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In Vitro Structural and Functional Characterization of the Small Heat Shock Proteins (sHSP) of the Cyanophage S-ShM2 and Its Host, Synechococcus sp. WH7803.

ABSTRACT: 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, malate dehydrogenase and luciferase under heat shock conditions through the formation of stable and soluble hetero-oligomeric complexes (sHSP:substrate). In contrast, the host cyanobacteria Synechococcus sp. WH7803 15 kDa sHSP (HspS-WH7803) aggregates when in the same conditions as HspSP-ShM2. However, its solubility can be maintained in the presence of non-ionic detergent Triton™X-100 and forms an oligomeric structure estimated to be between dimer and tetramer but exhibits no apparent inducible structural dynamics neither chaperon-like activity in all the assays and molar ratios tested. SEC/FPLC and thermal aggregation prevention assays results indicate no formation of hetero-oligomeric complex or functional interactions between both sHSPs. Taken together these in vitro results portray the phage HspSP-ShM2 as a classical sHSP and suggest that it may be functional at the in vivo level while behaving differently than its host amphitropic sHSP.

SUBMITTER: Bourrelle-Langlois M

PROVIDER: S-EPMC5028025 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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In Vitro Structural and Functional Characterization of the Small Heat Shock Proteins (sHSP) of the Cyanophage S-ShM2 and Its Host, Synechococcus sp. WH7803.

Bourrelle-Langlois Maxime M Morrow Geneviève G Finet Stéphanie S Tanguay Robert M RM

PloS one 20160919 9

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 ...[more]

PMID: 27643500

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Project description:BackgroundIn living organisms, small heat shock proteins (sHSPs) are triggered in response to stress situations. This family of proteins is large in plants and, in the case of tomato (Solanum lycopersicum), 33 genes have been identified, most of them related to heat stress response and to the ripening process. Transcriptomic and proteomic studies have revealed complex patterns of expression for these genes. In this work, we investigate the coregulation of these genes by performing a computational analysis of their promoter architecture to find regulatory motifs known as heat shock elements (HSEs). We leverage the presence of sHSP members that originated from tandem duplication events and analyze the promoter architecture diversity of the whole sHSP family, focusing on the identification of HSEs.ResultsWe performed a search for conserved genomic sequences in the promoter regions of the sHSPs of tomato, plus several other proteins (mainly HSPs) that are functionally related to heat stress situations or to ripening. Several computational analyses were performed to build multiple sequence motifs and identify transcription factor binding sites (TFBS) homologous to HSF1AE and HSF21 in Arabidopsis. We also investigated the expression and interaction of these proteins under two heat stress situations in whole tomato plants and in protoplast cells, both in the presence and in the absence of heat shock transcription factor A2 (HsfA2). The results of these analyses indicate that different sHSPs are up-regulated depending on the activation or repression of HsfA2, a key regulator of HSPs. Further, the analysis of protein-protein interaction between the sHSP protein family and other heat shock response proteins (Hsp70, Hsp90 and MBF1c) suggests that several sHSPs are mediating alternative stress response through a regulatory subnetwork that is not dependent on HsfA2.ConclusionsOverall, this study identifies two regulatory motifs (HSF1AE and HSF21) associated with the sHSP family in tomato which are considered genomic HSEs. The study also suggests that, despite the apparent redundancy of these proteins, which has been linked to gene duplication, tomato sHSPs showed different up-regulation and different interaction patterns when analyzed under different stress situations.

Project description:The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.

Dataset Information

In Vitro Structural and Functional Characterization of the Small Heat Shock Proteins (sHSP) of the Cyanophage S-ShM2 and Its Host, Synechococcus sp. WH7803.

Publications

In Vitro Structural and Functional Characterization of the Small Heat Shock Proteins (sHSP) of the Cyanophage S-ShM2 and Its Host, Synechococcus sp. WH7803.

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