The Arabidopsis Deubiquitylase OTU5 Suppresses Flowering by Histone Modification-Mediated Activation of the Major Flowering Repressors FLC, MAF4, and MAF5.

Distinct phylogeny and substrate specificities suggest that 12 Arabidopsis Ovarian Tumor domain-containing (OTU) deubiquitinases participate in conserved or plant-specific functions. The otu5-1 null mutant displayed a pleiotropic phenotype, including early flowering, mimicking that of mutants harboring defects in subunits (e.g., ARP6) of the SWR1 complex (SWR1c) involved in histone H2A.Z deposition. Transcriptome and RT-qPCR analyses suggest that downregulated FLC and MAF4-5 are responsible for the early flowering of otu5-1. qChIP analyses revealed a reduction and increase in activating and repressive histone marks, respectively, on FLC and MAF4-5 in otu5-1. Subcellular fractionation, GFP-fusion expression, and MNase treatment of chromatin showed that OTU5 is nucleus-enriched and chromatin-associated. Moreover, OTU5 was found to be associated with FLC and MAF4-5. The OTU5-associated protein complex(es) appears to be distinct from SWR1c, as the molecular weights of OTU5 complex(es) were unaltered in arp6-1 plants. Furthermore, the otu5-1 arp6-1 double mutant exhibited synergistic phenotypes, and H2A.Z levels on FLC/MAF4-5 were reduced in arp6-1 but not otu5-1. Our results support the proposition that Arabidopsis OTU5, acting independently of SWR1c, suppresses flowering by activating FLC and MAF4-5 through histone modification. Double-mutant analyses also indicate that OTU5 acts independently of the HUB1-mediated pathway, but it is partially required for FLC-mediated flowering suppression in autonomous pathway mutants and FRIGIDA-Col.

Genetic Alteration and Their Significance on Clinical Events in Small Cell Lung Cancer.

Introduction: Small cell lung cancer (SCLC), an aggressive subtype of lung cancer characterized by the development of neuroendocrine tumors, is prone to distant metastasis, resistant to platinum-based drugs and has a poor prognosis. The development of next-generation sequencing technology (NGS) has led to the identification of many genetic alterations in SCLC. Few druggable targeted molecules can be used in clinical practice. Currently, NGS is widely employed in routine clinical practice of non-small cell lung cancer to assist in therapeutic options and prognosis evaluation. This study aims to investigate genes involved in small cell lung cancer (SCLC), their occurrence and their significance in clinical events. Methods: Tumor tissue specimens from 18 Chinese SCLC patients were collected through a 520 cancer-related genes panel for next-generation sequencing. First, the association between sequence results and clinical outcomes was examined. Subsequently, data on clinical pathology and sequencing results were analyzed. Results: The Kaplan-Meier curve displayed a significant reduction in PFS for SCLC patients with LRP1B or MAP3K13 mutations. Overall survival (OS) of SCLC patients with MSH6 mutation was significantly higher than those with SPEN mutation. Conclusion: Next-generation sequencing demonstrates that the genetic landscape of SCLC. Mutation status of LRP1B, MAP3K13, MSH6 and SPEN has prognostic significance, which might be potential therapeutic targets. We found possible genes and related signaling pathways that affect metastasis. These results can improve our understanding of the mutation characteristics of SCLC and identify potential biomarkers to guide targeted therapies.

Dynamic changes of acquired T790M mutation and small cell lung cancer transformation in a patient with EGFR-mutant adenocarcinoma after first- and third-generation EGFR-TKIs: a case report.

Epithelial growth factor receptor (EGFR) T790M mutation and small cell lung cancer (SCLC) transformation are well-known resistance mechanisms acquired during treatment with EGFR tyrosine kinase inhibitors (TKIs). Various mechanisms sometimes coexist in patients. Here, we report a 57-year-old female diagnosed with stage IV lung adenocarcinoma, who harbored an EGFR exon 19 deletion mutation. This patient initially received gefitinib and progressed after 14 months. A repeat biopsy was performed, and the original EGFR exon 19 deletion and acquired exon 20 T790M mutation were identified. Then, pemetrexed plus carboplatin was administered as second-line and osimertinib as third-line treatment. Rapid progression and mixed response were observed after 2 months on osimertinib, with stable disease of the primary lung lesion but rapid growth of a right lower chest mass. The progressive chest lesion underwent biopsy, and the SCLC transformation was revealed. Furthermore, the patient was treated with etoposide and cisplatin, and she achieved disease control for 4 months. A fourth biopsy both for the primary lung lesion and the chest mass were finally conducted. Interestingly, the histopathology of the two different lesions showed adenocarcinoma and SCLC, respectively. The patient then rapidly suffered brain metastasis, and no EGFR mutations were detected in her cerebrospinal fluid (CSF). Overall survival (OS) of the patient was 29 months. This patient experienced concomitant resistance mechanisms of T790M mutation and SCLC transformation, which might have resulted from intra-tumor heterogeneity and drug-induced selection. Ultimately, this case reminds us that repeat biopsies are essential for patients receiving EGFR-TKIs in order to make appropriate treatment decisions according to the diverse mechanisms of acquired resistance.

PRAS40 hyperexpression promotes hepatocarcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers, whereas the molecular mechanism remains largely unknown. PRAS40 (encoded by AKT1S1) phosphorylation was increased in human melanoma, prostate cancer and lung cancer specimens, which was considered as the results of Akt activation. However the mechanism in detail and its role in HCC stay elusive. METHODS: PRAS40 expression and phosphorylation were analyzed in HCC specimens, and the survival rates of patients were investigated. Functional analyses of PRAS40 in HCC were performed in vivo and in vitro. The miR-124-3p binding sites in PRAS40 were investigated using luciferase assay. MiR-124-3p expression in HCC specimens was examined by In Situ hybridization, and the correlation to PRAS40 level was evaluated. FINDINGS: The phosphorylation, protein and mRNA levels of PRAS40 were increased significantly in HCC specimens from our cohorts and TCGA database, which was positively correlated to the poor prognosis of HCC patients. Compared to Akt1s1+/+ mice, hepatocarcinogenesis was suppressed in Akt1s1-/- mice, and the activation of Akt was impaired. PRAS40 depletion resulted in the inhibition of HCC cellular proliferation. Tumor suppressor miR-124-3p was found to downregulate PRAS40 expression by targeting its 3'UTR. MiR-124-3p levels were inversely correlated to PRAS40 protein and phosphorylation levels in HCC specimens. The proliferation inhibition by miR-124-3p mimics was partially reversed by exogenous PRAS40 introduction in HCC cells. INTERPRETATION: PRAS40 hyperexpression induced by loss of miR-124-3p contributes to PRAS40 hyperphosphorylation and hepatocarcinogenesis. These results could be expected to offer novel clues for understanding hepatocarcinogenesis and developing approaches.

The Deubiquitinase OTU5 Regulates Root Responses to Phosphate Starvation.

Phosphorus, taken up by plants as inorganic phosphate (Pi), is an essential but often growth-limiting mineral nutrient for plants. As part of an orchestrated response to improve its acquisition, insufficient Pi supply triggers alterations in root architecture and epidermal cell morphogenesis. Arabidopsis (Arabidopsis thaliana) mutants defective in the expression of the OVARIAN TUMOR DOMAIN-CONTAINING DEUBIQUITINATING ENZYME5 (OTU5) exhibited a constitutive Pi deficiency root phenotype, comprising the formation of long and dense root hairs and attenuated primary root growth. Quantitative protein profiling of otu5 and wild-type roots using the isobaric tag for relative and absolute quantification methodology revealed genotype- and Pi-dependent alterations in protein profiles. In otu5 plants, Pi starvation caused a short-root-hair phenotype and decreased abundance of a suite of Pi-responsive root hair-related proteins. Mutant plants also showed the accumulation of proteins involved in chromatin remodeling and altered distribution of reactive oxygen species along the root, which may be causative for the alterations in root hair morphogenesis. The root hair phenotype of otu5 was synergistic to that of actin-related protein6 (arp6), harboring a mutation in the SWR1 chromatin-remodeling complex. Genetic analysis of otu5/arp6 double mutants suggests independent but functionally related roles of the two proteins in chromatin organization. The root hair phenotype of otu5 is not caused by a general up-regulation of the Pi starvation response, indicating that OTU5 acts downstream of or interacts with Pi signaling. It is concluded that OTU5 is involved in the interpretation of environmental information, probably by altering chromatin organization and maintaining redox homeostasis.

Deubiquitinating Enzyme OTU5 Contributes to DNA Methylation Patterns and Is Critical for Phosphate Nutrition Signals.

Phosphate (Pi) starvation induces a suite of adaptive responses aimed at recalibrating cellular Pi homeostasis. Plants harboring a mutation in OVARIAN TUMOR DOMAIN-CONTAINING DEUBIQUITINATING ENZYME5 (OTU5) showed altered DNA methylation of root hair-related genes and altered Pi-responsive root traits. Unlike the wild type, homozygous otu5 mutants did not respond to Pi starvation by increased lateral root formation and increased root hair length but formed very short root hairs when grown on low-Pi media. Under Pi-replete conditions, otu5 plants developed more root hairs than the wild type due to attenuated primary root growth, a phenotype that resembled that of Pi-deficient plants. Growth of plants on low-Pi media altered both H3K4 and H3K27 trimethylation levels at the transcriptional start site of a subset of genes encoding key players in Pi homeostasis, which was correlated with mRNA abundance changes of these genes. Pi starvation had a minor impact on DNA methylation. Differentially methylated regions were enriched in transposable elements, suggesting that DNA methylation associated with low Pi supply is required for maintaining genome integrity. It is concluded that DNA methylation and histone methylation constitute critical, interdependent regulatory components that orchestrate the activity of a subset of Pi-responsive genes.

BMP6 Regulates Proliferation and Apoptosis of Human Sertoli Cells Via Smad2/3 and Cyclin D1 Pathway and DACH1 and TFAP2A Activation.

Sertoli cells are essential for regulating normal spermatogenesis. However, the mechanisms underlying human Sertoli cell development remain largely elusive. Here we examined the function and signaling pathways of BMP6 in regulating human Sertoli cells. RT-PCR, immunocytochemistry and Western blots revealed that BMP6 and its multiple receptors were expressed in human Sertoli cells. CCK-8 and EDU assays showed that BMP6 promoted the proliferation of Sertoli cells. Conversely, BMP6 siRNAs inhibited the division of these cells. Annexin V/PI assay indicated that BMP6 reduced the apoptosis in human Sertoli cells, whereas BMP6 knockdown assumed reverse effects. BMP6 enhanced the expression levels of ZO1, SCF, GDNF and AR in human Sertoli cells, and ELISA assay showed an increase of SCF by BMP6 and a reduction by BMP6 siRNAs. Notably, Smad2/3 phosphorylation and cyclin D1 were enhanced by BMP6 and decreased by BMP6 siRNAs in human Sertoli cells. The levels of DACH1 and TFAP2A were increased by BMP6 and reduced by BMP6 siRNAs, and the growth of human Sertoli cells was inhibited by these siRNAs. Collectively, these results suggest that BMP6 regulates the proliferation and apoptosis of human Sertoli cells via activating the Smad2/3/cyclin D1 and DACH1 and TFAP2A pathway.

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase.

Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3ß-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine.

Transdifferentiation of human male germline stem cells to hepatocytes in vivo via the transplantation under renal capsules.

Here we proposed a new concept that human spermatogonial stem cells (SSCs) can transdifferentiate into hepatocytes in vivo. We first established liver injury model of mice by carbon tetrachloride to provide proper environment for human SSC transplantation. Liver mesenchymal cells were isolated from mice and identified phenotypically. Human SSC line was recombined with liver mesenchymal cells, and they were transplanted under renal capsules of nude mice with liver injury. The grafts expressed hepatocyte hallmarks, including ALB, AAT, CK18, and CYP1A2, whereas germ cell and SSC markers VASA and GPR125 were undetected in these cells, implicating that human SSCs were converted to hepatocytes. Furthermore, Western blots revealed high levels of PCNA, AFP, and ALB, indicating that human SSCs-derived hepatocytes had strong proliferation potential and features of hepatocytes. In addition, ALB-, CK8-, and CYP1A2- positive cells were detected in liver tissues of recipient mice. Significantly, no obvious lesion or teratomas was observed in several important organs and tissues of recipient mice, reflecting that transplantation of human SSCs was safe and feasible. Collectively, we have for the first time demonstrated that human SSCs can be transdifferentiated to hepatocyte in vivo. This study provides a novel approach for curing liver diseases using human SSC transplantation.

Distinct phylogenetic relationships and biochemical properties of Arabidopsis ovarian tumor-related deubiquitinases support their functional differentiation.

The reverse reaction of ubiquitylation is catalyzed by different classes of deubiquitylation enzymes (DUBs), including ovarian tumor domain (OTU)-containing DUBs; experiments using Homo sapiens proteins have demonstrated that OTU DUBs modulate various cellular processes. With the exception of OTLD1, plant OTU DUBs have not been characterized. We identified 12 Arabidopsis thaliana OTU loci and analyzed 11 of the encoded proteins in vitro to determine their preferences for the ubiquitin (UB) chains of M1, K48, and K63 linkages as well as the UB-/RUB-/SUMO-GST fusions. The A. thaliana OTU DUBs were shown to be cysteine proteases and classified into four groups with distinct linkage preferences: OTU1 (M1 = K48 > K63), OTU3/4/7/10 (K63 > K48 > M1), OTU2/9 (K48 = K63), and OTU5/11/12/OTLD1 (inactive). Five active OTU DUBs (OTU3/4/7/9/10) also cleaved RUB fusion. OTU1/3/4 cleaved M1 UB chains, suggesting a possible role for M1 chains in plant cellular signaling. The different substrate specificities of the various A. thaliana OTU DUBs indicate the involvement of distinct structural elements; for example, the OTU1 oxyanion residue D89 is essential for cleaving isopeptide bond-linked chains but dispensable for M1 chains. UB-binding activities were detected only for OTU2 and OTLD1, with distinct linkage preferences. These differences in biochemical properties support the involvement of A. thaliana OTU DUBs in different functions. Moreover, based on the established phylogenetic tree, plant- and H. sapiens-specific clades exist, which suggests that the proteins within these clades have taxa-specific functions. We also detected five OTU clades that are conserved across species, which suggests that the orthologs in different species within each clade are involved in conserved cellular processes, such as ERAD and DNA damage responses. However, different linkage preferences have been detected among potential cross-species OTU orthologs, indicating functional and mechanistic differentiation.

IRT1 degradation factor1, a ring E3 ubiquitin ligase, regulates the degradation of iron-regulated transporter1 in Arabidopsis.

Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, iron-regulated transporter1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 degradation factor1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity.

Purification of the Arabidopsis 26 S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms.

The 26 S proteasome is a multisubunit protease complex responsible for degrading a wide range of intracellular proteins in eukaryotes, especially those modified with polyubiquitin chains. It is composed of a self-compartmentalized core protease (CP) that houses the peptidase active sites appended on either or both ends by a regulatory particle (RP) that identifies appropriate substrates and translocates them into the lumen of the CP for breakdown. Here, we describe the molecular and biochemical properties of the 26 S proteasome from the plant Arabidopsis thaliana. Like the CP and the ATPase ring of the RP, the RP non-ATPase subunits are often encoded by two transcriptionally active genes with some pairs displaying sufficient sequence divergence to suggest functional differences. Most RPN subunits could functionally replace their yeast counterparts, implying that they have retained their positions and activities within the complex. A method was developed to purify the 26 S proteasome intact from whole Arabidopsis seedlings. These preparations are biochemically indistinguishable from those from yeast and mammals, including the need for ATP to maintain integrity and a strong sensitivity to the inhibitors MG115, MG132, lactacystin, and epoxomicin. Mass spectrometric analysis of the complex detected the presence of almost all CP and RP subunits. In many cases, both products of paralogous genes were detected, demonstrating that each isoform assembles into the mature particle. As with the yeast and animal 26 S proteasomes, attenuation of individual RP genes induces a coordinated up-regulation of many of the other 26 S proteasome genes, suggesting that plants contain a negative feedback mechanism to regulate the 26 S proteasome levels. The incorporation of paralogous subunits into the Arabidopsis holoprotease raises the intriguing possibility that plants synthesize multiple 26 S proteasome types with unique properties and/or target specificities.

Molecular cloning of the carboxylesterase gene and biochemical characterization of the encoded protein from Pseudomonas citronellolis ATCC 13674.

A genomic library of Pseudomonas citronellolis ATCC 13674 was constructed and screened for esterase activity in Escherichia coli using tributyrin-containing medium. One positive transformant was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.1-kb DNA fragment potentially carrying an esterase gene. The deduced nucleotide sequence of the DNA was found to contain an open reading frame encoding carboxylesterase and designated estA. Amino acid sequence analysis of estA showed the serine conservative motif, GDSAG, located between residues 208 and 212. Together with Ser, residues 310 and 334 corresponding to aspartic acid and histidine, respectively, comprised the catalytic triad. With the aid of immobilized metal ion affinity chromatography, the carboxylesterase fused with poly His at its C-terminus was purified and shown to be strongly inhibited by the tryptophan modifier and mercuric ion, indicating the important role of conservative Trp (189) and cysteine (152 and/or 183) residues in maintaining the structural integrity of the protein. Further analyses showed that the carboxylesterase functioned optimally at 37-40 degrees C with pH ranging between 8 and 9 and displayed a broad substrate spectrum. The protein exhibited greater preference toward short-chain (C2-C4) than medium- and long-chain fatty acids. Higher substrate specificity on para-nitrophenol butyrate was observed in comparison with para-nitrophenol acetate as indicated by the higher kcat/Km value of the former.