The T cell repertoire in each individual includes T cell receptors (TCRs) of enormous sequence diversity through the pairing of diverse TCR α- and β-chains, each generated by somatic recombination of paralogous gene segments. Whether the TCR repertoire contributes to susceptibility to infectious or autoimmune diseases in concert with disease-associated major histocompatibility complex (MHC) polymorphisms is unknown. Due to a lack in high-throughput technologies to sequence TCR α–β pairs, current studies on whether the TCR repertoire is shaped by host genetics have so far relied only on single-chain analysis. Using a high-throughput single T cell sequencing technology, we obtained the largest paired TCRαβ dataset so far, comprising 965,523 clonotypes from 15 healthy individuals including 6 monozygotic twin pairs. Public TCR α- and, to a lesser extent, TCR β-chain sequences were common in all individuals. In contrast, sharing of entirely identical TCRαβ amino acid sequences was very infrequent in unrelated individuals, but highly increased in twins, in particular in CD4 memory T cells. Based on nucleotide sequence identity, a subset of these shared clonotypes appeared to be the progeny of T cells that had been generated during fetal development and had persisted for more than 50 y. Additional shared TCRαβ in twins were encoded by different nucleotide sequences, implying that genetic determinants impose structural constraints on thymic selection that favor the selection of TCR α–β pairs with entire sequence identities.

The level of individual plasma membrane proteins needs to be regulated strictly depending on the situation under which the cell is placed. To reduce the level of a specific plasma membrane protein in a short period, cells internalize the protein from the cell surface by endocytosis and degrade it in the lysosome. Internalized cargo proteins are transported to the limiting membrane of the early endosome, from which they are incorporated into the lumenal vesicles of the endosome. Such endosomes, called the late endosome or multivesicular body, fuse with the lysosome, thereby delivering cargo proteins to the lysosomal lumen and exposing them to acid hydrolases. During this lysosomal trafficking process, ubiquitination serves as a signal that drives internalization and endosome-to-lysosome transport of the cargo proteins. In this review, we discuss the types of ubiquitination that drive these trafficking processes, and how the ubiquitin (Ub) modifications are recognized by specific Ub-binding proteins.

The mechanism of ubiquitin-dependent endocytosis of cell surface proteins is not completely understood. Here we examine the role of the ankyrin repeat domain (Ankrd) 13A, 13B, and 13D proteins, which constitute a functionally unknown family of ubiquitin-interacting motif (UIM)–bearing proteins, in the process. Stimulation of human HeLa cells with epidermal growth factor (EGF) rapidly induced direct binding of Ankrd 13 proteins to ubiquitinated EGF receptor (EGFR) via the UIMs. The binding was inhibited when the Ankrd 13 proteins underwent UIM-dependent monoubiquitination, suggesting that their activity is regulated by ubiquitination of themselves. Ankrd 13 proteins bound specifically to Lys-63–linked ubiquitin chains, which was consistent with a previous report that EGFR mainly undergoes Lys-63–linked polyubiquitination. Ankrd 13 proteins were anchored, via the central region and UIMs, to the plasma membrane, where they colocalized with EGFR. Finally, overexpression of wild-type as well as truncated-mutant Ankrd 13 proteins strongly inhibited rapid endocytosis of ubiquitinated EGFR from the surface in EGF-treated cells. We conclude that by binding to the Lys-63–linked polyubiquitin moiety of EGFR at the plasma membrane, Ankrd 13 proteins regulate the rapid internalization of ligand-activated EGFR.

We report a simple and low-cost one-step high-throughput technology for sequencing the paired antibody/TCR repertoire.