The Roles of Ubiquitin-Binding Protein Shuttles in the Degradative Fate of Ubiquitinated Proteins in the Ubiquitin-Proteasome System and Autophagy

Abstract

The ubiquitin-proteasome system (UPS) and autophagy are the two major intracellular protein quality control (PQC) pathways that are responsible for cellular proteostasis (homeostasis of the proteome) by ensuring the timely degradation of misfolded, damaged, and unwanted proteins. Ubiquitination serves as the degradation signal in both these systems, but substrates are precisely targeted to one or the other pathway. Determining how and when cells target specific proteins to these two alternative PQC pathways and control the crosstalk between them are topics of considerable interest. The ubiquitin (Ub) recognition code based on the type of Ub-linked chains on substrate proteins was believed to play a pivotal role in this process, but an increasing body of evidence indicates that the PQC pathway choice is also made based on other criteria. These include the oligomeric state of the Ub-binding protein shuttles, their conformation, protein modifications, and the presence of motifs that interact with ATG8/LC3/GABARAP (autophagy-related protein 8/microtubule-associated protein 1A/1B-light chain 3/GABA type A receptor-associated protein) protein family members. In this review, we summarize the current knowledge regarding the Ub recognition code that is bound by Ub-binding proteasomal and autophagic receptors. We also discuss how cells can modify substrate fate by modulating the structure, conformation, and physical properties of these receptors to affect their shuttling between both degradation pathways.

Keywords: protein quality control; proteostasis; selective autophagy; ubiquitin-proteasome system; ubiquitination.

Figure 1

The ubiquitin code. A schematic representation of possible ubiquitin (Ub) chain formations on a target protein (substrate) from single mono-ubiquitination to mixed and multiply -branched Ub chains. Within polyubiquitin chains, which can exist on one of the seven intrinsic lysine (Lys) residues within the Ub sequence, or methionine (Met) at position 1, Ub can form eight different homogeneous or heterogeneous linkage types. However, only five linkage types have been structurally characterized (Met1-chains, Lys6-chains, Lys11-chains, Lys48-chains, and Lys63-chains) and classified as “open” or “compact” based on their three-dimensional topologies.

Figure 2

Ub-binding protein shuttles from yeast, mammals, and plants. Schematic representation of the domain/motif/region organization of the known Ub-binding shuttle receptors used by the proteasomal [59] and autophagic [24,65,66,67] protein quality control (PQC) pathways. The graphical depiction of Ub-binding modules is based on their identification using UniProt and extended by data in the published literature [68,69]. The domain/motif/regions are shown for the proteasomal (S. cerevisiae Rad23 (P32628), H. sapiens HR23A (P54725), H. sapiens HR23B (P54727), A. thaliana RAD23A (Q84L32), A. thaliana RAD23B (Q84L33), A. thaliana RAD23C (Q84L31), A. thaliana RAD23D (Q84L30), S. cerevisiae Dsk2 (P48510), H. sapiens ubiquilin1 (Q9UMX0), H. sapiens ubiquilin2 (Q9UHD9), H. sapiens ubiquilin3 (Q9H347), H. sapiens ubiquilin4 (Q9NRR5), A. thaliana DSK2A (Q9SII9), A. thaliana DSK2B (Q9SII8), S. cerevisiae Ddi1 (P40087), H. sapiens DDI1 (Q8WTU0), H. sapiens DDI2 (Q5TDH0), A. thaliana DDI1 (Q1EBV4)), and autophagic shuttles (S. cerevisiae Cue5 (Q08412), H. sapiens TOLLIP (Q9H0E2), H. sapiens SQSTM1 (Q13501), H. sapiens NBR1 (Q14596), A. thaliana NBR1 (Q9SB64), N. tabacum Joka2 (F8RP79), and H. sapiens OPTN (Q96CV9)). All proteins and domains are drawn to scale. UbL, Ubiquitin-like. UBA, Ubiquitin-associated. STI1, stress-inducible 1. UBXN4, Ubiquitin-regulatory X domain-containing protein 4. RVP, retroviral protease fold domain. PEST, protein rich in Pro, Glu, Ser and Thr. UIM, Ub-interacting motif. AIM/LIR, ATG8-interacting motif/LC3-interacting region. CUE, coupling of Ub-conjugation to ER degradation. C2, Ca⁺²-dependent membrane-targeting module. PB1, Phox and Bem1. ZZ, Zinc finger. TRAF6, TNF receptor-associated factor 6. NBR1, Neighbor of BRCA1. NES, Nuclear Export Signal. NLS, Nuclear Localization Signal. CC, coiled-coil. KIR, KEAP1 (Kelch-like ECH-associated protein 1)-interacting region. JUBA, Juxta-UBA. UBAN, UBD Ub-binding in A20-binding inhibitor of NF-κB (ABIN) proteins and NF-κB essential modulator (NEMO).

Figure 3

A model for the substrate fate based on the Ub code and an oligomeric state of the receptor and recognition of ATG8/LC3/GABARAP (autophagy-related protein 8/microtubule-associated protein 1A/1B-light chain 3/GABA type A receptor-associated protein) proteins. The PQC network targeting misfolded or unwanted proteins via ubiquitination for degradation is hierarchically organized, but retains flexibility and coordinates the Ub-proteasome system (UPS) and autophagy pathways, according to cellular needs. In the low aggregation (undisturbed) condition, unwanted proteins initially become substrates for the UPS. During ubiquitination, particular Ub labels are attached to the target proteins via the E1–E2–E3 enzyme cascade. This process is reversed by deubiquitinases (DUBs). When the Ub code is written on the substrates, the cargos are then primarily recognized by intrinsic or shuttling proteasomal receptors characterized by higher affinity to Ub in comparison to the affinity of autophagic receptors to Ub. Simultaneous binding of shuttle receptors (probably in monomeric states) to ubiquitinated cargo and proteasomal proteins permit the transfer of the degradation substrates to the proteasome. However, in high aggregation conditions such as when proteasomes are overloaded or when misfolded or redundant proteins accumulate and form aggregates on which Ub is locally concentrated. Such aggregates are mainly recognized by autophagic receptors, which in their oligomeric states have high avidity for Ub and spontaneously coalesce with ubiquitinated proteins into larger clusters that further promote substrate aggregation and delivery to the autophagic machinery. In times of cellular need, shuttling receptors from the proteasomal or autophagic clearance pathways adapt to the alternative, but functional, degradation system to support cellular proteostasis. Proteasomal shuttles with exposed AIM/LIR motif and/or with a tendency to oligomerization can support protein turnover via autophagy by directly driving substrates to autophagic machinery or indirectly by sequestering ubiquitinated proteins in aggregates and stimulating cluster formation. Conversely, autophagic receptors in monomeric forms with non-functional AIM/LIR motifs can promote proteasomal degradation, if they are able to recognize and engage proteasomal subunits.

Figure 4

Schematic representation of factors affecting conformational changes and function of Ub-binding receptors. The impact of shuttling Ub-binding receptors on the fate of ubiquitinated targets relies on the combined effect of several regulatory factors acting on those receptors and affecting their oligomerization tendency or proteasome recognition, availability of AIM/LIR motifs for ATG8/LC3/GABARAP-family member proteins decorating isolation membranes, and the receptor affinity for particular type(s) of Ub chains. Factors influencing these choices include post-translational modifications, intramolecular, intermolecular homo-oligomeric, and hetero-oligomeric interactions of each domain present in these receptors, chaperone assistance, levels of available free Ub and Ub-conjugates and effects of other proteins and molecules interacting with Ub-binding receptors and affecting their conformational changes and/or function.