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

Genome editing reemerged in 2012 with the development of CRISPR/Cas9 technology, which is a genetic manipulation tool derived from the defense system of certain bacteria against viruses and plasmids. This method is easy to apply and has been used in a wide variety of experimental models, including cell lines, laboratory animals, plants, and even in human clinical trials. The CRISPR/Cas9 system consists of directing the Cas9 nuclease to create a site‑directed double‑strand DNA break using a small RNA molecule as a guide. A process that allows a permanent modification of the genomic target sequence can repair the damage caused to DNA. In the present study, the basic principles of the CRISPR/Cas9 system are reviewed, as well as the strategies and modifications of the enzyme Cas9 to eliminate the off‑target cuts, and the different applications of CRISPR/Cas9 as a system for visualization and gene expression activation or suppression. In addition, the review emphasizes on the potential application of this system in the treatment of different diseases, such as pulmonary, gastrointestinal, hematologic, immune system, viral, autoimmune and inflammatory diseases, and cancer.

/pdf/genome-editing-a-perspective-on-the-application-of-crispr-3mu9bk6m05.pdf

Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review)

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.

PVT1 has been reported to be involved in the tumorigenesis and development of different cancers. However, the role of PVT1 in hepatocellular carcinoma (HCC) remains unclear. In this study, we applied gene microarray analysis to detect differentially expressed genes (DEGs) between PVT1 RNAi groups and controls. We initially investigated and confirmed PVT1 expression in HCC using The Cancer Genome Atlas (TCGA). The potential genes and pathways associated with PVT1 were also analyzed. We also performed bioinformatics analyses (Gene Ontology (GO), pathway, Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses) to explore the underlying pathways and networks of these potential genes. We selected DLC1 for further analysis. Based on the TCGA database, PVT1 was markedly up-regulated in HCC, whereas DLC1 was down-regulated. Moreover, PVT1 expression negatively correlated with DLC1 in HCC, an observation that has been further validated in different cohorts with Oncomine. High expression of PVT1 was positively associated with gender, race, vascular invasion and pathological grade in HCC. Additionally, the ROC curve indicated that both PVT1 and DLC1 have high diagnostic value in HCC. We speculated that PVT1 might play a significant role in HCC development and progression via regulation of various pathways and genes, especially DLC1 and the Hippo signaling pathway. However, this mechanism should be confirmed by functional experiments.

Comprehensive analysis of long non-coding RNA PVT1 gene interaction regulatory network in hepatocellular carcinoma using gene microarray and bioinformatics

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

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

Chronic hepatitis B virus (HBV) infection has been considered as the major cause of hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) has been reported to be oncogenic. The underlying mechanisms of HBV-related HCC are not fully understood, and the role played by the HBx protein in HBV induced carcinogenesis remains controversial. CDC42, a member of the Rho GTPase family, has been reported to be overexpressed in several different cancers, including HBV-related HCC. However, the specific role of CDC42 in HCC development remains unclear. Here, we investigated the cellular mechanisms by which CDC42 was responsible for the higher proliferation of HuH-7 cells mediated by HBx. We found that the expression level of CDC42 and its activity were significantly increased in HuH-7-HBx cells. The deficiency of CDC42 using the CRISPR/Cas9 system and inhibition by specific inhibitor CASIN led to the reduction of HBx-mediated proliferation. Furthermore, we observed that IQ Motif Containing GTPase Activating Protein 1 (IQGAP1), the downstream mediator of the CDC42 pathway, might be involved in the carcinogenesis induced by HBx. Therefore, the HBx/CDC42/IQGAP1 signaling pathway may potentially play an important role in HBx-mediated carcinogenesis.

https://www.mdpi.com/1422-0067/18/3/586/pdf

Hepatitis B Virus X Protein Stimulates Proliferation, Wound Closure and Inhibits Apoptosis of HuH-7 Cells via CDC42

Deinococcus radiodurans is a robust bacterium best known for its capacity to resist to radiation. In this study, the SDS-PAGE coupled with high-precision LC-MS/MS was used to study the D. radiodurans proteome. A total of 1951 proteins were identified which covers 63.18% protein-coding genes. Comparison of the identified proteins to the key enzymes in amino acid biosyntheses from KEGG database showed the methionine biosynthesis module is incomplete while other amino acid biosynthesis modules are complete, which indicated methionine auxotrophy in D. radiodurans. The subsequent amino acid-auxotrophic screening has verified methionine instead of other amino acids is essential for the growth of D. radiodurans. With molecular evolutionary genetic analysis, we found the divergence in methionine biosynthesis during the evolution of the common ancestor of bacteria. We also found D. radiodurans lost the power of synthesizing methionine because of the missing metA and metX in two types of methionine biosyntheses. For the first time, this study used high-coverage proteome analysis to identify D. radiodurans amino acid auxotrophy, which provides the important reference for the development of quantitative proteomics analysis using stable isotope labeling in metabolomics of D. radiodurans and in-depth analysis of the molecular mechanism of radiation resistance.

Chen Deng

Papers

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

Hepatitis B Virus X Protein Stimulates Proliferation, Wound Closure and Inhibits Apoptosis of HuH-7 Cells via CDC42

High-coverage Proteomics Reveals Methionine Auxotrophy in Deinococcus radiodurans