Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis.

Neddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9.

Smurf1 aggravates non-alcoholic fatty liver disease by stabilizing SREBP-1c in an E3 activity-independent manner.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver disorders which are characterized by the accumulation of excessive lipid in hepatocytes. The precise pathogenesis of NAFLD is very complicated and remains largely unknown. Smad ubiquitination regulatory factor 1 (Smurf1) is crucial for numerous processes including bone homeostasis, embryogenesis, and pathogenic autophagy. In this study, we found that liver steatosis was alleviated in Smurf1-deficient mice fed with high-fat diet (HFD) for 19 weeks. The deletion of Smurf1 reduced the accumulation of lipid droplets and triglycerides in hepatocytes. The stability of sterol regulatory element-binding protein-1c (SREBP-1c), a key transcription factor that mediates de novo lipogenesis, was markedly reduced in Smurf1-deficient mice. The mechanistic study showed that Smurf1 interacts with SREBP-1c and protects SREBP-1c from ubiquitination and degradation by preventing the binding of SREBP-1c to its ubiquitin E3 ligase Fbw7a. Thus, our study presented an E3 ligase catalytic activity-independent function of Smurf1 in the fatty liver development.

Nedd8-activating enzyme inhibitor MLN4924 (Pevonedistat), inhibits miR-1303 to suppress human breast cancer cell proliferation via targeting p27Kip1.

MLN4924/Pevonedistat, a Nedd8-activating enzyme (NAE, E1) inhibitor, has shown notable anti-cancer effect in pre-clinical trials, but it still faces tolerance resistance risk. Combination target therapy indicates a much better clinical effect than single target, and miRNAs are beneficial for easy detection in bodily fluids and tissues. Up to now, MLN4924 and miRNA-targeting combination approaching to treat breast cancer patients remains largely unknown. Here, microRNA-seq analysis showed that the expression of miR-1303 was significantly decreased after MLN4924 treatment in breast cancer cells. Moreover, miR-1303 was abnormally high in breast cancer tissues, and breast cancer patients with high miR-1303 showed poor prognosis. Functionally, excessive miR-1303 promoted the malignant phenotypes of breast cancer cells. Excessive miR-1303 accelerated cell cycle progression by promoting G2/M arrest. Furthermore, we revealed that miR-1303 targeted p27Kip1 to release G2/M arrest. Notably, excessive miR-1303 partially disturbed the anti-cancer effect of MLN4924. These findings provide potential evidences for combined anti-cancer target therapy of breast cancer patients in the future.

Towards efficient enzymatic conversion of D-galactose to D-tagatose: purification and characterization of L-arabinose isomerase from Lactobacillus brevis.

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1 kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75 °C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65 °C and 7.0, respectively. Besides, we found that Ca2+, Cu2+, and Ba2+ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg2+, Mn2+, or Co2+ ions. The optimum concentration of Mn2+ and Co2+ was determined to be 1 mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the Km (129 mM) and the Vmax (0.045 mM min- 1) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms.

[Low sap flow of Picea crassifolia and its influencing factors in Qilian Mountains, China]. / ç¥è¿žå±±é’æµ·äº‘æ‰ä½Žæ¶²æµç‰¹å¾åŠå ¶å½±å“å› ç´ .

As an important part of ecological hydrology, transpiration is the basis for analyzing forest water cycle and healthy growth, and important for forest protection and scientific management. We used thermal diffusion probes (TDP) to continuously monitor sap flow of Picea crassifolia in the Qilian Mountains from 2017 to 2018 to explore water consumption of P. crassifolia, and analyze the main controlling factors for the growth and transpiration of spruce. The results showed that the instantaneous change of P. crassifolia sap flow showed a single-peak curve in sunny days, a multi-peak or double-peak curve in cloudy days, and basically no obvious regularity in rainy days. The sap flow density of Qinghai spruce was consistent with the dynamics of solar radiation. The sap flow started earlier and ended later on sunny days, and lasted for 12 to 14 hours. Due to the high altitude (2700 m), low air temperature, and low vapor pressure difference (VPD) in this area, the overall sap flow density was low, with an average of (0.86±0.49) kg·d-1. On the hourly scale, the instantaneous rate of sap flow was significantly affected by solar radiation and VPD. On the daily scale, soil temperature and soil moisture content of the 0-40 cm layer were significantly related to sap flow density. The spruce sap flow density decreased with the decreases of solar radiation, air temperature, and VPD. In the high-altitude forest area of Qilian Mountains, lower soil and air temperature as well as lower VPD and solar radiation were the causes of low sap flow in Picea crassifolia in this area.

[Soil physical and chemical properties and vegetation characteristics of different types of grassland in Qilian Mountains, China]. / 祁连山不同类型草地的土壤理化性质与植被特征.

Grasslands in Qilian Mountains plays an important role in maintaining the ecological security of western China. To understand soil physical and chemical properties and the distribution characteristics of vegetation, as well as their correlation in different types of grasslands in Qilian Mountains, we measured soil moisture, nutrient content, bulk density, particle composition, and vegetation characteristics in seven types of grassland in Qilian Mountains. The fractal dimension of soil particles, soil organic carbon, total nitrogen and total phosphorus storages in 0-40 cm soil layer, and plant diversity index were calculated. The results showed that there were significant differences in soil physical and chemical properties and vegetation characteristics among different grassland types. Compared with other types of grassland, alpine meadow had higher soil water, nutrient and clay content, but lower bulk density and sand content. Soil organic carbon, total nitrogen and total phosphorus storages in 0-40 cm layer ranged from 3084 to 45247, 164 to 2358 and 100 to 319 g·m-2, respectively, with high contents of organic carbon and total nitrogen and low content of total phosphorus. There was a significant positive correlation between soil total phosphorus storage and plant diversity index, indicating that soil total phosphorus content was the key factor affec-ting grassland plant diversity in Qilian Mountains. Compared with other grassland types, alpine meadow in Qilian Mountains had better vegetation status, soil moisture, and nutrient conditions.

Neddylation of PTEN regulates its nuclear import and promotes tumor development.

PTEN tumor suppressor opposes the PI3K/Akt signaling pathway in the cytoplasm and maintains chromosomal integrity in the nucleus. Nucleus-cytoplasm shuttling of PTEN is regulated by ubiquitylation, SUMOylation and phosphorylation, and nuclear PTEN has been proposed to exhibit tumor-suppressive functions. Here we show that PTEN is conjugated by Nedd8 under high glucose conditions, which induces PTEN nuclear import without effects on PTEN stability. PTEN neddylation is promoted by the XIAP ligase and removed by the NEDP1 deneddylase. We identify Lys197 and Lys402 as major neddylation sites on PTEN. Neddylated PTEN accumulates predominantly in the nucleus and promotes rather than suppresses cell proliferation and metabolism. The nuclear neddylated PTEN dephosphorylates the fatty acid synthase (FASN) protein, inhibits the TRIM21-mediated ubiquitylation and degradation of FASN, and then promotes de novo fatty acid synthesis. In human breast cancer tissues, neddylated PTEN correlates with tumor progression and poor prognosis. Therefore, we demonstrate a previously unidentified pool of nuclear PTEN in the Nedd8-conjugated form and an unexpected tumor-promoting role of neddylated PTEN.

The Absence of PTEN in Breast Cancer Is a Driver of MLN4924 Resistance.

Background: Numerous studies have indicated that the neddylation pathway is closely associated with tumor development. MLN4924 (Pevonedistat), an inhibitor of the NEDD8-activating E1 enzyme, is considered a promising chemotherapeutic agent. Recently, we demonstrated that neddylation of the tumor suppressor PTEN occurs under high glucose conditions and promotes breast cancer development. It has been shown, however, that PTEN protein levels are reduced by 30-40% in breast cancer. Whether this PTEN deficiency affects the anti-tumor function of MLN4924 is unknown. Methods: In the present study, cell counting kit-8 and colony formation assays were used to detect cell proliferation, and a transwell system was used to quantify cell migration. A tumor growth assay was performed in BALB/c nude mice. The subcellular location of PTEN was detected by fluorescence microscopy. The CpG island of the UBA3 gene was predicted by the Database of CpG Islands and UCSC database. Western blotting and qRT-PCR were used to measure the expression of indicated proteins. The Human Protein Atlas database, the Cancer Genome Atlas and Gene Expression Omnibus datasets were used to validate the expression levels of UBA3 in breast cancer. Results: Our data show that the anti-tumor efficacy of MLN4924 in breast cancer cells was markedly reduced with the deletion of PTEN. PI3K/Akt signaling pathway activity correlated positively with UBA3 expression. Pathway activity correlated negatively with NEDP1 expression in PTEN-positive breast cancer patients, but not in PTEN-negative patients. We also demonstrate that high glucose conditions upregulate UBA3 mRNA by inhibiting UBA3 promoter methylation, and this upregulation results in the overactivation of PTEN neddylation in breast cancer cells. Conclusion: These data suggest a mechanism by which high glucose activates neddylation. PTEN is critical, if not indispensable, for MLN4924 suppression of tumor growth; PTEN status thus may help to identify MLN4924-responsive breast cancer patients.

USF2 inhibits the transcriptional activity of Smurf1 and Smurf2 to promote breast cancer tumorigenesis.

Smurf1 (Smad ubiquitylation regulatory factor 1) and Smurf2 are negative regulators of the TGF-ß (transforming growth factor-ß) pathway. The protein stability and ubiquitin E3 activity regulation of Smurfs have been well studied. However, the mechanism of Smurfs expression at the transcriptional level remains uncharacterized. Here, we reported that USF2 (upstream stimulatory factor 2), a basic helix-loop-helix-leucine-zip transcription factor, is necessary for the transcriptional activity of Smurf1 and Smurf2. The 5'-flanking sequences of the Smurfs gene have more than one E-box motifs, and USF2 bounds the Smurfs promoter in vitro and in vivo. Over-expression USF2 inhibited the transcriptional activity of the Smurfs, and Smurfs mRNA was markedly decreased. Therefore, the activity of TGF-ß was distinctly enhanced. Furthermore, in human breast cancers, USF2 was abnormally high expressed and correlated with cancer progression. USF2 was specifically inversely correlated with Smurfs in Luminal A subtype breast cancer patients. These findings suggest the mechanism regulation of Smurfs transcriptional activity, and shed new light on the cancer-promoting role of USF2.