Tagging the cell surface receptor with ubiquitin is believed to provide a signal for the endocytic pathway. E3 ubiquitin ligases such as Cbl-b and Itch have been implicated in T cell activation and tolerance induction. However, the underlying mechanisms remain unclear. We describe that in mice deficient in the E3 ubiquitin ligases Cbl-b and Itch, T cell activation was augmented, accompanied by spontaneous autoimmunity. The double-mutant T cells exhibited increased phosphorylation of the T cell receptor-zeta (TCR-zeta) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-zeta was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the zeta chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-zeta was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-zeta and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction.

K33-Linked Polyubiquitination of T Cell Receptor-? Regulates Proteolysis-Independent T Cell Signalin

Ubiquitination regulates nearly all cellular processes by coordinated activity of ubiquitin writers (E1, E2, and E3 enzymes), erasers (deubiquitinating enzymes) and readers (proteins that recognize ubiquitinated proteins by their ubiquitin-binding domains). By differentially modifying cellular proteome and by recognizing these ubiquitin modifications, ubiquitination machinery tightly regulates execution of specific cellular events in space and time. Dynamic and complex ubiquitin architecture, ranging from monoubiquitination, multiple monoubiquitination, eight different modes of homotypic and numerous types of heterogeneous polyubiquitin linkages, enables highly dynamic and complex regulation of cellular processes. We discuss available tools and approaches to study ubiquitin networks, including methods for the identification and quantification of ubiquitin-modified substrates, as well as approaches to quantify the length, abundance, linkage type and architecture of different ubiquitin chains. Furthermore, we also summarize the available approaches for the discovery of novel ubiquitin readers and ubiquitin-binding domains, as well as approaches to monitor and visualize activity of ubiquitin conjugation and deconjugation machineries. We also discuss benefits, drawbacks and limitations of available techniques, as well as what is still needed for detailed spatiotemporal dissection of cellular ubiquitination networks.

Resolving the Complexity of Ubiquitin Networks.

Down-regulation of RIP3 potentiates cisplatin chemoresistance by triggering HSP90-ERK pathway mediated DNA repair in esophageal squamous cell carcinoma.

Ubiquitin (UB) transfer cascades consisting of E1, E2, and E3 enzymes constitute a complex network that regulates a myriad of biologic processes by modifying protein substrates. Deubiquitinating enzymes (DUBs) reverse UB modifications or trim UB chains of diverse linkages. Additionally, many cellular proteins carry UB-binding domains (UBDs) that translate the signals encoded in UB chains to target proteins for degradation by proteasomes or in autophagosomes, as well as affect nonproteolytic outcomes such as kinase activation, DNA repair, and transcriptional regulation. Dysregulation of the UB transfer pathways and malfunctions of DUBs and UBDs play causative roles in the development of many diseases. A greater understanding of the mechanism of UB chain assembly and the signals encoded in UB chains should aid in our understanding of disease pathogenesis and guide the development of novel therapeutics. The recent flourish of protein-engineering approaches such as unnatural amino acid incorporation, protein semisynthesis by expressed protein ligation, and high throughput selection by phage and yeast cell surface display has generated designer proteins as powerful tools to interrogate cell signaling mediated by protein ubiquitination. In this study, we highlight recent achievements of protein engineering on mapping, probing, and manipulating UB transfer in the cell. SIGNIFICANCE STATEMENT: The post-translational modification of proteins with ubiquitin alters the fate and function of proteins in diverse ways. Protein engineering is fundamentally transforming research in this area, providing new mechanistic insights and allowing for the exploration of concepts that can potentially be applied to therapeutic intervention.

https://pharmrev.aspetjournals.org/content/pharmrev/72/2/380.full.pdf

Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond.

Fragile X syndrome (FXS) is the most prevalent inherited intellectual disability, resulting from a loss of fragile X mental retardation protein (FMRP). Patients with FXS suffer lifelong cognitive disabilities, but the function of FMRP in the adult brain and the mechanism underlying age-related cognitive decline in FXS is not fully understood. Here, we report that a loss of FMRP results in increased protein synthesis of histone acetyltransferase EP300 and ubiquitination-mediated degradation of histone deacetylase HDAC1 in adult hippocampal neural stem cells (NSCs). Consequently, FMRP-deficient NSCs exhibit elevated histone acetylation and age-related NSC depletion, leading to cognitive impairment in mature adult mice. Reducing histone acetylation rescues both neurogenesis and cognitive deficits in mature adult FMRP-deficient mice. Our work reveals a role for FMRP and histone acetylation in cognition and presents a potential novel therapeutic strategy for treating adult FXS patients.

/pdf/reducing-histone-acetylation-rescues-cognitive-deficits-in-a-2kffqs57uv.pdf

Reducing histone acetylation rescues cognitive deficits in a mouse model of Fragile X syndrome

Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)

The invention discloses artificially constructed hybrid ubiquitin conjugation structure domain polypeptide and an application thereof. The hybrid ubiquitin conjugation structure domain polypeptide is formed by connecting at least tow ubiquitin conjugation structures in series, and in all ubiquitin conjugation structure domains forming the hybrid ubiquitin conjugation structure domain polypeptide, at least two ubiquitin conjugation structure domain are different. In addition, the hybrid ubiquitin conjugation structure domain polypeptide can contain tag protein. The experiment shows that the tag protein disclosed by the invention can conjugate ubiquitin molecules and different ubiquitin chains carried in the ubiquitin molecules efficiently without unbiasedness, can improve the enrichment efficiency of on-scale ubiquitin protein, is used for developing efficient ubiquitin protein separation mediums and biologic reagents, and can provide technical methods for regulating in-vivo protein ubiquitination. The polypeptide is significant for prompting human disease sample ubiquitin proteomics study and development of health medicine.

Jingwei Wang

Papers

Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)

Artificially constructed hybrid ubiquitin conjugation structure domain polypeptide and application thereof