Nuclear Receptor FTZ-F1 Controls Locust Molt by Regulating the Molting Process of Locusta migratoria.

Fushi-tarazu factor 1 (FTZ-F1) is a class of transcription factors belonging to the nuclear receptor superfamily and an important molting regulator in insects; however, its detailed function in the molting process of Locusta migratoria is still unclear. This study identified two FTZ-F1 transcripts (LmFTZ-F1-X1 and LmFTZ-F1-X2) in L. migratoria. The classical domains of FTZ-F1 were present in their protein sequences and distinguished based on their variable N-terminal domains. Reverse-transcription quantitative polymerase chain reaction analysis revealed that LmFTZ-F1-X1 and LmFTZ-F1-X2 were highly expressed in the integument. RNA interference (RNAi) was used to explore the function of LmFTZ-F1s in the molting of the third-instar nymph. Separate LmFTZ-F1-X1 or LmFTZ-F1-X2 silencing did not affect the normal development of third-instar nymphs; however, the simultaneous RNAi of LmFTZ-F1-X1 and LmFTZ-F1-X2 caused the nymphs to be trapped in the third instar stage and finally die. Furthermore, the hematoxylin-eosin and chitin staining of the cuticle showed that the new cuticles were thickened after silencing the LmFTZ-F1s compared to the controls. RNA-seq analysis showed that genes encoding four cuticle proteins, two chitin synthesis enzymes, and cytochrome P450 303a1 were differentially expressed between dsGFP- and dsLmFTZ-F1s-injected groups. Taken together, LmFTZ-F1-X1 and LmFTZ-F1-X2 are involved in the ecdysis of locusts, possibly by regulating the expression of genes involved in cuticle formation, chitin synthesis, and other key molting processes.

Quantitative Proteomics-Based Substrate Screening Revealed Cyclophilin Stabilization Regulated by Deubiquitinase Ubp7.

Quantitative proteomics has emerged as a crucial approach to identifying ubiquitinated substrates to investigate the functions of ubiquitination in cells. In this regard, although the substrate screening of certain enzymes in the ubiquitin system has been based on proteome or ubiquitinome level measurements, the direct comparison of these two approaches has not been determined to date. To quantitatively compare the efficiency and effectiveness of substrate screening from the entire proteomics to the ubiquitinomics filter, we used yeast deubiquitinating enzyme, Ubp7, as an example to evaluate it in this study. A total of 112 potential ubiquitinated substrates were identified from the ubiquitinomics level, whereas only 27 regulated substrates were identified from the entire proteomic screening, demonstrating the increased efficiency of ubiquitinomics quantitative analysis. Subsequently, we selected cyclophilin A (Cpr1) protein as an example, which was filtered out at the proteomics level but was a promising candidate according to the ubiquitinomics filter. Additional investigations revealed that Cpr1 possessed a K48-linked ubiquitin chain regulated by Ubp7, which may affect its homeostasis and, consequently, sensitivity to the therapeutic drug cyclosporine (CsA).

[Quantitative proteomics reveal the potential biological functions of the deubiquitinating enzyme Ubp14 in Saccharomyces cerevisiae].

Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14∆ mutant was constructed by homologous recombination technique. The growth rate of ubp14∆ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14∆ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.

SMAD4, activated by the TCR-triggered MEK/ERK signaling pathway, critically regulates CD8+ T cell cytotoxic function.

Transforming growth factor-ß is well known to restrain cytotoxic T cell responses to maintain self-tolerance and to promote tumor immune evasion. In this study, we have investigated the role of SMAD4, a core component in the TGF-ß signaling pathway, in CD8+ T cells. Unexpectedly, we found that SMAD4 was critical in promoting CD8+ T cell function in both tumor and infection models. SMAD4-mediated transcriptional regulation of CD8+ T cell activation and cytotoxicity was dependent on the T cell receptor (TCR) but not TGF-ß signaling pathway. Following TCR activation, SMAD4 translocated into the nucleus, up-regulated genes encoding TCR signaling components and cytotoxic molecules in CD8+ T cells and thus reinforced T cell function. Biochemically, SMAD4 was directly phosphorylated by ERK at Ser367 residue following TCR activation. Our study thus demonstrates a critical yet unexpected role of SMAD4 in promoting CD8+ T cell-mediated cytotoxic immunity.

The Root Hair Development of Pectin Polygalacturonase PGX2 Activation Tagging Line in Response to Phosphate Deficiency.

Pectin, cellulose, and hemicellulose constitute the primary cell wall in eudicots and function in multiple developmental processes in plants. Root hairs are outgrowths of specialized epidermal cells that absorb water and nutrients from the soil. Cell wall architecture influences root hair development, but how cell wall remodeling might enable enhanced root hair formation in response to phosphate (P) deficiency remains relatively unclear. Here, we found that POLYGALACTURONASE INVOLVED IN EXPANSION 2 (PGX2) functions in conditional root hair development. Under low P conditions, a PGX2 activation tagged line (PGX2AT ) displays bubble-like root hairs and abnormal callose deposition and superoxide accumulation in roots. We found that the polar localization and trafficking of PIN2 are altered in PGX2AT roots in response to P deficiency. We also found that actin filaments were less compact but more stable in PGX2AT root hair cells and that actin filament skewness in PGX2AT root hairs was recovered by treatment with 1-N-naphthylphthalamic acid (NPA), an auxin transport inhibitor. These results demonstrate that activation tagging of PGX2 affects cell wall remodeling, auxin signaling, and actin microfilament orientation, which may cooperatively regulate root hair development in response to P starvation.

Pectin methyltransferase QUASIMODO2 functions in the formation of seed coat mucilage in Arabidopsis.

Pectin, cellulose, and hemicelluloses are major components of primary cell walls in plants. In addition to cell adhesion and expansion, pectin plays a central role in seed mucilage. Seed mucilage contains abundant pectic rhamnogalacturonan-I (RG-I) and lower amounts of homogalacturonan (HG), cellulose, and hemicelluloses. Previously, accumulated evidence has addressed the role of pectin RG-I in mucilage production and adherence. However, less is known about the function of pectin HG in seed coat mucilage formation. In this study, we analyzed a novel mutant, designated things fall apart2 (tfa2), which contains a mutation in HG methyltransferase QUASIMODO2 (QUA2). Etiolated tfa2 seedlings display short hypocotyls and adhesion defects similar to qua2 and tumorous shoot development2 (tsd2) alleles, and show seed mucilage defects. The diminished uronic acid content and methylesterification degree of HG in mutant seed mucilage indicate the role of HG in the formation of seed mucilage. Cellulosic rays in mutant mucilage are collapsed. The epidermal cells of seed coat in tfa2 and tsd2 display deformed columellae and reduced radial wall thickness. Under polyethylene glycol treatment, seeds from these three mutant alleles exhibit reduced germination rates. Together, these data emphasize the requirement of pectic HG biosynthesis for the synthesis of seed mucilage, and the functions of different pectin domains together with cellulose in regulating its formation, expansion, and release.

Deubiquitinase Ubp3 enhances the proteasomal degradation of key enzymes in sterol homeostasis.

Sterol homeostasis is tightly controlled by molecules that are highly conserved from yeast to humans, the dysregulation of which plays critical roles in the development of antifungal resistance and various cardiovascular diseases. Previous studies have shown that sterol homeostasis is regulated by the ubiquitin-proteasome system. Two E3 ubiquitin ligases, Hrd1 and Doa10, are known to mediate the proteasomal degradation of 3-hydroxy-3-methylglutaryl-CoA reductase Hmg2 and squalene epoxidase Erg1 with accumulation of the toxic sterols in cells, but the deubiquitinases (DUBs) involved are unclear. Here, we screened for DUBs responsible for sterol homeostasis using yeast strains from a DUB-deletion library. The defective growth observed in ubp3-deleted (ubp3Δ) yeast upon fluconazole treatment suggests that lack of Ubp3 disrupts sterol homeostasis. Deep-coverage quantitative proteomics reveals that ergosterol biosynthesis is rerouted into a sterol pathway that generates toxic products in the absence of Ubp3. Further genetic and biochemical analysis indicated that Ubp3 enhances the proteasome's ability to degrade the ergosterol biosynthetic enzymes Erg1 and Erg3. The retardation of ergosterol enzyme degradation in the ubp3Δ strain resulted in the severe accumulation of the intermediate lanosterol and a branched toxic sterol, and ultimately disrupted sterol homeostasis and led to the fluconazole susceptibility. Our findings uncover a role for Ubp3 in sterol homeostasis and highlight its potential as a new antifungal target.

Ubiquitin Linkage Specificity of Deubiquitinases Determines Cyclophilin Nuclear Localization and Degradation.

Ubiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profiling in vivo. We used quantitative proteomics to compare the seven lysine-linked ubiquitin chains between wild-type yeast and its 20 DUB-deletion strains, which may reflect the linkage specificity of DUBs in vivo. Utilizing the specificity and ubiquitination heterogeneity, we developed a method termed DUB-mediated identification of linkage-specific ubiquitinated substrates (DILUS) to screen the ubiquitinated lysine residues on substrates modified with certain chains and regulated by specific DUB. Then we were able to identify 166 Ubp2-regulating substrates with 244 sites potentially modified with K63-linked chains. Among these substrates, we further demonstrated that cyclophilin A (Cpr1) modified with K63-linked chain on K151 site was regulated by Ubp2 and mediated the nuclear translocation of zinc finger protein Zpr1. The K48-linked chains at non-K151 sites of Cpr1 were mainly regulated by Ubp3 and served as canonical signals for proteasome-mediated degradation.

Quantitative Proteomics Combined with Two Genetic Strategies for Screening Substrates of Ubiquitin Ligase Hrt3.

Ubiquitin ligases (E3s) serve as key regulators for the ubiquitylation-mediated pathway. The identification of the corresponding relationship between E3 and its substrates is challenging but required for understanding the regulatory network of ubiquitylation. The low abundance of ubiquitinated conjugates and high redundancy of E3 substrate regulation made the screening pretty hard. Herein, we combined SILAC-based quantitative proteomics with two contrary genetic methods (overexpression and knockout) in theory for E3 (Hrt3, the F-box subunit of the SCF complex) substrate screening. The knockout method could not overcome the constraint mentioned above, while the overexpression approach turned on the access to the potential substrates of E3. Subsequently, we obtained 77 candidates, which are involved in many critical biological processes and need to be verified in the future. Within these candidates, we confirmed the relationship between one of the candidates Nce103 and Hrt3 and linked Hrt3 with oxygen sensitivity and oxidative stress response in which Nce103 took part as well. This research is also beneficial for understanding the impact of oxygen supply on regulation of yeast growth through the ubiquitination of Nce103.

Proteomics Links Ubiquitin Chain Topology Change to Transcription Factor Activation.

A surprising complexity of ubiquitin signaling has emerged with identification of different ubiquitin chain topologies. However, mechanisms of how the diverse ubiquitin codes control biological processes remain poorly understood. Here, we use quantitative whole-proteome mass spectrometry to identify yeast proteins that are regulated by lysine 11 (K11)-linked ubiquitin chains. The entire Met4 pathway, which links cell proliferation with sulfur amino acid metabolism, was significantly affected by K11 chains and selected for mechanistic studies. Previously, we demonstrated that a K48-linked ubiquitin chain represses the transcription factor Met4. Here, we show that efficient Met4 activation requires a K11-linked topology. Mechanistically, our results propose that the K48 chain binds to a topology-selective tandem ubiquitin binding region in Met4 and competes with binding of the basal transcription machinery to the same region. The change to K11-enriched chain architecture releases this competition and permits binding of the basal transcription complex to activate transcription.

High-coverage proteomics reveals methionine auxotrophy in Deinococcus radiodurans.

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.

[Progress in ubiquitin, ubiquitin chain and protein ubiquitination].

Protein ubiquitination is one of the most important and widely exist protein post-translational modifications in eukaryotic cells, which takes the ubiquitin and ubiquitin chains as signal molecules to covalently modify other protein substrates. It plays an important roles in the control of almost all of the life processes, including gene transcription and translation, signal transduction and cell-cycle progression, besides classical 26S protesome degradation pathway. Varied modification sites in the same substrates as well as different types of ubiquitin linkages in the same modification sites contain different structural information, which conduct different signal or even determine the fate of the protein substrates in the cell. Any abnormalities in ubiquitin chain formation or its modification process may cause severe problem in maintaining the balance of intracellular environment and finally result in serious health problem of human being. In this review, we discussed the discovery, genetic characteristics and the crystal structure of the ubiquitin. We also emphasized the recent progresses of the assembly processes, structure and their biological function of ubiquitin chains. The relationship between the disregulation and related human diseases has also been discussed. These progress will shed light on the complexity of proteome, which may also provide tools in the new drug research and development processes.

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.

[Application of proteomics in deubiquitinases research].

As the major pathway mediating specific protein degradation in eukaryotes, ubiquitin-proteasome system (UPS) is involved in various physiological and pathological processes such as cell cycle regulation, immune response, signal transduction and DNA-repair. Deubiquitinases (DUB) maintain the balance of UPS and related physiological processes via reversibly removing ubiquitin from the covalently modified protein substrates, which have been implicated in various disease processes in case of their imbalance expression. Because DUB plays critical regulating roles in the UPS pathway, they may be also the ideal drug targets for severe and intractable human diseases, such as cancer and neurodegenerative disease. With the rapid development of proteomic technology, systematical investigation of specific substrates and interacting proteins of varied DUB via mass spectrometry approach may shed light on these DUB's biological function and regulating roles in the physiological and pathogenic states. In this review, we briefly introduce the characteristics of DUB and summarize the recent application and progresses of proteomics in DUB research.