GroE Chaperonin System Research Articles

Are Gastrointestinal Microorganisms Involved in the Onset and Development of Amyloid Neurodegenerative Diseases?

This review discusses a few examples of specific mechanisms mediating the contribution of the GIT microbiota to the development of amyloid neurodegenerative diseases caused by the pathologic transformation of prion protein, or alpha-synuclein. The effect of the bacterial GroE chaperonin system and phage chaperonins (single-ring OBP and double-ring EL) on prion protein transformation has been described. A number of studies have shown that chaperonins stimulate the formation of cytotoxic amyloid forms of prion protein in an ATP-dependent manner. Moreover, it was found that E. coli cell lysates have a similar effect on prion protein, and the efficiency of amyloid transformation correlates with the content of GroE in cells. Data on the influence of some metabolites synthesized by gut microorganisms on the onset of synucleinopathies, such as Parkinson’s disease, is provided. In particular, the induction of amyloid transformation of alpha-synuclein from intestinal epithelial cells with subsequent prion-like formation of its pathologic forms in nervous tissues featuring microbiota metabolites is described. Possible mechanisms of microbiota influence on the occurrence and development of amyloid neurodegenerative diseases are considered.

Contact Order Is a Determinant for the Dependence of GFP Folding on the Chaperonin GroEL

The GroE chaperonin system facilitates protein folding in an ATP-dependent manner. It has remained unclear why some proteins are obligate clients of the GroE system, whereas other closely related proteins are able to fold efficiently in its absence. Factors that cause folding to be slower affect kinetic partitioning between spontaneous folding and chaperone binding in favor of the latter. One such potential factor is contact order (CO), which is the average separation in sequence between residues that are in contact in the native structure. Here, we generated variants of enhanced green fluorescent protein with different COs using circular permutations. We found that GroE dependence in vitro and in vivo increases with increasing CO. Thus, our results show that CO is relevant not only for folding in vitro of relatively simple model systems but also for chaperonin dependence and folding in vivo.

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

The versatile mutational "repertoire" of Escherichia coli GroEL, a multidomain chaperonin nanomachine.

The bacterial chaperonins are highly sophisticated molecular nanomachines, controlled by the hydrolysis of ATP to dynamically trap and remove from the environment unstable protein molecules that are susceptible to denaturation and aggregation. Chaperonins also act to assist in the refolding of these unstable proteins, providing a means by which these proteins may return in active form to the complex environment of the cell. The Escherichia coli GroE chaperonin system is one of the largest protein supramolecular complexes known, whose quaternary structure is required for segregating aggregation-prone proteins. Over the course of more than two decades of research on GroE, it has become accepted that GroE, more specifically the GroEL subunit, is a "high-tolerance" molecular system, capable of accommodating numerous mutations, while retaining its molecular integrity. In some cases, a given site of mutation was revealed to be absolutely required for GroEL function, providing hints regarding the network of signals and triggers that propel this unique system. In other instances, however, a mutation has produced a more delicate response, altering only part of, or in some cases, only a single facet of, the molecular mechanism, and these mutants have often provided invaluable hints on the extent of the complexity underlying chaperonin-assisted protein folding. In this review, we highlight some examples of the latter type of GroEL mutants which compose the unique "mutational repertoire" of GroEL and touch upon the important clues that each mutant provided to the overall effort to elucidate the details of GroE action.

Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL

The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.Electronic supplementary materialThe online version of this article (doi:10.1007/s12154-013-0106-5) contains supplementary material, which is available to authorized users.

Transcriptional Regulation of Stress Response and Motility Functions inHelicobacter pyloriIs Mediated by HspR and HrcA

The hrcA and hspR genes of Helicobacter pylori encode two transcriptional repressor proteins that negatively regulate expression of the groES-groEL and hrcA-grpE-dnaK operons. While HspR was previously shown to bind far upstream of the promoters transcribing these operons, the binding sites of HrcA were not identified. Here, we demonstrate by footprinting analysis that HrcA binds to operator elements similar to the so-called CIRCE sequences overlapping both promoters. Binding of HspR and HrcA to their respective operators occurs in an independent manner, but the DNA binding activity of HrcA is increased in the presence of GroESL, suggesting that the GroE chaperonin system corepresses transcription together with HrcA. Comparative transcriptome analysis of the wild-type strain and hspR and hrcA singly and doubly deficient strains revealed that a set of 14 genes is negatively regulated by the action of one or both regulators, while a set of 29 genes is positively regulated. While both positive and negative regulation of transcription by HspR and/or HrcA could be confirmed by RNA primer extension analyses for two representative genes, binding of either regulator to the promoters could not be detected, indicating that transcriptional regulation at these promoters involves indirect mechanisms. Strikingly, 14 of the 29 genes which were found to be positively regulated by HspR or HrcA code for proteins involved in flagellar biosynthesis. Accordingly, loss of motility functions was observed for HspR and HrcA single or double mutants. The possible regulatory intersections of the heat shock response and flagellar assembly are discussed.

Analysis of orthologoushrcAgenes inEscherichia coliandBacillus subtilis

The hrcA gene codes for a transcriptional repressor protein interacting with the CIRCE operator thereby reducing expression of the groE operon of more than 120 bacterial species. At least in Bacillus subtilis, the activity of the HrcA protein is modulated by the GroE chaperonin system. We amplified the hrcA gene from five different bacterial species and analyzed its activity in Escherichia coli and Bacillus subtilis. While those from Clostridium acetobutylicum and Staphylococcus aureus turned out to be active, those of Helicobacter pylori, Lactococcus lactis and Thermotoga maritima were inactive in E. coli, but that of T. maritima turned out to repress expression of the reporter gene in B. subtilis. All these results strongly suggest to us a specific recognition of HrcA by the GroE chaperonin system.

Analysis of orthologous hrcA genes in Escherichia coli and Bacillus subtilis

The hrcA gene codes for a transcriptional repressor protein interacting with the CIRCE operator thereby reducing expression of the groE operon of more than 120 bacterial species. At least in Bacillus subtilis, the activity of the HrcA protein is modulated by the GroE chaperonin system. We amplified the hrcA gene from five different bacterial species and analyzed its activity in Escherichia coli and Bacillus subtilis. While those from Clostridium acetobutylicum and Staphylococcus aureus turned out to be active, those of Helicobacter pylori, Lactococcus lactis and Thermotoga maritima were inactive in E. coli, but that of T. maritima turned out to repress expression of the reporter gene in B. subtilis. All these results strongly suggest to us a specific recognition of HrcA by the GroE chaperonin system.

Introducing Professor Wolfgang Schumann, Microbial Stress Responses Section Editor

Dear Colleagues It is a pleasure to announce the opening of a new dedicated section in Cell Stress & Chaperones, “Microbial Stress Responses,” and to introduce our founding section editor, Wolfgang Schumann. Since 1993, Wolfgang has been Professor of Genetics at the Institute of Genetics of the University of Bayreuth, Germany. He studied biology at the Johannes-Gutenberg University in Mainz. His PhD thesis was an immunogenetic analysis of different strains of the mosquito Culex pipiens related to cytoplasmic incompatibility. Thereafter, he started to work on two different bacteriophages: first the Salmonella typhimurium phage P22 at the Institute of Microbiology, University of Goettingen, and then the Escherichia coli phage Mu at the Department of Biology, University of Konstanz. Wolfgang has worked on bacterial stress responses since 1989, searching for inducible promoters in the genome of Bacillus subtilis. While studying the regulation of the dnaK and groE operons of B. subtilis, he discovered a new regulation mechanism which turned out to be the most widespread heat shock regulation mechanism currently known in the eubacterial world (Schmidt et al 1992; Wetzstein et al 1992). It consists of the repressor protein HrcA, which binds to an operator sequence called the CIRCE element (Schulz et al 1995; Schulz et al 1996; Zuber and Schumann 1994). How is the activity of the HrcA repressor protein modulated during heat shock? Several lines of experimental data suggest that the GroE chaperonin system exerts an important role in this process. This leads to the working model that the GroE team participates in the conversion of inactive HRCA into its active form (Mogk et al 1997; Reischl et al 2002). According to this model, inactive HrcA is present immediately after de novo synthesis and after dissociation from its operator. Recently, Wolfgang started working with Thomas Wiegert to study the alkaline stress in B. subtilis (Wiegert et al 2001). To start the new section, Wolfgang has contributed a timely review article on the B. subtilis heat shock stimulon that includes a discussion of possible thermosensors (Schumann 2003). His review is joined in this issue by a second review article featuring the E. coli stress protein GrpE that functions as a nucleotide exchange factor for the molecular chaperone DnaK (Harrison 2003). This review, which emphasizes the structural and kinetic aspects of GrpE and also discusses the thermosensor hypothesis, is an excellent companion article arranged by our Reviews Editor, Avrom Caplan. Also, although not strictly a microbial stress response article, the paper by Morales et al (2003), also in this issue, speaks to the types of interactions between microbial cells, gram negative bacteria in this example, and cells of vertebrate immune systems that no doubt influenced the evolution of both. I would like to give our readers a taste of the articles that have appeared in recent issues of Cell Stress & Chaperones involving microbes and microbial stress responses. In a study of how yeast survive oxidative stress during dessication, Periera and colleagues (2003) showed that both cytosolic and mitochondrial forms of superoxide dismutase contribute to survival, as does the disaccharide trehalose. How do yeast survive cold stress? Somer et al (2002) showed that chaperonin CCT (cytoplasmic cpn60) is a cold shock protein that appears to be needed during recovery from low temperatures and growth transition at higher temperatures. Properties and functions of microbial stress proteins were described in a second group of articles. From a comparison of four evolutionarily diverse Hsp70s including E. coli DnaK, Kumaraguru et al (2003) showed that only human Hsp70 can stimulate protective immunity in mice against HSV1. In a similar vein, Lewthwaite et al (2002) showed that the Rhizobium chaperonin 60.3 induces cytokine production by human monocytes, but the very closely related Rhizobium chaperonin 60.1 does not. Maguire et al (2002) have contributed a very useful review comparing Cpn60 proteins to expand upon the point that these proteins can have quite distinct biological properties and discussing the evidence of their activities in cell signaling pathways. Using a set of monoclonal antibodies raised against E. coli DnaJ, Krzewski et al (2003) demonstrated significant immunological similarity between DnaJ and its human homolog HDJ-1, suggesting that HDJ-1 may be targeted in vivo by immune responses stimulated by a bacterial stress protein. A third area in which we hope this new section will stimulate activity is the study of interactions between microbial cells and animal or plant cells that stimulate stress responses. An example is a study by Malago et al (2003) in which stress proteins were induced by S. enteritidis 857, a human intestinal pathogen, in the human enterocyte cell line Caco-2. Manuscripts for the new Microbial Stress Response Section or any other section of Cell Stress & Chaperones can now be submitted electronically at our new website http://cellstress.allentrack.net.

Isolation and Analysis of Mutant Alleles of the Bacillus subtilis HrcA Repressor with Reduced Dependency on GroE Function

The hrcA gene of Bacillus subtilis codes for a transcriptional repressor protein that negatively regulates expression of the heptacistronic dnaK and the bicistronic groE operon by binding to an operator-element called CIRCE. Recently, we have published data suggesting that the activity of HrcA is modulated by the GroE chaperonin system. Biochemical analyses of the HrcA protein have been hampered so far by its strong tendency to aggregate. Here, a genetic method was used to isolate mutant forms of HrcA with increased activity under conditions of decreased GroE function. One of these mutant forms (HrcA114) containing five amino acid replacements exhibited enhanced solubility when overexpressed. HrcA114 purified under native conditions produced two retarded CIRCE-containing DNA fragments in band shift experiments. The amount of the larger fragment increased after addition of GroEL, GroES, and ATP but decreased when ATP was replaced by the nonhydrolyzable ATP analog ATPgammaS. DNase I footprinting experiments exhibited full protection of the CIRCE element and neighboring nucleotides in an asymmetric way. An in vitro binding assay using affinity chromatography showed direct and specific interaction between HrcA114 and GroEL. All these experimental data are in full agreement with our previously published model that HrcA needs the GroE chaperonin system for activation.

High Salt-induced Conversion of Escherichia coliGroEL into a Fully Functional Thermophilic Chaperonin

The GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5, 5')-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 degrees C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins.

Coupling between protein folding and allostery in the GroE chaperonin system.

GroEL is an allosteric protein that facilitates protein folding in an ATP-dependent manner. Herein, the relationship between cooperative ATP binding by GroEL and the kinetics of GroE-assisted folding of two substrates with different GroES dependence, mouse dihydrofolate reductase (mDHFR) and mitochondrial malate dehydrogenase, is examined by using cooperativity mutants of GroEL. Strong intra-ring positive cooperativity in ATP binding by GroEL decreases the rate of GroEL-assisted mDHFR folding owing to a slow rate of the ATP-induced transition from the protein-acceptor state to the protein-release state. Inter-ring negative cooperativity in ATP binding by GroEL is found to affect the kinetic partitioning of mDHFR, but not of mitochondrial malate dehydrogenase, between folding in solution and folding in the cavity underneath GroES. Our results show that protein folding by this "two-stroke motor" is coupled to cooperative ATP binding.

Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism

Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.

Minimal and optimal mechanisms for GroE-mediated protein folding.

We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i.e., one that has very low spontaneous refolding yield) for which rate data can be acquired. In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate. This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, 11-fold by ADP and GroES, and 465-fold by ATP and GroES. Optimal refolding activity was observed when the dissociation of GroES from the chaperonin complex was dramatically reduced. Although GroEL minichaperones were able to bind denatured mitochondrial malate dehydrogenase, they were ineffective in enhancing the refolding rate. The spectrum of mechanisms for GroE-mediated protein folding depends on the nature of the substrate. The minimal mechanism for permissive substrates (i.e., having significant yields of spontaneous refolding), requires only binding to the apical domain of GroEL. Slow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone. The optimal mechanism, which requires holoGroEL, physiological amounts of GroES, and ATP hydrolysis, is necessary for the chaperonin-mediated folding of nonpermissive substrates at physiologically relevant rates under conditions in which retention of bound GroES prevents the premature release of aggregation-prone folding intermediates from the chaperonin complex. The different mechanisms are described in terms of the structural features of mini- and holo-chaperones.

The GroE chaperonin machine is the major modulator of the CIRCE heat shock regulon ofBacillus subtilis

Regulation of the heat shock response in bacteria has been studied extensively inEscherichia coli where heat shock genes are classified into three classes and where each class is regulated by a different alternate sigma factor.Bacillus subtilis serves as a second model bacterium to study regulation of the heat shock response in detail. Here, four classes of heat shock genes have been described so far where two are controlled by two different repressor proteins and the third by the alternate sigma factorσ ᴮ. Class I heat shock genes consists of two operons, the heptacistronicdnaK and the bicistronicgroE operon. Transcription of thednaK operon is complex involving two promoters, premature termination of transcription, mRNA processing and different stabilities of the processed transcripts to ensure the appropriate amounts of heat shock proteins under different growth conditions. The translation product of thehrcA gene, the first gene of thednaK operon, binds to an operator designated CIRCE element, and its activity is modulated by the GroE chaperonin system. We assume that the HrcA protein, uponde novo synthesis and upon dissociation from its operator, is present in an inactive form and has to be activated by the GroE chaperonin system resulting in an HrcA-GroE reaction cycle. Induction of class I heat shock genes occurs by the appearance of denatured proteins within the cytoplasm which titrate the GroE system. This results in accumulation of inactive HrcA repressor and thereby in induction of class I heat shock genes. Upon removal of the non-native proteins from the cytoplasm, the GroE chaperonin will interact with HrcA and promote folding into its active conformation resulting in turning off of class I heat shock genes. This mechanism ensures adequate adjustment of class I heat shock proteins depending on their actual need.