Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression.

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid ß-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid ß-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid ß-oxidation.

The Effects of Graded Levels of Calorie Restriction: XIX. Impact of Graded Calorie Restriction on Protein Expression in the Liver.

Calorie restriction (CR) extends life span by modulating the mechanisms involved in aging. We quantified the hepatic proteome of male C57BL/6 mice exposed to graded levels of CR (0%-40% CR) for 3 months, and evaluated which signaling pathways were most affected. The metabolic pathways most significantly stimulated by the increase in CR, included the glycolysis/gluconeogenesis pathway, the pentose phosphate pathway, the fatty acid degradation pathway, the valine, leucine, and isoleucine degradation pathway, and the lysine degradation pathway. The metabolism of xenobiotics by cytochrome P450 pathway was activated and feminized by increased CR, while production in major urinary proteins (Mups) was strongly reduced, consistent with a reduced investment in reproduction as predicted by the disposable soma hypothesis. However, we found no evidence of increased somatic protection, and none of the 4 main pathways implied to be linked to the impact of CR on life span (insulin/insulin-like growth factor [IGF-1], nuclear factor-κB [NF-κB], mammalian Target of Rapamycin [mTOR], and sirtuins) as well as pathways in cancer, were significantly changed at the protein level in relation to the increase in CR level. This was despite previous work at the transcriptome level in the same individuals indicating such changes. On the other hand, we found Aldh2, Aldh3a2, and Aldh9a1 in carnitine biosynthesis and Acsl5 in carnitine shuttle system were up-regulated by increased CR, which are consistent with our previous work on metabolome of the same individuals. Overall, the patterns of protein expression were more consistent with a "clean cupboards" than a "disposable soma" interpretation.

Fu Fang Gang Liu aqueous extract inhibits the proliferation of HeLa cells by causing deoxyribonucleic acid damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Fu Fang Gang Liu (FFGL) is an effective formula for treating wart proliferation caused by human papillomavirus (HPV) infection and has the potential to treat HPV-related cancers. However, scientific evidence of its anti-tumor activity against cervical cancer, the most common cancer caused by HPV, is lacking. AIM OF THE STUDY: To clarify the anti-tumor effect of an FFGL aqueous extract on human cervical cancer and its possible mechanism of cell cycle arrest in HeLa cells. MATERIALS AND METHODS: The anti-proliferative effect of FFGL on cervical cancer cells was assessed using the cell counting kit-8 assay. The proportion of apoptotic cells, cell cycle distribution, and cell division rate were determined using flow cytometry. Quantitative proteomics was used to identify differentially expressed proteins after FFGL treatment, and bioinformatics analysis was used to identify key nodal proteins affected by FFGL. Immunofluorescence and western blot analyses were used to explore changes in the expression of related proteins in the cell cycle and DNA damage pathways to elucidate the potential mechanism of action of FFGL against HeLa cell proliferation. RESULTS: FFGL inhibited cervical cancer cell proliferation and caused cell cycle arrest. According to quantitative proteomics, CyclinB1 may play an important role in the anti-proliferative effect of FFGL on HeLa cells. Additional experiments showed that FFGL aqueous extract caused ATM-mediated DNA damage, further phosphorylated CHK2, led to the inactivation of Cdc25C, inhibited the activity of the CDK1/CyclinB1 complex, and resulted in cell cycle arrest. CONCLUSIONS: FFGL can inhibit cervical cancer cell proliferation. Furthermore, it can increase CDK1 phosphorylation, block the cell cycle by causing DNA damage, and inhibit HeLa cell proliferation.

O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection.

Human papillomaviruses (HPVs) cause a subset of head and neck squamous cell carcinomas (HNSCCs). Previously, we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT), but OGT substrates affected by this increase are unclear. Here, we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, Unc-51-like kinase 1 (ULK1), an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized, and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409, which is distinct from the previously reported Thr635/Thr754 sites. It has been demonstrated that PKCα mediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperone-mediated autophagy (CMA) pathway. Using biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at Ser423. Moreover, mutations of Ser409A and its neighboring site Ser410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperone HSC70 (heat shock cognate 70 kDa protein). Furthermore, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of The Cancer Genome Atlas (TCGA) database reveals that ULK1 is upregulated in HPV-positive HNSCCs, and its level positively correlates with HNSCC patient survival. Overall, our work demonstrates that O-GlcNAcylation of ULK1 is altered in response to environmental changes. O-GlcNAcylation of ULK1 at Ser409 and perhaps Ser410 stabilizes ULK1, which might underlie the molecular mechanism of HPV-positive HNSCC patient survival.

Scaling-up and proteomic analysis reveals photosynthetic and metabolic insights toward prolonged H2 photoproduction in Chlamydomonas hpm91 mutant lacking proton gradient regulation 5 (PGR5).

Clean and sustainable H2 production is crucial to a carbon-neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not large-scalable because of lacking desirable strains with both optimal H2 productivity and sufficient knowledge of underlying molecular mechanism. We hereby carried out extensive and in-depth investigations of H2 photoproduction of hpm91 mutant lacking PGR5 (Proton Gradient Regulation 5) toward its up-scaling and fundamental mechanism issues. We show that hpm91 is at least 100-fold scalable (up to 10 L) with continuous H2 collection of 7287 ml H2/10L-HPBR in averagely 26 days under sulfur deprivation. Also, we show that hpm91 is robust and active during sustained H2 photoproduction, most likely due to decreased intracellular ROS relative to wild type. Moreover, we obtained quantitative proteomic profiles of wild type and hpm91 at four representing time points of H2 evolution, leading to 2229 and 1350 differentially expressed proteins, respectively. Compared to wild type, major proteome alterations of hpm91 include not only core subunits of photosystems and those related to anti-oxidative responses but also essential proteins in photosynthetic antenna, C/N metabolic balance, and sulfur assimilation toward both cysteine biosynthesis and sulfation of metabolites during sulfur-deprived H2 production. These results reveal not only new insights of cellular and molecular basis of enhanced H2 production in hpm91 but also provide additional candidate gene targets and modules for further genetic modifications and/or in artificial photosynthesis mimics toward basic and applied research aiming at advancing solar-H2 technology.

The plastid-encoded protein Orf2971 is required for protein translocation and chloroplast quality control.

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

Modulation of nitrate-induced phosphate response by the MYB transcription factor RLI1/HINGE1 in the nucleus.

The coordinated utilization of nitrogen (N) and phosphorus (P) is vital for plants to maintain nutrient balance and achieve optimal growth. Previously, we revealed a mechanism by which nitrate induces genes for phosphate utilization; this mechanism depends on NRT1.1B-facilitated degradation of cytoplasmic SPX4, which in turn promotes cytoplasmic-nuclear shuttling of PHR2, the central transcription factor of phosphate signaling, and triggers the nitrate-induced phosphate response (NIPR) and N-P coordinated utilization in rice. In this study, we unveiled a fine-tuning mechanism of NIPR in the nucleus regulated by Highly Induced by Nitrate Gene 1 (HINGE1, also known as RLI1), a MYB-transcription factor closely related to PHR2. RLI1/HINGE1, which is transcriptionally activated by PHR2 under nitrate induction, can directly activate the expression of phosphate starvation-induced genes. More importantly, RLI1/HINGE1 competes with PHR2 for binding to its repressor proteins in the nucleus (SPX proteins), and consequently releases PHR2 to further enhance phosphate response. Therefore, RLI1/HINGE1 amplifies the phosphate response in the nucleus downstream of the cytoplasmic SPX4-PHR2 cascade, thereby enabling fine-tuning of N-P balance when nitrate supply is sufficient.

CDK4/6 regulate lysosome biogenesis through TFEB/TFE3.

Lysosomes are degradation and signaling organelles that adapt their biogenesis to meet many different cellular demands; however, it is unknown how lysosomes change their numbers for cell division. Here, we report that the cyclin-dependent kinases CDK4/6 regulate lysosome biogenesis during the cell cycle. Chemical or genetic inactivation of CDK4/6 increases lysosomal numbers by activating the lysosome and autophagy transcription factors TFEB and TFE3. CDK4/6 interact with and phosphorylate TFEB/TFE3 in the nucleus, thereby inactivating them by promoting their shuttling to the cytoplasm. During the cell cycle, lysosome numbers increase in S and G2/M phases when cyclin D turnover diminishes CDK4/6 activity. These findings not only uncover the molecular events that direct the nuclear export of TFEB/TFE3, but also suggest a mechanism that controls lysosome biogenesis in the cell cycle. CDK4/6 inhibitors promote autophagy and lysosome-dependent degradation, which has important implications for the therapy of cancer and lysosome-related disorders.

Evaluation of the Potential Risk of Advanced Peak Determination in Distorting Isobaric Labeling-Based Single-Shot Proteome Quantitation.

The recent development and implementation of the advanced peak determination (APD) algorithm with MS instrument dramatically increased the sampling of low abundance features for MS/MS fragmentation. After in-depth evaluation, it is found that with APD on, many chimeric spectra are acquired through co-fragmentation of high abundance contaminants with low abundance targets, and such co-fragmentations are largely avoided when APD is off. To evaluate whether such a co-fragmentation could significantly distort the accuracy of the isobaric-labeling based quantitation of the low abundance target, a single-shot TMT experiment is performed using a two-proteome model, whereby each TMT channel contains premixed peptides from human and a cyanobacterium with a known ratio. Unexpectedly, it is found that APD does not significantly distort TMT ratios, probably because the majority of the APD-specific chimeric spectra are not identifiable. Nevertheless, a few examples of significant distortion of TMT ratios of low abundance peptides caused by APD is found through manual inspection, and suggests that APD should be off in a single-shot TMT experiment to avoid the laborious and time-costing manual inspection.

Extensive protein S-nitrosylation associated with human pancreatic ductal adenocarcinoma pathogenesis.

NO (nitric oxide)-mediated protein S-nitrosylation has been established as one major signaling mechanism underlying cancer initiation and development, but its roles in PDAC (pancreatic ductal adenocarcinoma) pathogenesis still remain largely unexplored. In this study, we identified 585 unique S-nitrosylation sites among 434 proteins in PDAC patients and PANC-1 cell line by a site-specific proteomics. Larger number of S-nitrosylated proteins were identified in PDAC tissues and PANC-1 cells than adjacent non-cancerous tissues. These S-nitrosylated proteins are significantly enriched in a multitude of biological processes associated with tumorigenesis, including carbohydrate metabolism, cytoskeleton regulation, cell cycle, focal adhesion, adherent junctions, and cell migration. Components of the pancreatic cancer pathway were extensively S-nitrosylated, such as v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) and Signal transducer and activator of transcription 3 (STAT3). Moreover, NOS (NO synthase) inhibitor significantly repressed STAT3 S-nitrosylation in PANC-1 cells, which caused significant increase of STAT3 phosphorylation and PANC-1 cell viability, suggesting important roles of protein S-nitrosylation in PDAC development. These results revealed extensive protein S-nitrosylation associated with PDAC pathogenesis, which provided a basis for protein modification-based cancer diagnosis and targeted therapy.

Overdosage of Balanced Protein Complexes Reduces Proliferation Rate in Aneuploid Cells.

Cells with complex aneuploidies display a wide range of phenotypic abnormalities. However, the molecular basis for this has been mainly studied in trisomic (2n + 1) and disomic (n + 1) cells. To determine how karyotype affects proliferation in cells with complex aneuploidies, we generated 92 2n + x yeast strains in which each diploid cell has between 3 and 12 extra chromosomes. Genome-wide and, for individual protein complexes, proliferation defects are caused by the presence of protein complexes in which all subunits are balanced at the 3-copy level. Proteomics revealed that over 50% of 3-copy members of imbalanced complexes were expressed at only 2n protein levels, whereas members of complexes in which all subunits are stoichiometrically balanced at 3 copies per cell had 3n protein levels. We validated this finding using orthogonal datasets from yeast and from human cancers. Taken together, our study provides an explanation of how aneuploidy affects phenotype.

Substrate-independent immunomodulatory characteristics of mesenchymal stem cells in three-dimensional culture.

Mesenchymal stem cells (MSCs) play important roles in tissue regeneration, and multi-lineage differentiation and immunomodulation are two major characteristics of MSCs that are utilized in stem cell therapy. MSCs in vivo have a markedly different three-dimensional (3D) niche compared to the traditional two-dimensional (2D) culture in vitro. A 3D scaffold is predicted to provide an artificial 3D environment similar to the in vivo environment. Significant changes in MSC differentiation are shown to be occurred when under 3D culture. However, the immunomodulatory characteristics of MSCs under 3D culture remain unknown. In this study, 3D culture systems were constructed using different substrates to evaluate the common immunomodulatory characteristics of MSCs. Compared to the MSCs under 2D culture, the MSCs under 3D culture, which had higher stemness and maintained cell phenotype, showed altered immunophenotypic pattern. Gene expression profile analysis at mRNA and protein level detected by gene chip and protein chip, respectively, further revealed the difference between 3D cultured MSCs and 2D cultured MSCs, which was mainly concentrated in the immunoregulation related aspects. Moreover, the immunoregulatory role of 3D culture was confirmed by our immunosuppressive experiments. These findings demonstrated that the immunomodulatory capacities of MSCs were enhanced by the 3D geometry of substrates.

Phosphorylation of serine/arginine-rich splicing factor 1 at tyrosine 19 promotes cell proliferation in pediatric acute lymphoblastic leukemia.

Serine/arginine-rich splicing factor 1 (SRSF1) has been linked to various human cancers including pediatric acute lymphoblastic leukemia (ALL). Our previous study has shown that SRSF1 potentially contributes to leukemogenesis; however, its underlying mechanism remains unclear. In this study, leukemic cells were isolated from pediatric ALL bone marrow samples, followed by immunoprecipitation assays and mass spectrometry analysis specific to SRSF1. Subcellular localization of the SRSF1 protein and its mutants were analyzed by immunofluorescence staining. Cell growth, colony formation, cell apoptosis, and the cell cycle were investigated using stable leukemic cell lines generated with lentivirus-mediated overexpressed WT or mutant plasmids. Cytotoxicity of the Tie2 kinase inhibitor was also evaluated. Our results showed the phosphorylation of SRSF1 at tyrosine 19 (Tyr-19) was identified in newly diagnosed ALL samples, but not in complete remission or normal control samples. Compared to the SRSF1 WT cells, the missense mutants of the Tyr-19 phosphorylation affected the subcellular localization of SRSF1. In addition, the Tyr-19 phosphorylation of SRSF1 also led to increased cell proliferation and enhanced colony-forming properties by promoting the cell cycle. Remarkably, we further identified the kinase Tie2 as a potential therapeutic target in leukemia cells. In conclusion, we identify for the first time that the phosphorylation state of SRSF1 is linked to different phases in pediatric ALL. The Tyr-19 phosphorylation of SRSF1 disrupts its subcellular localization and promotes proliferation in leukemia cells by driving cell-cycle progression. Inhibitors targeting Tie2 kinase that could catalyze Tyr-19 phosphorylation of SRSF1 offer a promising therapeutic target for treatment of pediatric ALL.

cTAGE5/MEA6 plays a critical role in neuronal cellular components trafficking and brain development.

Normal neural development is essential for the formation of neuronal networks and brain function. Cutaneous T cell lymphoma-associated antigen 5 (cTAGE5)/meningioma expressed antigen 6 (MEA6) plays a critical role in the secretion of proteins. However, its roles in the transport of nonsecretory cellular components and in brain development remain unknown. Here, we show that cTAGE5/MEA6 is important for brain development and function. Conditional knockout of cTAGE5/MEA6 in the brain leads to severe defects in neural development, including deficits in dendrite outgrowth and branching, spine formation and maintenance, astrocyte activation, and abnormal behaviors. We reveal that loss of cTAGE5/MEA6 affects the interaction between the coat protein complex II (COPII) components, SAR1 and SEC23, leading to persistent activation of SAR1 and defects in COPII vesicle formation and transport from the endoplasmic reticulum to the Golgi, as well as disturbed trafficking of membrane components in neurons. These defects affect not only the transport of materials required for the development of dendrites and spines but also the signaling pathways required for neuronal development. Because mutations in cTAGE5/MEA6 have been found in patients with Fahr's disease, our study potentially also provides insight into the pathogenesis of this disorder.

Site-specific nitrosoproteomic identification of endogenously S-nitrosylated proteins in Arabidopsis.

Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.

Aged monkey brains reveal the role of ubiquitin-conjugating enzyme UBE2N in the synaptosomal accumulation of mutant huntingtin.

Although misfolded proteins are ubiquitinated and cleared by the proteasome, they can accumulate in synapses in aged neurons to promote synaptic dysfunction in a variety of neurodegenerative diseases, including Huntington's disease (HD), which is caused by polyglutamine expansion in huntingtin. The mechanism behind this aging-related phenomenon is unknown and has been difficult to investigate using animals with short life spans. With brain tissues from longer-lived rhesus monkeys of different ages, we found that aging reduces ubiquitin-proteasomal activity and also increases the level of ubiquitin-conjugating enzyme UBE2N (Ubc13) in synaptosomes. Synaptosomal fractions from aged monkey brain increase in vitro ubiquitinated huntingtin, whereas depletion of UBE2N markedly reduces this increase. Overexpressing UBE2N increases the aggregation of mutant huntingtin, and reducing UBE2N attenuates huntingtin aggregation in cellular and mouse models of HD. Our studies suggest that increased UBE2N plays a critical role in the synaptosomal accumulation of mutant huntingtin with age.

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

BSCTV C2 attenuates the degradation of SAMDC1 to suppress DNA methylation-mediated gene silencing in Arabidopsis.

Plant viruses are excellent tools for studying microbial-plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus-plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein-directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1.