mild hypertonicity dramatically increases the osmotic threshold for protein aggregation, demonstrating that protein aggregation-inhibiting pathways are activated by osmotic stress. Our studies demonstrate that hypertonic stress induces protein damage in vivo and that detection and degradation of damaged proteins are essential mechanisms for survival under hypertonic conditions.     

Ubiquitin-like protein 5 positively regulates chaperone gene expression in the mitochondrial unfolded protein response

Perturbation of the protein-folding environment in the mitochondrial matrix selectively upregulates the expression of nuclear genes encoding mitochondrial chaperones. To identify components of the signal transduction pathway(s) mediating this mitochondrial unfolded protein response (UPR(mt)), we first isolated a temperature-sensitive mutation (zc32) that conditionally activates the UPR(mt) in C. elegans and subsequently searched for suppressors by systematic inactivation of genes. RNAi of ubl-5, a gene encoding a ubiquitin-like protein, suppresses activation of the UPR(mt) markers hsp-60::gfp and hsp-6::gfp by the zc32 mutation and by other manipulations that promote mitochondrial protein misfolding. ubl-5 (RNAi) inhibits the induction of endogenous mitochondrial chaperone encoding genes hsp-60 and hsp-6 and compromises the ability of animals to cope with mitochondrial stress. Mitochondrial morphology and assembly of multi-subunit mitochondrial complexes of biotinylated proteins are also perturbed in ubl-5(RNAi) worms, indicating that UBL-5 also counteracts physiological levels of mitochondrial stress. Induction of mitochondrial stress promotes accumulation of GFP-tagged UBL-5 in nuclei of transgenic worms, suggesting that UBL-5 effects a nuclear step required for mounting a response to the threat of mitochondrial protein misfolding.     

Influence of chemical chaperones on the properties of lysozyme and the reaction center protein from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.     

Effect of chemical chaperones on properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and the functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. Selected chemical chaperones are strongly different by the structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described within the frames of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic nuclus) moieties. This fact provides additional regulation of functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in denaturation temperature T(d), and an increase in the amplitude of equilibrium fluctuation, which allows him to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in the case of methylresorcinol. Therefore, methylresorcinol does not effect the functional properties of the RC protein. It was concluded that various chaperones at one and the same concentration and chaperones at different concentrations form diverse 3D structures of proteins, which differ by dynamic and functional characteristics.     

A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint

A Werner syndrome protein homolog in C. elegans (WRN-1) was immunolocalized to the nuclei of germ cells, embryonic cells, and many other cells of larval and adult worms. When wrn-1 expression was inhibited by RNA interference (RNAi), a slight reduction in C. elegans life span was observed, with accompanying signs of premature aging, such as earlier accumulation of lipofuscin and tissue deterioration in the head. In addition, various developmental defects, including small, dumpy, ruptured, transparent body, growth arrest and bag of worms, were induced by RNAi. The frequency of these defects was accentuated by gamma-irradiation, implying that they were derived from spontaneous or induced DNA damage. wrn-1(RNAi) worms showed accelerated larval growth irrespective of gamma-irradiation, and pre-meiotic germ cells had an abnormal checkpoint response to DNA replication blockage. These observations suggest that WRN-1 acts as a checkpoint protein for DNA damage and replication blockage. This idea is also supported by an accelerated S phase in wrn-1(RNAi) embryonic cells. wrn-1(RNAi) phenotypes similar to those of Werner syndrome, such as premature aging and short stature, suggest wrn-1-deficient C. elegans as a useful model organism for Werner syndrome.     

Some aspects of the mechanism of thermal acclimation in the earthworm Lampito mauritii

Summary Inorganic ions (Ca, Mg, Na, K, Cl, SO₄) and free amino acids of the body fluids of the normal, cold and warm acclimated worms (laboratory as well as seasonal populations) are estimated. Calcium increased and chloride and sodium decreased on both cold and warm acclimation in relation to normal. But magnesium and sulphate and free amino acids increased on warm acclimation whereas potassium increased and magnesium decreased on cold acclimation. Changes in different ions in the same direction are observed in the seasonal populations. Attention is drawn to the adaptive significance of these changes in the different ions during thermal acclimation.Changes in the glycogen, RNA, protein and non-protein nitrogen, and water content in the tissues of normal and acclimated worms are studied. Glycogen increased on warm and cold acclimation, whereas RNA content, protein nitrogen and dry weight of the cold worms increased over normal. No change is observed in non-protein nitrogen on thermal acclimation. The role of these substances and the significance of the changes observed, in the operation of homeostatic mechanism compensating to temperature changes in the metabolic rate of the worms, are also discussed.Changes in the pattern of neurosecretory activity are followed with thermal acclimation and it is shown that the activity of the neurosecretory cells increased on cold and warm acclimation, but the positions of these cells, which are active, are different from normal worms in warm acclimated worms.Studies on the effect of the body fluids of acclimated worms on the tissues of normal and acclimated worms showed that the body fluids of cold acclimated worms increased the respiration of the tissues of normal and warm acclimated worms and vice-versa.     

Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling.

BACKGROUND: Molecular chaperones recognize nonnative proteins and orchestrate cellular folding processes in conjunction with regulatory cofactors. However, not every attempt to fold a protein is successful, and misfolded proteins can be directed to the cellular degradation machinery for destruction. Molecular mechanisms underlying the cooperation of molecular chaperones with the degradation machinery remain largely enigmatic so far. RESULTS: By characterizing the chaperone cofactors BAG-1 and CHIP, we gained insight into the cooperation of the molecular chaperones Hsc70 and Hsp70 with the ubiquitin/proteasome system, a major system for protein degradation in eukaryotic cells. The cofactor CHIP acts as a ubiquitin ligase in the ubiquitination of chaperone substrates such as the raf-1 protein kinase and the glucocorticoid hormone receptor. During targeting of signaling molecules to the proteasome, CHIP may cooperate with BAG-1, a ubiquitin domain protein previously shown to act as a coupling factor between Hsc/Hsp70 and the proteasome. BAG-1 directly interacts with CHIP; it accepts substrates from Hsc/Hsp70 and presents associated proteins to the CHIP ubiquitin conjugation machinery. Consequently, BAG-1 promotes CHIP-induced degradation of the glucocorticoid hormone receptor in vivo. CONCLUSIONS: The ubiquitin domain protein BAG-1 and the CHIP ubiquitin ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control.     

Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo

The budding of clathrin-coated vesicles is essential for protein transport. After budding, clathrin must be uncoated before the vesicles can fuse with other membranous structures. In vitro, the molecular chaperone Hsc70 uncoats clathrin-coated vesicles in an ATP-dependent process that requires a specific J-domain protein such as auxilin. However, there is little evidence that either Hsc70 or auxilin is essential in vivo. Here we show that C. elegans has a single auxilin homologue that is identical to mammalian auxilin in its in vitro activity. When RNA-mediated interference (RNAi) is used to inhibit auxilin expression in C. elegans, oocytes show markedly reduced receptor-mediated endocytosis of yolk protein tagged with green fluorescent protein (GFP). In addition, most of these worms arrest during larval development, exhibit defective distribution of GFP-clathrin in many cell types, and show a marked change in clathrin dynamics, as determined by fluorescence recovery after photobleaching (FRAP). We conclude that auxilin is required for in vivo clathrin-mediated endocytosis and development in C. elegans.     

Hypertonic stress induces rapid and widespread protein damage in C. elegans

Proteostasis is defined as the homeostatic mechanisms that maintain the function of all cytoplasmic proteins. We recently demonstrated that the capacity of the proteostasis network is a critical factor that defines the limits of cellular and organismal survival in hypertonic environments. The current studies were performed to determine the extent of protein damage induced by cellular water loss. Using worm strains expressing fluorescently tagged foreign and endogenous proteins and proteins with temperature-sensitive point mutations, we demonstrate that hypertonic stress causes aggregation and misfolding of diverse proteins in multiple cell types. Protein damage is rapid. Aggregation of a polyglutamine yellow fluorescent protein reporter is observable with <1 h of hypertonic stress, and aggregate volume doubles approximately every 10 min. Aggregate formation is irreversible and occurs after as little as 10 min of exposure to hypertonic conditions. To determine whether endogenous proteins are aggregated by hypertonic stress, we quantified the relative amount of total cellular protein present in detergent-insoluble extracts. Exposure for 4 h to 400 mM or 500 mM NaCl induced a 55-120% increase in endogenous protein aggregation. Inhibition of insulin signaling or acclimation to mild hypertonic stress increased survival under extreme hypertonic conditions and prevented aggregation of endogenous proteins. Our results demonstrate that hypertonic stress causes widespread and dramatic protein damage and that cells have a significant capacity to remodel the network of proteins that function to maintain proteostasis. These findings have important implications for understanding how cells cope with hypertonic stress and other protein-damaging stressors.     

Tat-mediated protein delivery in living Caenorhabditis elegans

The Tat protein from HIV-1 fused with heterologous proteins traverses biological membranes in a transcellular process called: protein transduction. This has already been successfully exploited in various biological models, but never in the nematode worm Caenorhabditis elegans. TAT-eGFP or GST-eGFP proteins were fed to C. elegans worms, which resulted in the specific localization of Tat-eGFP to epithelial intestinal cells. This system represents an efficient tool for transcellular transduction in C. elegans intestinal cells. Indeed, this approach avoids the use of tedious purification steps to purify the TAT fusion proteins and allows for rapid analyses of the transduced proteins. In addition, it may represent an efficient tool to functionally analyze the mechanisms of protein transduction as well as to complement RNAi/KO in the epithelial intestinal system. To sum up, the advantage of this technology is to combine the potential of bacterial expression system and the Tat-mediated transduction technique in living worm.     

Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase

In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL/GroES-assisted refolding of denatured protein, the yield of correctly folded protein from glycerol- or proline-assisted spontaneous refolding process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

From the cradle to the grave: molecular chaperones that may choose between folding and degradation

Molecular chaperones are known to facilitate cellular protein folding. They bind non-native proteins and orchestrate the folding process in conjunction with regulatory cofactors that modulate the affinity of the chaperone for its substrate. However, not every attempt to fold a protein is successful and chaperones can direct misfolded proteins to the cellular degradation machinery for destruction. Protein quality control thus appears to involve close cooperation between molecular chaperones and energy-dependent proteases. Molecular mechanisms underlying this interplay have been largely enigmatic so far. Here we present a novel concept for the regulation of the eukaryotic Hsp70 and Hsp90 chaperone systems during protein folding and protein degradation.     

Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.     

Chaperone signalling complexes in Alzheimer's disease

Molecular chaperones and heat shock proteins (Hsp) have emerged as critical regulators of proteins associated with neurodegenerative disease pathologies. The very nature of the chaperone system, which is to maintain protein quality control, means that most nascent proteins come in contact with chaperone proteins. Thus, amyloid precursor protein (APP), members of the gamma-secretase complex (presenilin 1 [PS1] collectively), the microtubule-associated protein tau (MAPT) as well as a number of neuroinflammatory components are all in contact with chaperones from the moment of their production. Chaperones are often grouped together as one machine presenting abnormal or mutant proteins to the proteasome for degradation, but this is not at all the case. In fact, the chaperone family consists of more than 100 proteins in mammalian cells, and the primary role for most of these proteins is to protect clients following synthesis and during stress; only as a last resort do they facilitate protein degradation. To the best of our current knowledge, the chaperone system in eukaryotic cells revolves around the ATPase activities of Hsp70 and Hsp90, the two primary chaperone scaffolds. Other chaperones and co-chaperones manipulate the ATPase activities of Hsp70 and Hsp90, facilitating either folding of the client or its degradation. In the case of Alzheimer's disease (AD), a number of studies have recently emerged describing the impact that these chaperones have on the proteotoxic effects of tau and amyloid-β accumulation. Here, we present the current understandings of chaperone biology and examine the literature investigating these proteins in the context of AD.