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

Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community.

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

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.

Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

Dynamic FMR1 granule phase switch instructed by m6A modification contributes to maternal RNA decay.

Maternal RNA degradation is critical for embryogenesis and is tightly controlled by maternal RNA-binding proteins. Fragile X mental-retardation protein (FMR1) binds target mRNAs to form ribonucleoprotein (RNP) complexes/granules that control various biological processes, including early embryogenesis. However, how FMR1 recognizes target mRNAs and how FMR1-RNP granule assembly/disassembly regulates FMR1-associated mRNAs remain elusive. Here we show that Drosophila FMR1 preferentially binds mRNAs containing m6A-marked "AGACU" motif with high affinity to contributes to maternal RNA degradation. The high-affinity binding largely depends on a hydrophobic network within FMR1 KH2 domain. Importantly, this binding greatly induces FMR1 granule condensation to efficiently recruit unmodified mRNAs. The degradation of maternal mRNAs then causes granule de-condensation, allowing normal embryogenesis. Our findings reveal that sequence-specific mRNAs instruct FMR1-RNP granules to undergo a dynamic phase-switch, thus contributes to maternal mRNA decay. This mechanism may represent a general principle that regulated RNP-granules control RNA processing and normal development.