Abstract
26S proteasome is a macromolecular multi-subunit complex responsible for recognizing, unfolding, and ultimately destroying proteins. It remains poorly understood how 26S proteasome activity is regulated. The present study was to investigate if AMP-activated protein kinase (AMPK) functions as a physiological suppressor of the 26S proteasome in endothelial cells. 26S proteasome assembly, activity, and O-GlcNAcylation of P700 were assayed in cultured human umbilical vein endothelial cells (HUVEC) and mouse aortas isolated from C57BL6 wild type and AMPKα2 knockout mice with or without being exposed to selective AMPK activators or inhibitors. Pharmacological and genetic activation of AMPK effectively suppresses 26S proteasomes in endothelial cells. Conversely, inactivation of AMPK either pharmacologically or genetically increases 26S proteasome activity; furthermore, the inactivation decreases the O-GlcNAcylation of PA700/S10B (the regulatory complex in 26S proteasomes) and increases the assembly of 26S proteasomes. In contrast, AMPK activation increases levels of O-GlcNAcylated PA700/S10B, likely through enhanced association of PA700 with O-GlcNAc transferase (OGT), the enzyme that catalyzes protein O-GlcNAcylation. Finally, aortas from AMPK-KO vs wild type mice exhibit elevated 26S proteasome activity in parallel with decreased PA700/S10B O-GlcNAcylation and PA700/S10B-OGT association. Taken together, we conclude that AMPK functions as a physiological suppressor of 26S proteasomes.
Highlights
The ubiquitin proteasome system (UPS) is the major nonlysosomal degradative machinery for most intracellular proteins [1,2]
The present study has demonstrated that inactivation of AMPK elevates 26S proteasome activity and that this effect is associated with decreased PA700/S10B O-GlcNAcylation and increased 26S proteasome assembly
O-GlcNAc transferase (OGT) appears to be crucial for this event, as AMPK-mediated 26S proteasome inhibition is blocked by siRNA-mediated OGT knockdown
Summary
The ubiquitin proteasome system (UPS) is the major nonlysosomal degradative machinery for most intracellular proteins [1,2]. A key component of this machinery is the 26S proteasome [3], a macromolecular multi-subunit complex that is responsible for recognizing, unfolding, and destroying proteins. The 20S proteasome is a cylindrical protease complex consisting of 28 subunits configured into four stacks of heptameric rings. The 26S proteasome is known to require ATP hydrolysis to degrade ubiquitinated substrates and for its assembly [7]. The UPS is recognized as a regulator of the cell cycle and cell division [8,9], immune responses and antigen presentation [10,11], apoptosis [12], and cell signaling [13,14]. The UPS has been shown to be either activated in certain cancers (e.g., multiple myeloma) [15,16] or dysfunctional in neurodegenerative disorders (e.g., Alzheimer’s disease, Huntington’s disease [17], and amyotrophic lateral sclerosis [18,19])
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