Functions and substrates of NEDDylation during cell cycle in the silkworm,Bombyx mori

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm.     

Identification and functional analysis of outer kinetochore genes in the holocentric insect Bombyx mori

The kinetochore creates chromosomal attachment sites for microtubules. The kinetochore-microtubule interface plays an important role in ensuring accurate transmission of genetic information to daughter cells. Bombyx mori is known to possess holocentric chromosomes, where spindle microtubules attach along the entire length of the chromosome. Recent evidence suggests that CENP-A and CENP-C, which are essential for centromere structure and function in other species, have lost in holocentric insects, implying that B. mori is able to build its kinetochore regardless of the lack of CENP-A and CENP-C. Here we report the identification of three outer kinetochore genes in the silkworm B. mori by using bioinformatics and RNA interference-based screening. While the homologs of Ndc80 and Mis12 have strong similarity with those of other organisms, the five encoded proteins (BmNuf2, BmSpc24, BmSpc25, BmDsn1 and BmNnf1) are highly diverged from their counterparts in other species. Microscopic studies show that the outer kinetochore protein is distributed along the entire length of the chromosomes, which is a key feature of holocentric chromosomes. We also demonstrate that BmDsn1 forms a heterotrimeric complex with BmMis12 and BmNnf1, which acts as a receptor of the Ndc80 complex. In addition, our study suggests that a small-scale RNAi-based candidate screening is a useful approach to identify genes which may be highly divergent among different species.     

Post-translational modifications of the N-terminal tail of histone H3 in holocentric chromosomes of Bombyx mori

Epigenetic information is encoded in post-translational modifications (PTMs) of histones. Various combinations of these marks contribute to the regulation of chromatin-templated DNA metabolisms. The histone code is gradually translated into biological responses in model organisms. However, in the silkworm, the modifications of histones with unique holocentric chromosomes have not yet been analyzed. TAU-PAGE analysis of the silkworm histone variants H2A, H2B, and H3, separated by RP-HPLC, suggested silkworm specific modification. Detailed mass spectrometry analyses of the peptides derived from the N-terminus of the silkworm H3.2 generated by glutamyl endopeptidase, lysyl endopeptidase, and trypsin digestions revealed global modifications around H3K9.     

The implication of the SUMOylation pathway in breast cancer pathogenesis and treatment

Abstract

Breast cancer is the most commonly diagnosed malignancy in woman worldwide, and is the second most common cause of death in developed countries. The transformation of a normal cell into a malignant derivate requires the acquisition of diverse genomic and proteomic changes, including enzymatic post-translational modifications (PTMs) on key proteins encompassing critical cell signaling events. PTMs occur on proteins after translation, and regulate several aspects of proteins activity, including their localization, activation and turnover. Deregulation of PTMs can potentially lead to tumorigenesis, and several de-regulated PTM pathways contribute to abnormal cell proliferation during breast tumorigenesis. SUMOylation is a PTM that plays a pivotal role in numerous aspects of cell physiology, including cell cycle regulation, protein trafficking and turnover, and DNA damage repair. Consistently with this, the deregulation of the SUMO pathway is observed in different human pathologies, including breast cancer. In this review we will describe the role of SUMOylation in breast tumorigenesis and its implication for breast cancer therapy.     

Ecdysteroid promotes cell cycle progression in the Bombyx wing disc through activation of c-Myc

Developmental switching from growth to metamorphosis in imaginal primordia is an essential process of adult body planning in holometabolous insects. Although it is disciplined by a sequential action of the ecdysteroid, molecular mechanisms linking to cell proliferation are poorly understood. In the present study, we investigated the expression control of cell cycle–related genes by the ecdysteroid using the wing disc of the final-instar larvae of the silkworm, Bombyx mori. We found that the expression level of c-myc was remarkably elevated in the post-feeding cell proliferation phase, which coincided with a small increase in ecdysteroid titer. An in vitro wing disc culture showed that supplementation of the moderate level of the ecdysteroid upregulated c-myc expression within an hour and subsequently increased the expression of cell cycle core regulators, including A-, B-, D-, and E-type cyclin genes, Cdc25 and E2F1. We demonstrated that c-myc upregulation by the ecdysteroid was not inhibited in the presence of a protein synthesis inhibitor, suggesting a possibility that the ecdysteroid directly stimulates c-myc expression. Finally, results from the administration of a c-Myc inhibitor demonstrated that c-Myc plays an essential role in 20E-inducible cell proliferation. These findings suggested a novel pathway for ecdysteroid-inducible cell proliferation in insects, and it is likely to be conserved between insects and mammals in terms of steroid hormone regulation.     

Growth factor-dependent signaling and cell cycle progression

There are three central ideas contained within this review. Firstly, growth factor-stimulated signaling is not restricted to a 30–60 min window, but occurs at a much later time as well. Secondly, the second wave of signaling overlaps temporally with the cell cycle program and may be directly responsible for engaging it. Thirdly, the G1 to S interval appears to encompass two distinct phases of the cell cycle, during which the coordinated activation of distinct sets of signaling enzymes drives cell cycle progression. Each of these concepts is likely to initiate new investigation and hence provide additional insight into the fundamental question of how growth factors drive cell proliferation.     

家蚕核型多角体病毒orf25基因在病毒感染过程中 编码一个30kDa的晚期蛋白

首次对家蚕核型多角体orf25基因进行了描述.扩增Bm25基因,亚克隆到原核表达载体pGEX-4T-2,在大肠杆菌BL21(DE3)中表达含有GST标签的融合蛋白.IPTG诱导后高效表达GST-Bm25融合蛋白.纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体.利用制备的抗GST-Bm25融合蛋白的多克隆抗体进行表达时相分析显示:24 h p.i.检测到30 kDa的蛋白条带.RT-PCR方法,在18-72 h p.i 检测到Bm25基因的转录本.结论:以上数据表明Bm25基因编码一晚期表达的30kDa蛋白.     

A single structurally conserved SUMOylation site in CRMP2 controls NaV1.7 function

The neuronal collapsin response mediator protein 2 (CRMP2) undergoes several posttranslational modifications that codify its functions. Most recently, CRMP2 SUMOylation (addition of small ubiquitin like modifier (SUMO)) was identified as a key regulatory step within a modification program that codes for CRMP2 interaction with, and trafficking of, voltage-gated sodium channel NaV1.7. In this paper, we illustrate the utility of combining sequence alignment within protein families with structural analysis to identify, from several putative SUMOylation sites, those that are most likely to be biologically relevant. Co-opting this principle to CRMP2, we demonstrate that, of 3 sites predicted to be SUMOylated in CRMP2, only the lysine 374 site is a SUMOylation client. A reduction in NaV1.7 currents was the corollary of the loss of CRMP2 SUMOylation at this site. A 1.78-Å-resolution crystal structure of mouse CRMP2 was solved using X-ray crystallography, revealing lysine 374 as buried within the CRMP2 tetramer interface but exposed in the monomer. Since CRMP2 SUMOylation is dependent on phosphorylation, we postulate that this state forces CRMP2 toward a monomer, exposing the SUMO site and consequently, resulting in constitutive regulation of NaV1.7.     

Calcium signaling and cell cycle: Progression or death

Cytosolic Ca²⁺ concentration levels fluctuate in an ordered manner along the cell cycle, in line with the fact that Ca²⁺ is involved in the regulation of cell proliferation. Cell proliferation should be an error-free process, yet is endangered by mistakes. In fact, a complex network of proteins ensures that cell cycle does not progress until the previous phase has been successfully completed. Occasionally, errors occur during the cell cycle leading to cell cycle arrest. If the error is severe, and the cell cycle checkpoints work perfectly, this results into cellular demise by activation of apoptotic or non-apoptotic cell death programs. Cancer is characterized by deregulated proliferation and resistance against cell death. Ca²⁺ is a central key to these phenomena as it modulates signaling pathways that control oncogenesis and cancer progression. Here, we discuss how Ca²⁺ participates in the exogenous and endogenous signals controlling cell proliferation, as well as in the mechanisms by which cells die if irreparable cell cycle damage occurs. Moreover, we summarize how Ca²⁺ homeostasis remodeling observed in cancer cells contributes to deregulated cell proliferation and resistance to cell death. Finally, we discuss the possibility to target specific components of Ca²⁺ signal pathways to obtain cytostatic or cytotoxic effects.     

Insulin action mechanism for redox signaling in the cell cycle: Its alterations in diabetes

Several reports describe the existence of a redox cycle within the normal cell cycle that helps control the process of cell proliferation. According to some of these reports, this redox cycle comprises an intracellular redox potential E that oscillates above and below θ during the cell cycle process. θ is the threshold for dephosphorylation of protein regulators associated with serine residues such as the retinoblastoma protein. This article describes how insulin action may be the source of the redox cycle within the cell cycle. The relative lack of insulin action as a consequence of oxidative stress results in the hallmarks of type 2 diabetes.     

System-level design of bacterial cell cycle control

Understanding of the cell cycle control logic in Caulobacter has progressed to the point where we now have an integrated view of the operation of an entire bacterial cell cycle system functioning as a state machine. Oscillating levels of a few temporally-controlled master regulator proteins in a cyclical circuit drive cell cycle progression. To a striking degree, the cell cycle regulation is a whole cell phenomenon. Phospho-signaling proteins and proteases dynamically deployed to specific locations on the cell wall are vital. An essential phospho-signaling system integral to the cell cycle circuitry is central to accomplishing asymmetric cell division.     

14-3-3 proteins as signaling integration points for cell cycle control and apoptosis

14-3-3 proteins play critical roles in the regulation of cell fate through phospho-dependent binding to a large number of intracellular proteins that are targeted by various classes of protein kinases. 14-3-3 proteins play particularly important roles in coordinating progression of cells through the cell cycle, regulating their response to DNA damage, and influencing life-death decisions following internal injury or external cytokine-mediated cues. This review focuses on 14-3-3-dependent pathways that control cell cycle arrest and recovery, and the influence of 14-3-3 on the apoptotic machinery at multiple levels of regulation. Recognition of 14-3-3 proteins as signaling integrators that connect protein kinase signaling pathways to resulting cellular phenotypes, and their exquisite control through feedforward and feedback loops, identifies new drug targets for human disease, and highlights the emerging importance of using systems-based approaches to understand signal transduction events at the network biology level.     

Studies on the control of the cell cycle in cultured plant cells

Summary Patterns of cell cycle arrest or temporal modification have been investigated using suspension cultures ofAcer pseudoplatanus under nutrient limiting and nutrient starvation conditions. The results of nitrogen, phosphorous and carbohydrate starvation have been compared and contrasted with reference to the Principal Control Point hypothesis ofVan't Hof andKovacs (1972). Whilst cells suffering phosphorus or carbohydrate starvation arrest in the G 1 and G 2 phases in the approximate ratio of 4 to 1, nitrogen starved cells accumulate virtually exclusively in G 1.     

Functional analysis of a survivin-like gene in Bombyx mori

The survivin (svv) gene is a newly discovered member of the inhibitors of apoptosis gene family. In recent years, svv has been confirmed to have an anti-apoptosis function and to play a critical role in cell division. We identified a survivin-like gene in the silkworm, Bombyx mori (Bm-svv). In this study, to gain insight into its function, a baculovirus expression system was used to express the Bm-svv gene in insect cell lines. The recombinant viruses were then used as a vector to transform insect cells, and cell activity was determined using the Cell Counting Kit-8 (CCK-8), which is usually employed for detecting mammalian cell number. The results indicated that the Bm-svv gene plays a role in the cell growth arrest or apoptosis induced by viruses. Furthermore, the CCK-8 kit is effective in determining the activity of insect cells.     

Artificial parthenogenesis and control of voltinism to manage transgenic populations in Bombyx mori

In order to improve the management of transformed populations in a routine application of transgenesis technology in Bombyx mori, we modified its mode of reproduction and its voltinism. On one hand, after a stable integration of the gene of interest by transgenesis, it is preferable to maintain this gene in an identical genomic context through successive generations. This can be obtained by artificial parthenogenetic reproduction (ameiotic parthenogenesis) giving isogenic females identical to their transformed mother. On the other hand, it is essential to obtain continuous generations (polyvoltinism) after microinjection, in order to screen positive transgenic insects and study genetics and insertion of the transgene. Thereafter, it is more convenient to store these populations, as diapause eggs before their use in biotechnology application. We obtained such polyvoltine parthenoclones, first by selection for a parthenogenetic character in polyvoltine races, and second, by selection for a polyvoltine character in a parthenogenetic, but diapausing clone of B. mori. As diapause was directly under the control of diapause hormone (DH), we also tested direct injection of DH in female pupae of polyvoltine strains, as well as anti-DH antibody treatment to eliminate diapause in univoltine strains. We discussed the advantages and limitations of these methods and proved the feasibility in obtaining polyvoltine parthenoclones and determining the voltinism in B. mori. These methods would permit us to improve the management of populations used in transgenesis technology.     

Linking cell division to cell growth in a spatiotemporal model of the cell cycle

Cell division must be tightly coupled to cell growth in order to maintain cell size, yet the mechanisms linking these two processes are unclear. It is known that almost all proteins involved in cell division shuttle between cytoplasm and nucleus during the cell cycle; however, the implications of this process for cell cycle dynamics and its coupling to cell growth remains to be elucidated. We developed mathematical models of the cell cycle which incorporate protein translocation between cytoplasm and nucleus. We show that protein translocation between cytoplasm and nucleus not only modulates temporal cell cycle dynamics, but also provides a natural mechanism coupling cell division to cell growth. This coupling is mediated by the effect of cytoplasmic-to-nuclear size ratio on the activation threshold of critical cell cycle proteins, leading to the size-sensing checkpoint (sizer) and the size-independent clock (timer) observed in many cell cycle experiments.     

A checkpoint-oriented cell cycle simulation model

Modeling and in silico simulations are of major conceptual and applicative interest in studying the cell cycle and proliferation in eukaryotic cells. In this paper, we present a cell cycle checkpoint-oriented simulator that uses agent-based simulation modeling to reproduce the dynamics of a cancer cell population in exponential growth. Our in silico simulations were successfully validated by experimental in vitro supporting data obtained with HCT116 colon cancer cells. We demonstrated that this model can simulate cell confluence and the associated elongation of the G1 phase. Using nocodazole to synchronize cancer cells at mitosis, we confirmed the model predictivity and provided evidence of an additional and unexpected effect of nocodazole on the overall cell cycle progression. We anticipate that this cell cycle simulator will be a potential source of new insights and research perspectives.     

Clusterin and DNA repair: a new function in cancer for a key player in apoptosis and cell cycle control

The glycoprotein clusterin (CLU), has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, while a secretory form (sCLU) is pro-survival. Both forms are implicated in various cell functions, including DNA repair, cell cycle regulation, and apoptotic cell death. CLU expression has been associated with tumorigenesis and the progression of various malignancies. In response to DNA damage, cell survival can be enhanced by activation of DNA repair mechanisms, while simultaneously stimulating energy-expensive cell cycle checkpoints that delay the cell cycle progression to allow more time for DNA repair. This review summarizes our current understanding of the role of clusterin in DNA repair, apoptosis, and cell cycle control and the relevance.