iProPhos: A Web-Based Interactive Platform for Integrated Proteome and Phosphoproteome Analysis.

Large-scale omics studies have generated a wealth of mass spectrometry-based proteomics data, which provide additional insights into disease biology spanning genomic boundaries. However, there is a notable lack of web-based analysis and visualization tools that facilitate the reutilization of these data. Given this challenge, we present iProPhos, a user-friendly web server to deliver interactive and customizable functionalities. iProPhos incorporates a large number of samples, including 1444 tumor samples and 746 normal samples across 12 cancer types, sourced from the Clinical Proteomic Tumor Analysis Consortium. Additionally, users can also upload their own proteomics/phosphoproteomics data for analysis and visualization. In iProPhos, users can perform profiling plotting and differential expression, patient survival, clinical feature-related, and correlation analyses, including protein-protein, mRNA-protein, and kinase-substrate correlations. Furthermore, functional enrichment, protein-protein interaction network, and kinase-substrate enrichment analyses are accessible. iProPhos displays the analytical results in interactive figures and tables with various selectable parameters. It is freely accessible at http://longlab-zju.cn/iProPhos without login requirement. We present two case studies to demonstrate that iProPhos can identify potential drug targets and upstream kinases contributing to site-specific phosphorylation. Ultimately, iProPhos allows end-users to leverage the value of big data in cancer proteomics more effectively and accelerates the discovery of novel therapeutic targets.

Oxidative Stress and Endoplasmic Reticulum Stress Contributes to Arecoline and Its Secondary Metabolites-Induced Dyskinesia in Zebrafish Embryos.

Areca nut has been listed as one of the most addictive substances, along with tobacco, alcohol and caffeine. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on embryonic development and motor behavior are rarely addressed in zebrafish embryo-larvae. Herein, we investigated the effects of exposure to three alkaloids (arecoline and secondary metabolites-arecaidine and arecoline N-oxide) on the developmental parameters, locomotive behavior, oxidative stress and transcriptome of zebrafish embryos. Zebrafish embryos exposed to different concentrations (0, 0.1, 1, 10, 100 and 1000 µM) of arecoline, arecaidine and arecoline N-oxide showed no changes in mortality and hatchability rates, but the malformation rate of zebrafish larvae was significantly increased in a dose-dependent manner and accompanied by changes in body length. Moreover, the swimming activity of zebrafish larvae decreased, which may be due to the increase in reactive oxygen species and the imbalance between oxidation and antioxidation. Meanwhile, transcriptome analysis showed that endoplasmic reticulum stress and the apoptosis p53 signaling pathway were significantly enriched after exposure to arecoline and arecoline N-oxide. However, arecaidine exposure focuses on protein synthesis and transport. These findings provide an important reference for risk assessment and early warning of areca nut alkaloid exposure.

Four novel mutations identification in 17 beta-hydroxysteroid dehydrogenase-3 deficiency and our clinical experience: possible benefits of early treatment.

Introduction: Individuals with 17-beta-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) deficiency face a multitude of challenges, primarily concerning genital appearance, potential malignancy risks, and fertility issues. This study reports our findings from an investigation involving five individuals affected by 17ß-HSD3 deficiency, ranging in age from pre-adolescence to adolescence. Notably, we identified four previously unreported mutations in these subjects. Methods: Our study included a comprehensive evaluation to determine the potential occurrence of testicular tumors. The methods involved clinical examinations, genetic testing, hormone profiling, and patient history assessments. We closely monitored the progress of the study subjects throughout their treatment. Results: The results of this evaluation conclusively ruled out the presence of testicular tumors among our study subjects. Moreover, four of these individuals successfully underwent gender transition. Furthermore, we observed significant improvements in genital appearance following testosterone treatment, particularly among patients in the younger age groups who received appropriate treatment interventions. Discussion: These findings underscore the critical importance of early intervention in addressing concerns related to genital appearance, based on our extensive clinical experience and assessments. In summary, our study provides insights into the clinical aspects of 17ß-HSD3 deficiency, emphasizing the vital significance of early intervention in addressing genital appearance concerns. This recommendation is supported by our comprehensive clinical assessments and experience.

Identification of rare PTCH1 nonsense variant causing orofacial cleft in a Chinese family and an up-to-date genotype-phenotype analysis.

The Patched 1 (PTCH1) gene encodes a membrane receptor involved in the Hedgehog (Hh) signaling pathway, an abnormal state of which may result in congenital defects or human tumors. In this study, we conducted whole-exome sequencing on a three-generation Chinese family characterized with variable penetrance of orofacial clefts. A rare heterozygous variant in the PTCH1 gene (c.2833C > T p.R945X) was identified as a disease-associated mutation. Structural modeling revealed a truncation starting from the middle of the second extracellular domain of PTCH1 protein. This may damage its ligand recognition and sterol transportation abilities, thereby affecting the Hh signaling pathway. Biochemical assays indicated that the R945X protein had reduced stability compared to the wild-type in vitro. In addition, we reviewed the locations and mutation types of PTCH1 variants in individuals with clefting phenotypes, and analyzed the associations between clefts and locations or types of variants within PTCH1. Our findings provide further evidence that PTCH1 variants result in orofacial clefts, and contributed to genetic counseling and clinical surveillance in this family.

Palmitic Acid-Induced Podocyte Apoptosis via the Reactive Oxygen Species-Dependent Mitochondrial Pathway.

BACKGROUND/AIMS: Chronic kidney disease (CKD) is often accompanied by hyperlipidemia, which accelerates progression of the disease. Podocyte injury can lead to dysfunction of the glomerular filtration barrier, which is associated with proteinuria, a risk marker for the progression of CKD. Our previous studies demonstrated that palmitic acid (PA) can induce podocyte apoptosis; however, the underlying mechanisms are unclear. In the present study, we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes. METHODS: We cultured mouse podocytes and treated them with PA. Then, cell viability was measured using the Cell Counting Kit-8 colorimetric assay, lipid uptake was assessed by Oil Red O staining and boron-dipyrromethene staining, apoptosis was measured by flow cytometry, mitochondrial injury was assessed by JC-1 staining and transmission electron microscopy, and mitochondrial production of reactive oxygen species (ROS) was evaluated by fluorescence microscopy using the MitoSOX Red reagent. The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax, Bid, cytochrome c, and Fas-associated protein with death domain (FADD) using western blotting. The translocation of Bax and cytochrome c were detected by immunofluorescence. RESULTS: PA treatment significantly increased lipid accumulation and induced podocyte apoptosis. We investigated whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) were involved in the execution of PA-induced podocyte apoptosis, and found that the levels of FADD, caspase-8, and Bid did not significantly change during this process. Meanwhile, PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression, with Bax translocation to the mitochondria. Furthermore, PA treatment induced mitochondrial impairment, and triggered the release of cytochrome c from the mitochondria to cytosol, with a concomitant dose-dependent increase in the levels of cleaved caspase-9, cleaved caspase-3, and PARP. Meanwhile, PA treatment increased mitochondrial production of ROS, and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis. CONCLUSION: Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway, and mitochondrial ROS production participated in this process, thus potentially contributing to podocyte injury.

Berberine Protects Against Palmitate-Induced Apoptosis in Tubular Epithelial Cells by Promoting Fatty Acid Oxidation.

BACKGROUND Increased lipid accumulation in renal tubular epithelial cells (TECs) contributes to their injury and dysfunction and progression of tubulointerstitial fibrosis. Berberine (BBR), a natural plant alkaloid isolated from traditional medicine herbs, is effective in lowing serum lipid, and has a protective effect on chronic kidney disease (CKD) with dyslipidemia, including diabetic nephropathy. The aim of this study was to investigate the effect of BBR on palmitate (PA)-induced lipid accumulation and apoptosis in TECs. MATERIAL AND METHODS Human kidney proximal tubular epithelial cell line (HK-2) cells were treated with PA, BBR, and/or palmitoyltransferase 1A (CPT1A) inhibitor Etomoxir. Intracellular lipid content was assessed by Oil Red O and Nile Red staining. Cell apoptosis rate was evaluated by flow cytometry assay. The expression of apoptosis-related protein cleaved-caspase3 and fatty acid oxidation (FAO)-regulating proteins, including CPT1A, peroxisome proliferator-activated receptor α (PPARα), and PPARγ co-activator-1α (PGC1α), was measured by Western blot analysis and immunofluorescence. RESULTS In the present study, PA treatment increased intracellular lipid deposition accompanied by elevated apoptosis in TECs compared with control group, whereas the protein expression of CPT1A, PPARα, and PGC1α, did not correspondingly increase in TECs. BBR significantly up-regulated the protein expression of CPT1A, PPARα, and PGC1α in TECs treated with or without PA, and reversed PA-induced intracellular lipid accumulation and apoptosis. Moreover, the CPT1A inhibitor Etomoxir counteracted the protective effect of BBR in TECs. CONCLUSIONS These in vitro findings suggest that PA can induce intracellular lipid accumulation and apoptosis in TECs, and the mechanism may be associated with inducing defective FAO, whereas BBR can protect TECs against PA-induced intracellular lipid accumulation and apoptosis by promoting FAO.

Tumor-derived exosomes antagonize innate antiviral immunity.

Malignancies can compromise innate immunity, but the mechanisms of this are largely unknown. Here we found that, via tumor-derived exosomes (TEXs), cancers were able to transfer activated epidermal growth factor receptor (EGFR) to host macrophages and thereby suppress innate antiviral immunity. Screening of the human kinome identified the kinase MEKK2 in macrophages as an effector of TEX-delivered EGFR that negatively regulated the antiviral immune response. In the context of experimental tumor implantation, MEKK2-deficient mice were more resistant to viral infection than were wild-type mice. Injection of TEXs into mice reduced innate immunity, increased viral load and increased morbidity in an EGFR- and MEKK2-dependent manner. MEKK2 phosphorylated IRF3, a transcription factor crucial for the production of type I interferons; this triggered poly-ubiquitination of IRF3 and blocked its dimerization, translocation to the nucleus and transcriptional activity after viral infection. These findings identify a mechanism by which cancer cells can dampen host innate immunity and potentially cause patients with cancer to become immunocompromised.

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling.

SMAD4 is a common intracellular effector for TGF-ß family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-ß family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.

Rnf25/AO7 positively regulates wnt signaling via disrupting Nkd1-Axin inhibitory complex independent of its ubiquitin ligase activity.

Wnt signaling components have been shown to control key events in embryogenesis and to maintain tissue homeostasis in the adult. Nkd1/2 and Axin1/2 protein families are required for feedback regulation of Wnt signaling. The mechanisms by which Nkd1 and Nkd2 exhibit significant differences in signal transduction remain incompletely understood. Here we report that Rnf25/AO7, a previously identified E3 ubiquitin ligase for Nkd2, physically interacts with Nkd1 and Axin in an E3 ligase-independent manner to strengthen Wnt signalling. To determine the biological role of Rnf25 in vivo, we found that the renal mesenchymal cell, in which rnf25 was knocked-down, also exhibited more epithelial characters than MOCK control. Meanwhile, the transcriptional level of rnf25 was elevated in three separate tumor tissues more than that in paracarcinomatous tissue. Depletion of Rnf25 in zebrafish embryos attenuated transcriptions of maternal and zygotic Wnt target genes. Our results indicated that Rnf25 might serve as a molecular device, controlling the different antagonizing functions against canonical Wnt signaling between Nkd1 and Nkd2 cooperated with Axin.

Characterization of zebrafish Pax1b and Pax9 in fin bud development.

Both Pax1 and Pax9 belong to the important paired box gene family (PAX), which mainly participates in animal development and sclerotome differentiation. To date, the precise molecular mechanism and related signaling pathway of Pax1 remain unclear. In our study, microinjection of morpholino- (MO-) modified antisense oligonucleotides against pax1b induced pectoral fin bud defects. Furthermore, we demonstrate that the phenotypes caused by the knockdown of Pax1b in zebrafish could not be phenocopied by pax9 MO and could not be rescued by either Pax1a or Pax9 overexpression. We further find that Pax1b affects the expression of col2a1, Uncx4.1, Noggin3, and aggrecan, confirming the role of Pax1b in chondrocyte differentiation and bone maturation. Moreover, we identify an interaction between PAX1 and FOXO1 and find that the interaction was enhanced under hypoxia stress. Together, this evidence for cell death caused by pax1b knockdown provides new insight into the role of the Pax protein family in cell fate determination and tissue specification.

GPR126 protein regulates developmental and pathological angiogenesis through modulation of VEGFR2 receptor signaling.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for development, wound healing, and tumor progression. The VEGF pathway plays irreplaceable roles during angiogenesis, but how other signals cross-talk with and modulate VEGF cascades is not clearly elucidated. Here, we identified that Gpr126, an endothelial cell-enriched gene, plays an important role in angiogenesis by regulating endothelial cell proliferation, migration, and tube formation. Knockdown of Gpr126 in the mouse retina resulted in the inhibition of hypoxia-induced angiogenesis. Interference of Gpr126 expression in zebrafish embryos led to defects in intersegmental vessel formation. Finally, we identified that GPR126 regulated the expression of VEGFR2 by targeting STAT5 and GATA2 through the cAMP-PKA-cAMP-response element-binding protein signaling pathway during angiogenesis. Our findings illustrate that GPR126 modulates both physiological and pathological angiogenesis through VEGF signaling, providing a potential target for the treatment of angiogenesis-related diseases.

TRAF4 mediates activation of TGF-ß signaling and is a biomarker for oncogenesis in breast cancer.

The tumor-promoting arm of transforming growth factor beta (TGF-ß) receptor signaling contributes to advanced cancer progression and is considered a master regulator of breast cancer metastasis. In mammals, there are six distinct members in the tumor-necrosis factor receptor (TNFR)-associated factor (TRAF) family (TRAF1-TRAF6), with the function of TRAF4 not being extensively studied in the past decade. Although numerous studies have suggested that there is elevated TRAF4 expression in human cancer, it is still unknown in which oncogenic pathway TRAF4 is mainly implicated. This review highlights TGF-ß-induced SMAD-dependent signaling and non-SMAD signaling as the major pathways regulated by TRAF4 involved in breast cancer metastasis.

Suppression of GSK3ß by ERK mediates lipopolysaccharide induced cell migration in macrophage through ß-catenin signaling.

We investigate the role of ß-catenin signaling in the response of macrophage to lipopolysaccharide (LPS) using RAW264.7 cells. LPS rapidly stimulated cytosolic ß-catenin accumulation. ß-catenin-mediated transcription was showed to be required for LPS induced gene expression and cell migration. Mechanically, ERK activation-primed GSK3ß inactivation by Akt was demonstrated to mediate the LPS induced ß-catenin accumulation. Overall, our findings suggest that suppression of GSK3ß by ERK stimulates ß-catenin signaling therefore contributes to LPS induced cell migration in macrophage activation.

USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-ß type I receptor.

The stability and membrane localization of the transforming growth factor-ß (TGF-ß) type I receptor (TßRI) determines the levels of TGF-ß signalling. TßRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-ß signalling. USP4 was found to directly interact with TßRI and act as a deubiquitylating enzyme, thereby controlling TßRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-ß-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TßRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-ß and AKT signalling pathways.

Bisindoylmaleimide I enhances osteogenic differentiation.

The Wnt/ß-catenin and bone morphogenetic proteins (BMPs) pathways play important roles in controlling osteogenesis. Using a cell-based kinase inhibitor screening assay, we identified the compound bisindoylmaleimide I (BIM) as a potent agonist of the cytosolic ß-catenin accumulation in preosteoblast cells. Through suppressing glycogen synthase kinase 3ß enzyme activities, BIM upregulated ß-catenin responsive transcription and extended duration of BMP initiated signal. Functional analysis revealed that BIM promoted osteoblast differentiation and bone formation. The treatment of human mesenchymal stem cells with BIM promoted osteoblastogenesis. Our findings provide a new strategy to regulate mesenchymal stem cell differentiation by integration of the cellular signaling pathways.

Study on the effect of IRE1a on cell growth and apoptosis via modulation PLK1 in ER stress response.

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1a by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1a induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1a can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1a-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1a in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1a may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.

FLNA and PGK1 are two potential markers for progression in hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most deadly diseases; metastasis and recurrence are the most important factors that affect the therapy of HCC chronically. Until now, the prognosis for the metastasis of HCC had not improved. Recently, several proteins that are related to metastasis and invasion of HCC were identified, but the effective markers still remain to be elucidated. METHODS: In this study, comparative proteomics was used to study the differentially expressed proteins in two HCC cell lines MHCC97L and HCCLM9, which have low and high metastatic potentials, respectively. RESULTS: Our findings indicated that filamin A (FLNA) and phosphoglycerate kinase 1 (PGK1) were two significantly differentially expressed proteins, with high expression in HCCLM9 cells, and may influence the metastasis of HCC cells. CONCLUSION: Taken together with the confirmation of expression on the mRNA level, we propose the use of FLNA and PGK1 as potential markers for the progression of HCC.

Protein encoded by the Axin(Fu) allele effectively down-regulates Wnt signaling but exerts a dominant negative effect on c-Jun N-terminal kinase signaling.

Axin plays an architectural role in many important signaling pathways that control various aspects of development and tumorigenesis, including the Wnt, transforming growth factor-beta, MAP kinase pathways, as well as p53 activation cascades. It is encoded by the mouse Fused (Fu) locus; the Axin(Fu) allele is caused by insertion of an IAP transposon. Axin(Fu/Fu) mice display varying phenotypes ranging from embryonic lethality to relatively normal adulthood with kinky tails. However, the protein product(s) has not been identified or characterized. In the present study, we conducted immunoprecipitation using brain extracts from the Axin(Fu) mice with specific antibodies against different regions of Axin and found that a truncated Axin containing amino acids 1-596 (designated as Axin(Fu-NT)) and the full-length complement of Axin (Axin(WT)) can both be generated from the Axin(Fu) allele. When tested for functionality changes, Axin(Fu-NT) was found to abolish Axin-mediated activation of JNK, which plays a critical role in dorsoventral patterning. Together with a proteomics approach, we found that Axin(Fu-NT) contains a previously uncharacterized dimerization domain and can form a heterodimeric interaction with Axin(WT). The Axin(Fu-NT)/Axin(WT) is not conducive to JNK activation, providing a molecular explanation for the dominant negative effect of Axin(Fu-NT) on JNK activation by wild-type Axin. Importantly, Axin(Fu-NT) exhibits no difference in the inhibition of Wnt signaling compared with Axin(WT) as determined by reporter gene assays, interaction with key Wnt regulators, and expression of Wnt marker genes in zebrafish embryos, suggesting that altered JNK signaling contributes, at least in part, to the developmental defects seen in Axin(Fu) mice.

Tob1 controls dorsal development of zebrafish embryos by antagonizing maternal beta-catenin transcriptional activity.

Maternal beta-catenin and Nodal signals are essential for the formation of the dorsal organizer, which, in turn, induces neural and other dorsal tissue development in vertebrate embryos. Tob (Transducer of ErbB2) proteins possess antiproliferative properties and are known to influence BMP signaling, but their relationship to other signaling pathways and to embryonic patterning in general was unclear. In this study, we demonstrate that zebrafish tob1a is required for correct dorsoventral patterning. Mechanistically, Tob1a inhibits beta-catenin transcriptional activity by physically associating with beta-catenin and preventing the formation of beta-catenin/LEF1 complexes. Although Tob1a can also inhibit the transcriptional activity of the Nodal effector Smad3, its role in limiting dorsal development is executed primarily by antagonizing the beta-catenin signal. We further demonstrate that Tob family members across species share similar biochemical properties and biological activities.