Dataset Information

Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine.

ABSTRACT: Many organisms can adapt to changes in the solute content of their surroundings (i.e., the osmolarity). Hyperosmotic shock causes water efflux and a concomitant reduction in cell volume, which is countered by the accumulation of osmolytes. This volume reduction increases the crowded nature of the cytoplasm, which is expected to affect protein stability. In contrast to traditional theory, which predicts that more crowded conditions can only increase protein stability, recent work shows that crowding can destabilize proteins through transient attractive interactions. Here, we quantify protein stability in living Escherichia coli cells before and after hyperosmotic shock in the presence and absence of the osmolyte, glycine betaine. The 7-kDa N-terminal src-homology 3 domain of Drosophila signal transduction protein drk is used as the test protein. We find that hyperosmotic shock decreases SH3 stability in cells, consistent with the idea that transient attractive interactions are important under physiologically relevant crowded conditions. The subsequent uptake of glycine betaine returns SH3 to the stability observed without osmotic shock. These results highlight the effect of transient attractive interactions on protein stability in cells and provide a new explanation for why stressed cells accumulate osmolytes.

SUBMITTER: Stadmiller SS

PROVIDER: S-EPMC5985519 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine.

Stadmiller Samantha S SS Gorensek-Benitez Annelise H AH Guseman Alex J AJ Pielak Gary J GJ

Journal of molecular biology 20170303 8

Many organisms can adapt to changes in the solute content of their surroundings (i.e., the osmolarity). Hyperosmotic shock causes water efflux and a concomitant reduction in cell volume, which is countered by the accumulation of osmolytes. This volume reduction increases the crowded nature of the cytoplasm, which is expected to affect protein stability. In contrast to traditional theory, which predicts that more crowded conditions can only increase protein stability, recent work shows that crowd ...[more]

PMID: 28263768

Similar Datasets

Project description:Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.

Project description:Lead (Pb) is a widespread and nondegradable environmental pollutant and affects several organs through oxidative mechanisms. This study was conducted to investigate the antioxidant protective effect of glycine betaine (GB) against Pb-induced renal and hepatic injury. Male albino rats (n = 45) were divided into three groups: G1 untreated control, G2 Pb-acetate (50 mg/kg/day), and G3 Pb-acetate (50 mg/kg/day) plus GB (250 mg/kg/day) administered for 6 weeks. For G3, Pb-acetate was administered first and followed by GB at least 4 h after. Pb-acetate treatment (G2) resulted in a significant decrease in renal function, including elevated creatinine and urea levels by 17.4% and 23.7%, respectively, and nonsignificant changes in serum uric acid levels. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphates (ALP) activities were significantly increased with Pb treatment by 37.6%, 59.3%, and 55.1%, respectively. Lipid peroxidation level was significantly increased by 7.8 times after 6 weeks of Pb-acetate treatment. The level of reduced glutathione (GSH-R) significantly declined after Pb-acetate treatment. Pb-acetate treatment also reduced the activities of superoxide dismutase (SOD), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-PX) by 74.1%, 85.0%, and 40.8%, respectively. Treatment of Pb-intoxicated rats with GB resulted in a significant reduction in creatinine, urea, ALT, AST, and lipid peroxidation, as well as a significant increase in the level of GSH-R and in the activities of ALP, SOD, GST, and GSH-PX. The molecular interaction between GB and GSH-PX indicated that the activation of GSH-PX in Pb-intoxicated rats was not the result of GB binding to the catalytic site of GSH-PX. The affinity of GB to bind to the catalytic site of GSH-PX is lower than that of H2O2. Thus, GB significantly mitigates Pb-induced renal and liver injury through the activation of antioxidant enzymes and the prevention of Pb-induced oxidative damage in the kidney and liver.

Project description:Bacillus subtilis adjusts to high osmolarity surroundings through the amassing of compatible solutes. It synthesizes the compatible solute glycine betaine from prior imported choline and scavenges many pre-formed osmostress protectants, including glycine betaine, from environmental sources. Choline is imported through the substrate-restricted ABC transporter OpuB and the closely related, but promiscuous, OpuC system, followed by its GbsAB-mediated oxidation to glycine betaine. We have investigated the impact of two MarR-type regulators, GbsR and OpcR, on gbsAB, opuB, and opuC expression. Judging by the position of the previously identified OpcR operator in the regulatory regions of opuB and opuC [Lee et al. (2013) Microbiology 159, 2087-2096], and that of the GbsR operator identified in the current study, we found that the closely related GbsR and OpcR repressors use different molecular mechanisms to control transcription. OpcR functions by sterically hindering access of RNA-polymerase to the opuB and opuC promoters, while GbsR operates through a roadblock mechanism to control gbsAB and opuB transcription. Loss of GbsR or OpcR de-represses opuB and opuC transcription, respectively. With respect to the osmotic control of opuB and opuC expression, we found that this environmental cue operates independently of the OpcR and GbsR regulators. When assessed over a wide range of salinities, opuB and opuC exhibit a surprisingly different transcriptional profile. Expression of opuB increases monotonously in response to incrementally increase in salinity, while opuC transcription levels decrease after an initial up-regulation at moderate salinities. Transcription of the gbsR and opcR regulatory genes is up-regulated in response to salt stress, and is also affected through auto-regulatory processes. The opuB and opuC operons have evolved through a gene duplication event. However, evolution has shaped their mode of genetic regulation, their osmotic-stress dependent transcriptional profile, and the substrate specificity of the OpuB and OpuC ABC transporters in a distinctive fashion.

Dataset Information

Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine.

Publications

Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets