PA28γ–20S proteasome is a proteolytic complex committed to degrade unfolded proteins

Abstract

PA28γ is a nuclear activator of the 20S proteasome that, unlike the 19S regulatory particle, stimulates hydrolysis of several substrates in an ATP-and ubiquitin-independent manner and whose exact biological functions and molecular mechanism of action still remain elusive. In an effort to shed light on these important issues, we investigated the stimulatory effect of PA28γ on the hydrolysis of different fluorogenic peptides and folded or denatured full-length proteins by the 20S proteasome. Importantly, PA28γ was found to dramatically enhance breakdown rates by 20S proteasomes of several naturally or artificially unstructured proteins, but not of their native, folded counterparts. Furthermore, these data were corroborated by experiments in cell lines with a nucleus-tagged myelin basic protein. Finally, mass spectrometry analysis of the products generated during proteasomal degradation of two proteins demonstrated that PA28γ does not increase, but rather decreases, the variability of peptides that are potentially suitable for MHC class I antigen presentation. These unexpected findings indicate that global stimulation of the degradation of unfolded proteins may represent a more general feature of PA28γ and suggests that this proteasomal activator might play a broader role in the pathway of protein degradation than previously believed.

Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes

“…In vitro, PA28γ preferentially promotes the degradation of unstructured proteins over their native/folded counterparts, including unfolded variants of β-casein and insulin-like growth factor 1 (IGF-1) 40 . Moreover, in vitro experiments demonstrated that PA28γ promotes the degradation of peptides containing expanded polyglutamine repeats (polyQ) 41 , which are linked with different human diseases.…”

Proteasome activator 28γ (PA28γ) allosterically activates trypsin-like proteolysis by binding to the α-ring of the 20S proteasome

“…The PA28γ-20S complex has recently been demonstrated to degrade unfolded proteins ( 29 ) and is known to play a role in nuclear processes such as: cell cycle progression and cell proliferation ( 44 , 48 ), apoptosis ( 48 ), formation of nuclear speckles ( 49 ), and DNA repair ( 50 ). Within these processes, PA28γ-20S complex has also been demonstrated to facilitate the degradation of many nuclear proteins, such as SRC-3/AIB1 ( 51 ), c-Myc ( 52 ), p21 ( 53 ), hepatitis C virus ( 54 ), and MDM2 ( 55 ).…”

“…used ammonium sulfate precipitation to purify recombinant PA28γ and concluded that it had 20S gate-opening activity and did not observe activation of a specific proteolytic site ( 27 ). Two other groups used a different purification strategy of recombinant PA28γ and also observed gate-opening 20S activity ( 28 , 29 ). Alternatively, recombinant PA28γ classically purified using ion-exchange and size-exclusion chromatography, N-terminally His-tagged recombinant PA28γ purified using NiNTA resin, and endogenous and transiently overexpressed N-terminally FLAG-tagged PA28γ from cell lines indicates that PA28γ specifically upregulates the cleavage of T-L peptides and does not demonstrate 20S gate opening activity ( 10 , 30 ).…”

Structure, Function, and Allosteric Regulation of the 20S Proteasome by the 11S/PA28 Family of Proteasome Activators

“…Enigmatically, alterations such as a single-point mutation and changes in purification strategies have been found to shift PA28γ’s function from a trypsin-like activator to a gate-opener [ 44 , 46 , 47 ]. In a physiological context, PA28γ has been shown to promote the degradation of unstructured proteins via the 20S CP [ 48 ], though the mechanism is yet to be fully elucidated. Gaining insights into these biochemical mechanisms is crucial, as PA28γ expression has been associated with certain disease states, a topic detailed thoroughly in the discussion of Stadtmueller and Hill, 2011 [ 42 ].…”