Entamoeba histolytica activation of caspase-1 degrades cullin that attenuates NF-κB dependent signaling from macrophages

While Entamoeba histolytica (Eh)-induced pro-inflammatory responses are critical in disease pathogenesis, the downstream signaling pathways that subsequently dampens inflammation and the immune response remains unclear. Eh in contact with macrophages suppresses NF-κB signaling while favoring NLRP3-dependent pro-inflammatory cytokine production by an unknown mechanism. Cullin-1 and cullin-5 (cullin-1/5) assembled into a multi-subunit RING E3 ubiquitin ligase complex are substrates for neddylation that regulates the ubiquitination pathway important in NF-κB activity and pro-inflammatory cytokine production. In this study, we showed that upon live Eh contact with human macrophages, cullin-1/4A/4B/5 but not cullin-2/3, were degraded within 10 minutes. Similar degradation of cullin-1/5 were observed from colonic epithelial cells and proximal colonic loops tissues of mice inoculated with live Eh. Degradation of cullin-1/5 was dependent on Eh-induced activation of caspase-1 via the NLRP3 inflammasome. Unlike cullin-4B, the degradation of cullin-4A was partially dependent on caspase-1 and was inhibited with a pan caspase inhibitor. Cullin-1/5 degradation was dependent on Eh cysteine proteinases EhCP-A1 and EhCP-A4, but not EhCP-A5, based on pharmacological inhibition of the cysteine proteinases and EhCP-A5 deficient parasites. siRNA silencing of cullin-1/5 decreased the phosphorylation of pIκ-Bα in response to Eh and LPS stimulation and downregulated NF-κB-dependent TNF-α mRNA expression and TNF-α and MCP-1 pro-inflammatory cytokine production. These results unravel a unique outside-in strategy employed by Eh to attenuate NF-κB-dependent pro-inflammatory responses via NLRP3 activation of caspase-1 that degraded cullin-1/5 from macrophages.     

Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex

Poxviruses are notorious for encoding multiple proteins that regulate cellular signaling pathways, including the ubiquitin-proteasome system. Bioinformatics indicated that ectromelia virus, the causative agent of lethal mousepox, encoded four proteins, EVM002, EVM005, EVM154, and EVM165, containing putative F-box domains. In contrast to cellular F-box proteins, the ectromelia virus proteins contain C-terminal F-box domains in conjunction with N-terminal ankyrin repeats, a combination that has not been previously reported for cellular proteins. These observations suggested that the ectromelia virus F-box proteins interact with SCF (Skp1, cullin-1, and F-box) ubiquitin ligases. We focused our studies on EVM005, since this protein had only one ortholog in cowpox virus. Using mass spectrometry, we identified cullin-1 as a binding partner for EVM005, and this interaction was confirmed by overexpression of hemagglutinin (HA)-cullin-1. During infection, Flag-EVM005 and HA-cullin-1 colocalized to distinct cellular bodies. Significantly, EVM005 coprecipitated with endogenous Skp1, cullin-1, and Roc1 and associated with conjugated ubiquitin, suggesting that EVM005 interacted with the components of a functional ubiquitin ligase. Interaction of EVM005 with cullin-1 and Skp1 was abolished upon deletion of the F-box, indicating that the F-box played a crucial role in interaction with the SCF complex. Additionally, EVM002 and EVM154 interacted with Skp1 and conjugated ubiquitin, suggesting that ectromelia virus encodes multiple F-box-containing proteins that regulate the SCF complex. Our results indicate that ectromelia virus has evolved multiple proteins that interact with the SCF complex.     

Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response

Regeneration is widespread throughout the animal kingdom, but our molecular understanding of this process in adult animals remains poorly understood. Wnt/β-catenin signaling plays crucial roles throughout animal life from early development to adulthood. In intact and regenerating planarians, the regulation of Wnt/β-catenin signaling functions to maintain and specify anterior/posterior (A/P) identity. Here, we explore the expression kinetics and RNAi phenotypes for secreted members of the Wnt signaling pathway in the planarian Schmidtea mediterranea. Smed-wnt and sFRP expression during regeneration is surprisingly dynamic and reveals fundamental aspects of planarian biology that have been previously unappreciated. We show that after amputation, a wounding response precedes rapid re-organization of the A/P axis. Furthermore, cells throughout the body plan can mount this response and reassess their new A/P location in the complete absence of stem cells. While initial stages of the amputation response are stem cell independent, tissue remodeling and the integration of a new A/P address with anatomy are stem cell dependent. We also show that WNT5 functions in a reciprocal manner with SLIT to pattern the planarian mediolateral axis, while WNT11-2 patterns the posterior midline. Moreover, we perform an extensive phylogenetic analysis on the Smed-wnt genes using a method that combines and integrates both sequence and structural alignments, enabling us to place all nine genes into Wnt subfamilies for the first time.     

Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5–1 and mll5–2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5–2 are required for regeneration and that set1, trr-1 and mll5–2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.     

涡虫生殖生物学研究进展

涡虫由于具有极强的再生能力而成为发育生物学及再生生物学研究的模式生物。此外,其在有性生殖方面所表现出来的独特性也备受人们关注。目前,涡虫生殖生物学研究领域主要围绕两个热点问题开展工作：1.无性生殖向有性生殖转化的诱因及机制的探讨;2.生殖相关基因的克隆、表达及功能分析。有关生殖转化机制方面的研究主要集中在涡虫的性化相关事件以及性化物质的本质探索;截至目前已克隆并对其表达和功能进行探讨的涡虫生殖相关基因主要有DjPTK1、vasa-like 基因、DeY1、Dryg、nanos相关基因以及Drpiwi-1等。此外,本文也对有关涡虫生殖生物学方面存在的问题及未来该领域的发展趋势进行了总结和展望。     

The structure and regulation of Cullin 2 based E3 ubiquitin ligases and their biological functions

Background

Cullin-RING E3 ubiquitin ligase complexes play a central role in targeting cellular proteins for ubiquitination-dependent protein turnover through 26S proteasome. Cullin-2 is a member of the Cullin family, and it serves as a scaffold protein for Elongin B and C, Rbx1 and various substrate recognition receptors to form E3 ubiquitin ligases.

Main body of the abstract

First, the composition, structure and the regulation of Cullin-2 based E3 ubiquitin ligases were introduced. Then the targets, the biological functions of complexes that use VHL, Lrr-1, Fem1b, Prame, Zyg-11, BAF250, Rack1 as substrate targeting subunits were described, and their involvement in diseases was discussed. A small molecule inhibitor of Cullins as a potential anti-cancer drug was introduced. Furthermore, proteins with VHL box that might bind to Cullin-2 were described. Finally, how different viral proteins form E3 ubiquitin ligase complexes with Cullin-2 to counter host viral defense were explained.

Conclusions

Cullin-2 based E3 ubiquitin ligases, using many different substrate recognition receptors, recognize a number of substrates and regulate their protein stability. These complexes play critical roles in biological processes and diseases such as cancer, germline differentiation and viral defense. Through the better understanding of their biology, we can devise and develop new therapeutic strategies to treat cancers, inherited diseases and viral infections.

     

Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria

Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but they have not historically been accessible to extensive genetic manipulation. Here we utilize RNA-mediated genetic interference (RNAi) to introduce large-scale gene inhibition studies to the classic planarian system. 1065 genes were screened. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. We assessed the effects of inhibiting genes with RNAi on tissue homeostasis in intact animals and stem cell (neoblast) proliferation in amputated animals identifying candidate stem cell, regeneration, and homeostasis regulators. Our study demonstrates the great potential of RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.     

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.     

A functional genomics screen identifies an Importin-α homolog as a regulator of stem cell function and tissue patterning during planarian regeneration

Background

Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea.

Methods

We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes.

Results

Along with five previously characterized genes (Smed-cycD, Smed-morf41/mrg-1, Smed-pdss2/dlp1, Smed-slbp, and Smed-tph), we identified 20 additional genes necessary for stem cell maintenance (Smed-sart3, Smed-smarcc-1, Smed-espl1, Smed-rrm2b-1, Smed-rrm2b-2, Smed-dkc1, Smed-emg1, Smed-lig1, Smed-prim2, Smed-mcm7, and a novel sequence) or general regenerative capability (Smed-rbap46/48-2, Smed-mcm2, Smed-ptbp1, and Smed-fen-1) or that caused tissue-specific defects upon knockdown (Smed-ddc, Smed-gas8, Smed-pgbd4, and Smed-b9d2). We also found that a homolog of the nuclear transport factor Importin-α plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration.

Conclusions

Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

     

Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.     

Physiological functions of FBW7 in cancer and metabolism

FBW7 is one of the most well characterized F-box proteins that serve as substrate recognition subunits of SCF (Skp1-Cullin 1-F-box proteins) E3 ubiquitin ligase complexes. SCF^FBW7 plays key roles in regulating cell cycle progression, differentiation, and stem cell maintenance largely through targeting a broad range of oncogenic substrates for proteasome-dependent degradation. The identification of an increasing number of FBW7 substrates for ubiquitination, and intensive in vitro and in vivo studies have revealed a network of signaling components controlled by FBW7 that contributes to metabolic regulation as well as its tumor suppressor role. Here we mainly focus on recent findings that highlight a critical role for FBW7 in cancer and metabolism.     

A systematic phenotypic screen of F-box genes through a tissue-specific RNAi-based approach in Drosophila

F-box proteins are components of the SCF(SkpA-Cullin 1-F-box) E3 ligase complexes,acting as the specificity-determinants in targeting substrate proteins for ubiquitination and degradation.In humans,at least 22 out of 75 F-box proteins have experimentally documented substrates,whereas in Drosophila 12 F-box proteins have been characterized with known substrates.To systematically investigate the genetic and molecular functions of F-box proteins in Drosophila,we performed a survey of the literature and databases.We identified 45 Drosophila genes that encode proteins containing at least one F-box domain.We collected publically available RNAi lines against these genes and used them in a tissue-specific RNAi-based phenotypic screen.Here,we present our systematic phenotypic dataset from the eye,the wing and the notum.This dataset is the first of its kind and represents a useful resource for future studies of the molecular and genetic functions of F-box genes in Drosophila.Our results show that,as expected,F-box genes in Drosophila have regulatory roles in a diverse array of processes including cell proliferation,cell growth,signal transduction,and cellular and animal survival.     

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.