Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation

Abstract

Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbRproteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a gen- Proteasomes are multisubunit protein complexes that are responsible for the degradation of ubiquitinated substrates to maintain cell viability and homeostasis. The 26S proteasome is composed of at least 33 subunits (1-3), which can be divided into two subcomplexes: the 20S catalytic core particle (CP) 1 and the 19S regulatory particle (RP). The 20S CP is responsible for various proteolytic activities, and has a highly conserved "barrel"-like structure consisting of two copies each of 14 nonidentical subunits (␣1-7, ␤1-7), which are arranged into four heptameric rings stacked in the order of ␣ 7 ␤ 7 ␤ 7 ␣ 7 (4, 5). The 19S RP intimately interacts with the 20S CP, regulating its activity. In addition, the 19S RP carries diverse functions including substrate recognition and deubiquitination, protein unfolding, and substrate translocation to the 20S CP for degradation (2, 3, 6 -8). In contrast to the highly ordered and stable structure of the 20S CP, the 19S RP appears to be much more flexible and dynamic (3, 9 -11). Current structural analyses have revealed that six Rpt subunits of the 19S RP form a hexameric AAA-ATPase ring to associate with the cylinder ends of the 20S CP, and are surrounded by a shell of Rpn subunits (9 -11). Apart from the

Structures of Rpn1 T1:Rad23 and hRpn13:hPLIC2 Reveal Distinct Binding Mechanisms between Substrate Receptors and Shuttle Factors of the Proteasome

“…It is conceivable that strengthening the affinity of Rpn1 or Rpn13 for ubiquitin itself may interfere with subsequent release of ubiquitin chains following their cleavage from substrate. hHR23b and hPLIC2 co-purify with proteasomes from mammalian cells (Besche et al, 2009; Hiyama et al, 1999; Kleijnen et al, 2003; Kleijnen et al, 2000; Wang et al, 2007; Wang and Huang, 2008; Yu et al, 2016). Thus, their affinity for the proteasome receptors apparently leads to a certain level of basal occupancy of these shuttle factors at the proteasome.…”

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…Over the years, we have developed several new HB (i.e. Histidine-Biotin)-tag based affinity strategies to effectively isolate protein complexes from human cells for XL-MS studies 79,99,152,153 . Similarly, we have also employed on-bead cross-linking and digestion of HB-tagged proteasome complexes, which was proven robust and effective with single-step Streptavidin purification 98,99 .…”

“…Recently, we have developed two affinity purification strategies, namely XAP ( in vivo cross-linking (X) assisted Affinity Purification) 98 and XBAP ( in vivo cross-linking (X) assisted Bimolecular tandem Affinity Purification) 153 to study dynamic interactors of protein complexes and subcomplexes, respectively. These approaches employ mild in vivo FA (<0.1%) cross-linking, which enables better preservation of dynamic interactions for affinity purification under native conditions.…”

Disabling the Protease DDI2 Attenuates the Transcriptional Activity of NRF1 and Potentiates Proteasome Inhibitor Cytotoxicity

“…pQCXIP-6xHis-Flag-DDI2 (referred to as Flag-DDI2 in this paper), a construct expressing human DDI2 is a gift from Lan Huang (University of California Irvine, Irvine, CA, USA) and has been described previously [36]. We generated a vector control by removing the DDI2 coding sequence and re-ligating the plasmid.…”