A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.     

Ubiquitin-independent proteasomal degradation during oncogenic viral infections

Most eukaryotic proteins destined for imminent destruction are first tagged with a chain of ubiquitin molecules and are subsequently dismantled by the proteasome. Ubiquitin-independent degradation of substrates by the proteasome, however, also occurs. The number of documented proteasome-dependent, ubiquitin-independent degradation events remains relatively small but continues to grow. Proteins involved in oncogenesis and tumor suppression make up the majority of the known cases for this type of protein destruction. Provocatively, viruses with confirmed or suspected oncogenic properties are also prominent participants in the pantheon of ubiquitin-independent proteasomal degradation events. In this review, we identify and describe examples of proteasome-dependent, ubiquitin-independent protein degradation that occur during tumor virus infections, speculate why this type of protein destruction may be preferred during oncogenesis, and argue that this uncommon type of protein turnover represents a prime target for antiviral and anticancer therapeutics.     

Cooperation between an intrinsically disordered region and a helical segment is required for ubiquitin-independent degradation by the proteasome

The 26 S proteasomal complex, which is responsible for the bulk of protein degradation within the cell, recognizes its target substrates via covalently linked polyubiquitin moieties. However, a small but growing number of proteasomal substrates are degraded without a requirement for ubiquitinylation. One such substrate is the pyrimidine biosynthetic enzyme thymidylate synthase (EC 2.1.1.45), which catalyzes the synthesis of TMP and is the sole de novo source of TTP for DNA replication and repair. Previous work showed that intracellular proteolysis of human thymidylate synthase is directed by a degron at the polypeptide's N-terminal end, composed of an intrinsically disordered region (IDR) followed by a highly conserved amphipathic α-helix (hA). In the present report, we show that the hA helix does not function simply as an extension or scaffold for the IDR; rather, it provides a specific structural component that is necessary for degradation. Furthermore, its helical conformation is required for this function. We demonstrate that small domains from heterologous proteins can substitute for the IDR and the hA helix of human thymidylate synthase, indicating that the degradation-promoting function of these regions is not sequence-specific. The results, in general, indicate that cooperation between intrinsically disordered domains and α-helical segments is required for ubiquitin-independent degradation by the proteasome. There appears to be little sequence constraint on the ability of these regions to function as degron constituents. Rather, it is the overall conformation (or lack thereof) that is critical.