Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.Sequestosome 1/p62 is a cellular protein which was initially identified as a phosphotyrosine-independent ligand of the Src homology 2 (SH2) domain of p56 lck (27). The protein has been cloned by two independent groups as a cointeracting protein of the atypical protein kinase C (aPKC) and is also named ZIP for PKC-interacting protein (47, 50). p62 has been shown to bind ubiquitin noncovalently (56), and its overexpression results in large cytoplasmic aggregates (47). We have recently determined that p62 possesses sequence homology with other proteins possessing a ubiquitin-associated (UBA) domain at their C terminus, amino acids 386 to 434 (16), in p62. Interestingly, p62 contains several structural motifs which suggest that it might participate in the formation of multimeric signaling complexes. These domains include an acidic interaction domain (AID/ORCA/PC/PB1) that binds the aPKC, a ZZ finger, a binding site for the RING finger protein TRAF6, two PEST sequences, and the UBA domain (16).Ubiquitin is a small polypeptide of 76 amino acids that can be covalently attached to other proteins. Monoubiquitination serves as a novel endocytosis signal (19), whereas polyubiquitin chains target substrates for degradation by the proteasome (45). p62 has been shown to interact in a noncovalent fashion with polyubiquitin chains (5, 53), which is consistent with reports demonstrating that proteins possessing UBA domains are more likely to bind polyubiquitin chains over monoubiquitin (59). Conjugation of ubiquitin to substrate proteins requires three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Initially, E1 activates ubiquitin by forming a high-energy thioester intermediate with the C-terminal glycine using ATP. The activated ubiquitin is sequentially transferred to E2 and then to E3 which catalyzes isopeptide bond formation between the activated C-terminal glycine of ubiquitin and an ε-amino group of a lysine residue in the substrate (45). However, only HECT-type E3s can form thioester bon...

After homing to lymph nodes, CD8 + T cells are primed by dendritic cells (DCs) in three phases. During phase one, T cells undergo brief serial contacts with DCs for several hours, whereas phase two is characterized by stable T cell-DC interactions. We show here that the duration of phase one and T cell activation kinetics correlated inversely with the number of complexes of cognate peptide and major histocompatibility complex (pMHC) per DC and with the density of antigen-presenting DCs per lymph node. Very few pMHC complexes were necessary for the induction of full-fledged T cell activation and effector differentiation. However, neither T cell activation nor transition to phase two occurred below a threshold antigen dose determined in part by pMHC stability. Thus, phase one permits T cells to make integrated 'measurements' of antigen dose that determine subsequent T cell participation in immune responses.The naive T cell population expresses a broad array of unique T cell antigen receptors (TCRs), each with a discrete affinity for a given complex of cognate peptide and major histocompatibility complex (pMHC). Naive T cells constantly survey and sample antigenpresenting cells (APCs) in secondary lymphoid tissues in search of rare cognate pMHC complexes 1 . Due to the diversity of the TCR repertoire, only one in 1 × 10 5 to 1 × 10 6 T cells expresses a TCR with sufficient affinity for any given antigen to transmit an activating Correspondence should be addressed to U. H.v.A. (E-mail: uva@cbr.med.harvard.edu). Note: Supplementary information is available on the Nature Immunology website. AUTHOR CONTRIBUTIONSS.E.H. designed the study, did and analyzed experiments, and wrote the manuscript; U.H.v.A designed the study and wrote the manuscript; T.R.M., I.B.M., A.P., M.P.F., B.S. and T.J. did experiments; B.L. and H.C.W. provided reagents; and M.N.A., H.Z. and A.K.C. modeled the experimental data. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/natureimmunology/.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript stimulus 2,3 . Activation of naive T cells also requires costimulatory and cytokine signals 4,5 , which are typically provided by mature dendritic cells (DCs) in secondary lymphoid tissues 6 . As they 'hunt' for their cognate antigen, naive T cells recirculate between the blood and lymph nodes and spend less than 1 d in any given secondary lymphoid tissue 1,7 . When T cells encounter antigen in the proper context, they become activated and upregulate the activation marker CD69, which causes their retention in lymph nodes 8 . The trapped cells proliferate and acquire effector functions. Effector cells egress from lymph nodes and travel to peripheral tissues to seek out cells presenting cognate antigen 9 .Although this chain of events is well establ...

Adoptive cell therapy (ACT) with antigen-specific T cells has shown remarkable clinical success; however, approaches to safely and effectively augment T cell function, especially in solid tumors, remain of great interest. Here we describe a strategy to 'backpack' large quantities of supporting protein drugs on T cells by using protein nanogels (NGs) that selectively release these cargos in response to T cell receptor activation. We designed cell surface-conjugated NGs that responded to an increase in T cell surface reduction potential after antigen recognition and limited drug release to sites of antigen encounter, such as the tumor microenvironment. By using NGs that carried an interleukin-15 super-agonist complex, we demonstrated that, relative to systemic administration of free cytokines, NG delivery selectively expanded T cells 16-fold in tumors and allowed at least eightfold higher doses of cytokine to be administered without toxicity. The improved therapeutic window enabled substantially increased tumor clearance by mouse T cell and human chimeric antigen receptor (CAR)-T cell therapy in vivo.

Chirality-controlling chelate (CCC) ligands are a class of chiral diamine ligands with one or two chiral secondary amine ligating groups. Analogues of platinum anticancer agents containing CCC ligands exhibit unusual steric and dynamic features. In this study NMR and CD methods were used to evaluate the influence of the N9 substituent in guanine derivatives (G) on conformer distribution in one class of (CCC)PtG(2)() complexes. We employed the CCC ligand, N,N'-dimethyl-2,3-diaminobutane [Me(2)()DAB with S,R,R,S or R,S,S,R configurations at the four asymmetric centers, N, C, C, and N]. For each Me(2)()DABPtG(2) complex, the presence of four G H8 signals demonstrated formation of all three possible atropisomers: DeltaHT (head-to-tail), LambdaHT, and HH (head-to-head). Different G ligands (5'-GMP, 3'-GMP, 1-MeGuo, Guo, or 9-EtG) were chosen to assess the effect of the N9 substituent on the relative stability and spectral properties of the atropisomers. The conformations of the atropisomers of Me(2)()DABPtG(2) were determined from CD spectra and from NOE cross-peaks (assigned via COSY spectra) between G H8 signals and those for the Me(2)()DAB protons. Regardless of the N9 substituent, the major form was HT. However, this form had the opposite chirality, LambdaHT and DeltaHT, and base tilt direction, left- and right-handed, respectively, for the S,R,R,S and R,S,S,R configurations of the Me(2)()DAB ligand. Thus, the chirality of the CCCligand, not hydrogen bonding, is the most important determinant of conformation. For each Me(2)()DABPtG(2) complex, the tilt direction of all three atropisomers is the same and, except for 5'-GMP, the order of abundance was major HT > minor HT > HH. For 5'-GMP, the HH atropisomer was three times as abundant as the minor HT species, suggesting that phosphate-NH(Me(2)()DAB) hydrogen bonds could be present since such bonding is possible only for the 5'-GMP derivatives. However, if such phosphate-NH hydrogen bonds exist, they are weak since the percentage of the major HT form of 5'-GMP complexes is similar and indeed can be smaller compared to this percentage for complexes with other G's. The CD spectra of all (S,R,R,S)-Me(2)()DABPtG(2) complexes were similar and opposite to those of all (R,S,S,R)-Me(2)()DABPtG(2) complexes, indicating the CD signature is characteristic of the dominant HT conformer, which has a chirality dictated by the chirality of the CCC ligand and not the N9 substituent.