Changes in Oligomerization Are Essential for the Chaperone Activity of a Small Heat Shock Protein in Vivo and in Vitro (original) (raw)
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Proceedings of the …, 2005
To investigate the mechanism of small heat shock protein (sHsp) function, unbiased by current models of sHsp chaperone activity, we performed a screen for mutations of Synechocystis Hsp16.6 that reduced the ability of the protein to provide thermotolerance in vivo. Missense mutations at 17 positions throughout the protein and a C-terminal truncation of 5 aa were identified, representing the largest collection of sHsp mutants impaired in function in vivo. Ten mutant proteins were purified and tested for alterations in native oligomeric structure and in vitro chaperone activity. These biochemical assays separated the mutants into two groups. The C-terminal truncation and six mutations in the ␣-crystallin domain destabilized the sHsp oligomer and reduced in vitro chaperone activity. In contrast, the other three mutations had little effect on oligomer stability or chaperone activity in vitro. These mutations were clustered in the N terminus of Hsp16.6, pointing to a previously unrecognized, important function for this evolutionarily variable domain. Furthermore, the fact that the N-terminal mutations were impaired in function in vivo, but active as chaperones in vitro, indicates that current biochemical assays do not adequately measure essential features of the sHsp mechanism of action. crystallin ͉ dimer ͉ Synechocystis ͉ thermotolerance ͉ heat stress S mall heat shock proteins (sHsps) are a ubiquitous family of chaperones defined by their conserved, Ϸ90-aa ␣-crystallin domain (1), which is flanked by a variable N-terminal region and a short C-terminal extension. In their native state the majority of sHsps are found as large oligomers of 9 to Ͼ30 subunits, depending on the sHsp (2-4). Biochemical studies with purified components have demonstrated that sHsps act as chaperones by binding and holding denaturing proteins in refoldable states until ATP-dependent chaperones, primarily DnaK͞Hsp70 (but enhanced by ClpB͞Hsp100), are supplied to refold them (5-12). Evidence that sHsps bind denaturing proteins in vivo comes from a number of studies. In the cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis), Hsp16.6 interacts during heat stress in vivo with a large number of heat-labile proteins that can subsequently be released by DnaK in cell extracts (13). sHsps in Escherichia coli, plants, and yeast enter an insoluble fraction of the cell during heat stress, presumably bound to substrates, and release to the soluble fraction is dependent on the ClpB͞ Hsp100 chaperones (14-16). In human diseases that are linked to sHsp gene mutations, sHsps have been found colocalized with misfolded cellular proteins (17-19). However, sHsps have been shown to interact with an increasing number of diverse cellular proteins, implicating them in everything from ubiquitinmediated proteolysis to modulation of translation (4). sHsps can also interact with lipids (20). Thus, it remains to be demonstrated that sHsp chaperone activity, as currently defined biochemically, fully accounts for the role of sHsps in vivo. Definition of sHsp function has suffered from lack of a genetic model system in which to test sHsp action in vivo. In both E. coli and Saccharomyces cerevisiae, phenotypes associated with sHsp deletions are difficult to measure (21, 22). However, the single sHsp in Synechocystis, Hsp16.6, is essential for tolerance to high Screen for Thermotolerance-Defective Mutants in Vivo. Pools of pNaive containing randomly mutated hsp16.6 were isolated from E. coli and transformed into the Synechocystis strain HK-1͞ ⌬ClpB1, which is null for both hsp16.6 and clpB1 (24). Clonal populations were replica plated to BG-11͞agar (27) with 140 mM MgSO 4 and 5 mM glucose, and heated at 44°C for 8 h. Colonies that did not survive were isolated from duplicate, unstressed plates and screened for Hsp16.6 accumulation by Conflict of interest statement: No conflicts declared.
Biotechnology & Biotechnological Equipment, 2015
The most important role of molecular chaperones, especially under extreme temperature, is in handling with the impact of such stress on proteins and avoiding loss of their native conformation. Molecular chaperones play roles in the folding, transport, synthesis and quality control of other proteins under extreme temperature. The genome of the cyanobacterium Synechocystis PCC 6803 includes three dnaK genes (encoding heat-shock protein 70, Hsp70). The role of the second Hsp70 protein (Sll0170; DnaK2) in the acquisition of thermotolerance in Escherichia coli cells was investigated. Escherichia coli strain BL21 (DE3) was transformed with an expression vector (pTYB21) carrying the coding sequence of the Sll0170 gene under the control of the lac promoter. The recombinant cells which showed inducible expression of DnaK2 were observed to have improved viability compared to the wild-type strain upon transfer from 37 C to 52 C for up to 2, 4 and 6 min. Moreover, the growth of the wild-type cell culture decreased to 64% of that of the recombinant cell culture, following a transient (15 min) heat shock at 45 C after which the culture was subsequently transferred back to 37 C. Interestingly, the recombinant E. coli cells showed significantly faster culture growth than the wild-type cells at 37 C.
Current Microbiology, 2000
The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47°C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43°C for 1 h and subsequent challenge exposure at 49°C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress.
Cyanobacterial heat-shock response: role and regulation of molecular chaperones
Cyanobacteria constitute a morphologically diverse group of oxygenic photoautotrophic microbes which range from unicellular to multicellular, and non-nitrogen-fixing to nitrogen-fixing types. Sustained long-term exposure to changing environmental conditions, during their three billion years of evolution, has presumably led to their adaptation to diverse ecological niches. The ability to maintain protein conformational homeostasis (folding-misfolding-refolding or aggregationdegradation) by molecular chaperones holds the key to the stress adaptability of cyanobacteria. Although cyanobacteria possess several genes encoding DnaK and DnaJ family proteins, these are not the most abundant heat-shock proteins (Hsps), as is the case in other bacteria. Instead, the Hsp60 family of proteins, comprising two phylogenetically conserved proteins, and small Hsps are more abundant during heat stress. The contribution of the Hsp100 (ClpB) family of proteins and of small Hsps in the unicellular cyanobacteria (Synechocystis and Synechococcus) as well as that of Hsp60 proteins in the filamentous cyanobacteria (Anabaena) to thermotolerance has been elucidated. The regulation of chaperone genes by several cis-elements and trans-acting factors has also been well documented. Recent studies have demonstrated novel transcriptional and translational (mRNA secondary structure) regulatory mechanisms in unicellular cyanobacteria. This article provides an insight into the heat-shock response: its organization, and ecophysiological regulation and role of molecular chaperones, in unicellular and filamentous nitrogen-fixing cyanobacterial strains.
PloS one, 2016
We previously reported the in silico characterization of Synechococcus sp. phage 18 kDa small heat shock protein (HspSP-ShM2). This small heat shock protein (sHSP) contains a highly conserved core alpha crystalline domain of 92 amino acids and relatively short N- and C-terminal arms, the later containing the classical C-terminal anchoring module motif (L-X-I/L/V). Here we establish the oligomeric profile of HspSP-ShM2 and its structural dynamics under in vitro experimental conditions using size exclusion chromatography (SEC/FPLC), gradient native gels electrophoresis and dynamic light scattering (DLS). Under native conditions, HspSP-ShM2 displays the ability to form large oligomers and shows a polydisperse profile. At higher temperatures, it shows extensive structural dynamics and undergoes conformational changes through an increased of subunit rearrangement and formation of sub-oligomeric species. We also demonstrate its capacity to prevent the aggregation of citrate synthase, mala...
Current Microbiology, 1998
The low molecular weight (LMW) heat shock protein (HSP) gene hsp16.6 was identified and cloned from the unicellular cyanobacterium Synechocystis sp. PCC 6803 through comparisons of genomic sequences and conserved gene sequences of the LMW HSPs. Hsp16.6 was isolated using PCR and cloned into the pGEMT plasmid. Hsp16.6 showed a significant increase in transcription after heat shock at 42°C that indicated hsp16.6 was a heat shock gene. To determine the role that hsp16.6 plays in the heat shock response, a mutant Synechocystis cell line was generated. Cell growth and oxygen evolution rates of wild type and mutant cells were compared after heat shock. Results showed significantly decreased cell growth rates and a 40% reduction in oxygen evolution rates in mutants after heat shock treatments. These data indicate a protective role for hsp16.6 in the heat shock response.
Investigation of the chaperone function of the small heat shock protein — AgsA
BMC Biochemistry, 2010
Background: A small heat shock protein AgsA was originally isolated from Salmonella enterica serovar Typhimurium. We previously demonstrated that AgsA was an effective chaperone that could reduce the amount of heataggregated proteins in an Escherichia coli rpoH mutant. AgsA appeared to promote survival at lethal temperatures by cooperating with other chaperones in vivo. To investigate the aggregation prevention mechanisms of AgsA, we constructed N-or C-terminal truncated mutants and compared their properties with wild type AgsA. Results: AgsA showed significant overall homology to wheat sHsp16.9 allowing its three-dimensional structure to be predicted. Truncations of AgsA until the N-terminal 23 rd and C-terminal 11 th amino acid (AA) from both termini preserved its in vivo chaperone activity. Temperature-controlled gel filtration chromatography showed that purified AgsA could maintain large oligomeric complexes up to 50°C. Destabilization of oligomeric complexes was observed for N-terminal 11-and 17-AA truncated AgsA; C-terminal 11-AA truncated AgsA could not form large oligomeric complexes. AgsA prevented the aggregation of denatured lysozyme, malate dehydrogenase (MDH) and citrate synthase (CS) but did not prevent the aggregation of insulin at 25°C. N-terminal 17-AA truncated AgsA showed no chaperone activity towards MDH. C-terminal 11-AA truncated AgsA showed weak or no chaperone activity towards lysozyme, MDH and CS although it prevented the aggregation of insulin at 25°C. When the same amount of AgsA and C-terminal 11-AA truncated AgsA were mixed (half of respective amount required for efficient chaperone activities), good chaperone activity for all substrates and temperatures was observed. Detectable intermolecular exchanges between AgsA oligomers at 25°C were not observed using fluorescence resonance energy transfer analysis; however, significant exchanges between AgsA oligomers and C-terminal truncated AgsA were observed at 25°C.
Cell Stress and Chaperones, 2012
Small heat shock proteins are ubiquitous in all three domains (Archaea, Bacteria and Eukarya) and possess molecular chaperone activity by binding to unfolded polypeptides and preventing aggregation of proteins in vitro. The functions of a small heat shock protein (S.so-HSP20) from the hyperthermophilic archaeon, Sulfolobus solfataricus P2 have not been described. In the present study, we used real-time polymerase chain reaction analysis to measure mRNA expression of S.so-HSP20 in S. solfataricus P2 and found that it was induced by temperatures that were substantially lower (60°C) or higher (80°C) than the optimal temperature for S. solfataricus P2 (75°C). The expression of S.so-HSP20 mRNA was also up-regulated by cold shock (4°C). Escherichia coli cells expressing S.so-HSP20 showed greater thermotolerance in response to temperature shock (50°C, 4°C). By assaying enzyme activities, S.so-HSP20 was found to promote the proper folding of thermodenatured citrate synthase and insulin B chain. These results suggest that S.so-HSP20 promotes thermotolerance and engages in chaperone-like activity during the stress response.