Distinct amino acids of  the Oenococcus oeni small heat shock protein Lo18 are essential for damaged protein protection and membrane stabilization

Abstract

The small heat shock protein (smHsp) Lo18 from lactic acid bacteria Oenococcus oeni reduces in vitro thermal aggregation of proteins and modulates the membrane fluidity of native liposomes. An absence of information relating to the way in which the smHsp demonstrates a stabilizing effect for both proteins and membranes prompted this study. We expressed three Lo18 proteins with amino acid substitutions in Escherichia coli to investigate their ability to prevent E. coli protein aggregation and their capacity to stabilize E. coli whole-cell membranes. Our results showed that the alanine 123 to serine substitution induces a decrease in chaperone activity in denaturated proteins, and that the tyrosine 107 is required for membrane stabilization. Moreover, this study revealed that the oligomeric structures of proteins with amino acid substitutions do not appear to be modified. Our data strongly suggest that different amino acids are involved in the thermostabilization of proteins and in membrane fluidity regulation and are localized in the alpha-crystallin domain.

Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

“…One strategy involves the induction of the expression of the hsp18 gene, encoding the sHsp Lo18. Lo18 was previously shown to participate in the modulation of membrane and liposome fluidity in response to stress (17,26,28), thus protecting membranes from the deleterious effects of ethanol. To characterize further the interaction between Lo18 and the O. oeni membrane, we used two types of liposomes prepared from O. oeni grown under optimal growth conditions or grown in 8% ethanol.…”

“…Cells were harvested in the exponential growth phase (A 600 of 1.5; 1 absorbance unit corresponds to 6 ϫ 10 8 CFU · ml Ϫ1 ). Lo18 was produced in Escherichia coli BL21 Star(DE3) containing the plasmid pET28a-hsp18 (28). E. coli cells were grown under aerobic conditions at 37°C in LB broth supplemented with kanamycin (50 g · ml Ϫ1 ).…”

“…We previously showed that the role of Lo18 in both membrane stabilization and protection of proteins from aggregation is dependent on its oligomer plasticity (17,28,29). However, the identity of the oligomeric form active in membrane stabilization remains elusive.…”

“…As it is not yet possible to construct the appropriate mutants in O. oeni or to overexpress the proteins to verify their in vivo function, many studies have been performed with heterologous models (27,28) or in vitro with purified proteins (17). In vitro experiments with purified Lo18 have characterized both its molecular chaperone (protection of proteins from aggregation) and lipochaperone (stabilization of liposome fluidity) activities (17,26,29).…”

The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium

“…Several studies have investigated Lo18 function in vitro using either heterologous (11,16) or in vitro (17) systems. Under stress conditions, Lo18 is produced in large amounts (10), notably during ethanol stress (5,10).…”