18 research outputs found
Joint Loop End Modeling Improves Covariance Model Based Non-coding RNA Gene Search
The effect of more detailed modeling of the interface between stem and loop in non-coding RNA hairpin structures on efficacy of covariance-model-based non-coding RNA gene search is examined. Currently, the prior probabilities of the two stem nucleotides and two loop-end nucleotides at the interface are treated the same as any other stem and loop nucleotides respectively. Laboratory thermodynamic studies show that hairpin stability is dependent on the identities of these four nucleotides, but this is not taken into account in current covariance models. It is shown that separate estimation of emission priors for these nucleotides and joint treatment of substitution probabilities for the two loop-end nucleotides leads to improved non-coding RNA gene search
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Mutants in a small heat shock proteins that affect the oligomeric state: analysis and allele specific suppression.
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Mutants in a Small Heat Shock Protein That Affect the Oligomeric State ANALYSIS AND ALLELE-SPECIFIC SUPPRESSION*
Kim C. Giese and Elizabeth Vierling‡
+ Author Affiliations
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721
‡ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, University of Arizona, 1007 E. Lowell St., Tucson, AZ 85721. Tel.: 520-621-1601; Fax: 520-621-3709; E-mail: [email protected].
Next Section Abstract
Oligomerization is an essential property of small heat shock proteins (sHSPs) that appears to regulate their chaperone activity. We have examined the role of conserved hydrophobic residues that are postulated to stabilize sHSP oligomers. We identified a mutation of Synechocystis Hsp16.6 that impairs function in vivo and in vitro. The V143A mutation is in the C-terminal extension, a region predicted to form an oligomeric interaction with a hydrophobic region that includes the site of a previously characterized mutation, L66A. Both mutants were dimeric, but V143A had a stronger oligomerization defect than L66A. However, V143A protected a model substrate better than L66A. This suggests that although the two regions both play a role in oligomerization, they are not equivalent. Nevertheless, the addition of either dimeric sHSP enhanced the in vitro chaperone activity of wild type Hsp16.6, consistent with models that the sHSP dimers initiate interactions with substrates. Suppressor analysis of V143A identified mutations in the N terminus that restored activity by restabilizing the oligomer. These mutants were allele-specific and unable to suppress L66A, although they suppressed a dimeric C-terminal truncation of Hsp16.6. Conversely, suppressors of L66A were unable to suppress either V143A or the truncation, although they, like suppressors of V143A, stabilize the Hsp16.6 oligomer. We interpret these data as evidence that the mutations V143A and L66A stabilize two different dimeric structures and as further support that sHSP dimers are active species. Previous SectionNext Section
Small heat shock proteins (sHSPs)1 are a family of ATP-independent chaperones that, in vitro, can protect denaturing proteins from irreversible aggregation by forming large, soluble complexes with their substrates (for reviews, see Refs. 1-3). sHSPs do not refold substrates, but sHSP-protected substrates can be refolded by ATP-dependent chaperones such as Hsp70/DnaK (4, 5). Although the role of sHSPs in vivo is not well defined, established in vitro assays for sHSP activity (i.e. protection of model substrates from aggregation and their subsequent refolding by ATP-dependent chaperones) appear to be closely related to sHSP functions in vivo. In Synechocystis sp. strain PCC 6803 (Synechocystis), a missense mutation of the single sHSP in this organism, Hsp16.6, causes loss of both protection of a model substrate in vitro and the ability of cells to survive heat treatment (thermotolerance). Both activities are restored by intragenic suppressors of this mutant (6). Mogk et al. (7) demonstrated that the sHSPs in Escherichia coli act with both DnaK and ClpB to facilitate the removal of aggregated protein after heat stress, in good agreement with in vitro observations of Hsp70/DnaK-dependent reactivation of sHSP-protected substrates (8, 9). Thus, it appears that sHSPs function to prevent or reduce irreversible protein aggregation during heat stress in vivo.
The mechanism of the chaperone activity of sHSPs is unknown. Current models suggest that changes in sHSP oligomerization are essential. Almost all sHSPs form oligomers (2), and these can be destabilized at high temperatures (10, 11). Disassembly of the oligomer into dimers has been proposed to be a required step in the chaperone process (10). Disassembly could produce an active state of the sHSP that can bind unfolding proteins (10), perhaps by exposure of hydrophobic residues that bind substrates (5, 11, 12). However, the oligomeric state is also essential, as it appears that reassembly of sHSP dimers into a complex with substrate is required for protection of substrates (6, 13-16). Therefore, identification of contacts involved in oligomerization is important for understanding the mechanism of sHSP chaperone activity.
The crystal structures of two sHSPs provide information about regions involved in oligomerization. The structures of Methanococcus jannaschii Hsp16.5 (17) and Triticum aestivum Hsp16.9 from wheat (11) reveal that despite the difference in the size of their oligomers (24 and 12 subunits, respectively), there is a high degree of similarity between the two sHSPs. Both proteins form oligomers that are composed of dimers, and the conserved ∼100 amino acid α-crystallin domains (18) have very similar β-sandwich structures. Oligomerization of M. jannaschii Hsp16.5 and T. aestivum Hsp16.9 involves a conserved interdimer interface between two hydrophobic regions: a “patch” of hydrophobicity on one surface of the β-sandwich of the α-crystallin domain and the hydrophobic residues in a basic-X(I/V)X(I/V) motif of the C-terminal extension (β-strand 10). The conservation of these residues among sHSPs (18) is consistent with essential functions of these regions.
The hypothesis that interactions between the C-terminal extension and the hydrophobic patch are essential to sHSP oligomerization and function is supported by some, but not all, mutagenesis experiments in vitro and has not been tested in vivo. Mutation of either Ile residue to Ala in the basic-X(I/V)X(I/V) motif of HspH from Bradyrhizobium japonicum destabilized the oligomer and led to loss of protection of citrate synthase from aggregation (15). A truncation of the C terminus that deleted this motif also reduced the oligomerization of M. jannaschii Hsp16.5 (19) but not of Hsp16.2 from Caenorhabditis elegans (13). Furthermore, while Kim et al. (19) found that the C-terminal truncation of M. jannaschii Hsp16.5 led to formation of a tetramer, it was still able to maintain substrate solubility. The role of the hydrophobic patch is even less well examined. We identified a mutation of a conserved hydrophobic residue in the patch, L66A of Synechocystis Hsp16.6, that severely reduced both oligomeric stability and chaperone activity (6), but this region has not been investigated in other sHSPs. Although on the whole, these studies argue for a role of the hydrophobic C-terminal motif and patch in both oligomerization and chaperone activity, the requirements for either are not certain. Furthermore, these studies have primarily assayed sHSP activity using model substrates in vitro, making it possible that the results do not reflect cellular needs for sHSP activity.
We took advantage of the requirement of Hsp16.6 for thermotolerance in Synechocystis to dissect genetically the role of the C-terminal motif and to compare it with that of the hydrophobic patch. The relationship of in vivo phenotypes to oligomeric stability and chaperone activity was also compared. Point mutants at Val143, the second hydrophobic residue in the basic-X(I/V)X(I/V) motif of Hsp16.6, decreased the viability of heat-stressed cells, providing the first direct evidence for the importance of this motif in vivo. The V143A protein was dimeric and had impaired chaperone activity in vitro. V143A disrupted oligomeric stability more, but chaperone activity less, than L66A, a mutation in the hydrophobic patch, suggesting the two hydrophobic regions do not have equivalent functions. Intragenic suppressors of V143A were then selected in Synechocystis as second-site mutations that restored cellular thermotolerance. These suppressors were located in the N terminus and greatly stabilized the Hsp16.6 oligomer. Contrary to expectations, the suppressors of V143A were not able to suppress L66A. Likewise, suppressors of L66A, which also stabilize the Hsp16.6 oligomer (6), did not suppress V143A. In total, although both V143A and L66A form stable dimers, there are structural and/or functional differences between the two mutants. We suggest that they may stabilize two distinct functional states of Hsp16.6
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Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro.
The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacterium Synechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystis depends on HSP16.6, a phenotype that is enhanced in a deltaClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligomerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restore in vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance
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Interactions between small heat shock protein subunits and substrate in small heat shock protein/substrate complexes.
Small heat shock proteins (sHSPs) are dynamic oligomeric proteins that bind unfolding proteins and protect them from irreversible aggregation. This binding results in the formation of sHSP-substrate complexes from which substrate can later be refolded. Interactions between sHSP and substrate in sHSP-substrate complexes and the mechanism by which substrate is transferred to ATP-dependent chaperones for refolding are poorly defined. We have established C-terminal affinity-tagged sHSPs from a eukaryote (pea HSP18.1) and a prokaryote (Synechocystis HSP16.6) as tools to investigate these issues. We demonstrate that sHSP subunit exchange for HSP18.1 and HSP16.6 is temperature-dependent and rapid at the optimal growth temperature for the organism of origin. Increasing the ratio of sHSP to substrate during substrate denaturation decreased sHSP-substrate complex size, and accordingly, addition of substrate to pre-formed sHSP-substrate complexes increased complex size. However, the size of pre-formed sHSP-substrate complexes could not be reduced by addition of more sHSP, and substrate could not be observed to transfer to added sHSP, although added sHSP subunits continued to exchange with subunits in sHSP-substrate complexes. Thus, although some number of sHSP subunits within complexes remain dynamic and may be important for complex structure/solubility, association of substrate with the sHSP does not appear to be similarly dynamic. These observations are consistent with a model in which ATP-dependent chaperones associate directly with sHSP-bound substrate to initiate refolding
Are there links between genes for phosphate uptake and quantitative trait loci for yield in barley?
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The identity of proteins associated with a small heat shock protein during heat stress in vivo indicates these chaperones protect a wide range of cellular functions.
The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and co-chaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function
The impact of solar ultraviolet radiation upon the skin of rainbow trout, Salmo gairdneri Richardson, farmed at high altitude in Bolivia
A successive SDP-NSDP approach to a robust optimization problem in finance
Robust optimization, Nonlinear semidefinite programming, Static hedging, Barrier options, Eigenvalue minimization,