Presentation Title

Heat-shock triggers the binding between HspA1A, a 70-kDA molecular chaperone, and phosphatidylserine in human cells.

Start Date

November 2016

End Date

November 2016

Location

HUB 302-53

Type of Presentation

Poster

Abstract

HspA1A is a seventy-kilodalton heat shock protein (Hsp70) and is essential for maintaining cellular homeostasis. Apart from its indispensable roles in protein homeostasis HspA1A also localizes at the plasma membrane and binds to several lipids, including phosphatidylserine (PS). The interaction of HspA1A with PS has direct physiological outcomes including activation of the immune system and regulation of cell death. However, the conditions that promote the interaction between HspA1A and PS remain unknown. To shed light to this newly described property of HspA1A, we tested whether heat-shock, a stress known to affect both HspA1A expression and membrane lipid composition, triggers the binding of HspA1A to PS. To test this prediction, we first determined whether HspA1A and the C2 domain of lactadherin (Lact-C2), a known PS-biosensor, compete for binding to intracellular PS. Specifically, HeLa cells were heat-shocked and the competition was assessed by quantifying the amount of membrane localized HspA1A in the presence or absence of Lact-C2. These imaging experiments revealed that in the absence of Lact-C2 HspA1A’s membrane localization increases continuously after heat-shock and reaches a maximal value at 8 hours during recovery. In the presence of Lact-C2, however, HspA1A’s membrane localization was minimal and did not increase. These results were verified using cell surface biotinylation. In these experiments, HeLa cells, after heat-shock were incubated with membrane impermeable biotin, lysed, and incubated with streptavidin agarose beads. Then the biotinylated proteins were analyzed by western blot and quantified using densitometry. Collectively, these results strongly suggest that HspA1A bind to intracellular PS, and that HspA1A’s membrane localization and anchorage depends on its interaction with PS. This discovery institutes PS as a new and dynamic partner in the cellular stress response and establish the required foundation to directly assess the biological implications of this newly described and largely uncharacterized function of Hsp70s.

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Heat-shock triggers the binding between HspA1A, a 70-kDA molecular chaperone, and phosphatidylserine in human cells.

HUB 302-53

HspA1A is a seventy-kilodalton heat shock protein (Hsp70) and is essential for maintaining cellular homeostasis. Apart from its indispensable roles in protein homeostasis HspA1A also localizes at the plasma membrane and binds to several lipids, including phosphatidylserine (PS). The interaction of HspA1A with PS has direct physiological outcomes including activation of the immune system and regulation of cell death. However, the conditions that promote the interaction between HspA1A and PS remain unknown. To shed light to this newly described property of HspA1A, we tested whether heat-shock, a stress known to affect both HspA1A expression and membrane lipid composition, triggers the binding of HspA1A to PS. To test this prediction, we first determined whether HspA1A and the C2 domain of lactadherin (Lact-C2), a known PS-biosensor, compete for binding to intracellular PS. Specifically, HeLa cells were heat-shocked and the competition was assessed by quantifying the amount of membrane localized HspA1A in the presence or absence of Lact-C2. These imaging experiments revealed that in the absence of Lact-C2 HspA1A’s membrane localization increases continuously after heat-shock and reaches a maximal value at 8 hours during recovery. In the presence of Lact-C2, however, HspA1A’s membrane localization was minimal and did not increase. These results were verified using cell surface biotinylation. In these experiments, HeLa cells, after heat-shock were incubated with membrane impermeable biotin, lysed, and incubated with streptavidin agarose beads. Then the biotinylated proteins were analyzed by western blot and quantified using densitometry. Collectively, these results strongly suggest that HspA1A bind to intracellular PS, and that HspA1A’s membrane localization and anchorage depends on its interaction with PS. This discovery institutes PS as a new and dynamic partner in the cellular stress response and establish the required foundation to directly assess the biological implications of this newly described and largely uncharacterized function of Hsp70s.