GenEpi: gene-based epistasis discovery using machine learning.

BackgroundGenome-wide association studies (GWAS) provide a powerful means to identify associations between genetic variants and phenotypes. However, GWAS techniques for detecting epistasis, the interactions between genetic variants associated with phenotypes, are still limited. We believe that developing an efficient and effective GWAS method to detect epistasis will be a key for discovering sophisticated pathogenesis, which is especially important for complex diseases such as Alzheimer's disease (AD).ResultsIn this regard, this study presents GenEpi, a computational package to uncover epistasis associated with phenotypes by the proposed machine learning approach. GenEpi identifies both within-gene and cross-gene epistasis through a two-stage modeling workflow. In both stages, GenEpi adopts two-element combinatorial encoding when producing features and constructs the prediction models by L1-regularized regression with stability selection. The simulated data showed that GenEpi outperforms other widely-used methods on detecting the ground-truth epistasis. As real data is concerned, this study uses AD as an example to reveal the capability of GenEpi in finding disease-related variants and variant interactions that show both biological meanings and predictive power.ConclusionsThe results on simulation data and AD demonstrated that GenEpi has the ability to detect the epistasis associated with phenotypes effectively and efficiently. The released package can be generalized to largely facilitate the studies of many complex diseases in the near future

The effects of Nedd8 on cullin-RING ubiquitin ligases and their substrates

泛素是一種小分子胜肽,泛素—蛋白解體系統負責許多細胞生理所必需的蛋白質降解.整個蛋白質降解的過程先是由泛素接合脢把泛素專一性地結合在即將被降解的標的蛋白上,再經過重複的接合脢作用,標的蛋白上聚合出長鏈型泛素聚合物,帶有長鏈型泛素聚合物的標的蛋白隨即被蛋白解體所辨識,並且被降解成短胜肽產物.細胞內有很多泛素接合脢,其中有一類稱為cullin-RING接合脢(簡稱CRL).完整的CRL是個由cullin蛋白為基石所建構的蛋白複合體.負責延攬受質的受質受器在cullin 的N端,而負責銜接泛素配合酵素(E2)的Roc1則座落在cullin的C端. 接合脢的酵素反應可視為CRL上的泛素藉由地緣之便,傳遞到同在CRL上的受質.Nedd8是胺基酸序列和三級結構都與泛素十分類似的一種小分子胜肽.它具有類似泛素的性質夠過neddylation的酵素反應能夠結合在其他蛋白如cullin之上.相反地透過CSN複合體催化的deneddylation酵素反應,Nedd8又可以從neddylated cullin上被切下來.過去的研究指出,CRL經過neddylation之後才能有效率的執行泛素接合脢的功能,可是有關deneddylation如何影響CRL酵素活性的了解甚少.本論文主要內容在於探討CSN突變果蠅的活體細胞內,cullin1和cullin3的穩定性變差.cullin穩定性變差的主要原因是在CSN突變果蠅裡,cullin一旦發生neddylation後就無法經由deneddylation回復到穩定的原態.結果細胞不再能夠保有足夠的cullin儲備,於是CRL的活性不但沒有因為neddylation而提高,反而因為cullin普遍不足而下降.在CSN突變果蠅細胞中,Nedd8和受質受器Slimb也變得特別不穩定.CSN突變和Nedd8突變,或和cullin突變,或和Slimb突變互相加成對果蠅的存活率影響,表示CSN調節CRL組成成員以及Nedd8的穩定性具有生理意義.但是對於特定的CRL受質如Ci,在CSN突變細胞中增加cullin1和Nedd8的量,不但不能藉由彌補CSN突變細胞中cullin1,Nedd8的儲備量而提高CRL的酵素活性,反而弔詭地減低了CRL的酵素活性,表示cullin1, Nedd8的量對CRL的酵素活性在不同deneddylation程度下會出現兩種截然不同的影響.論文最後探討了同樣是SCFSlimb接合脢受質的兩種蛋白質,被泛素--蛋白解體系統降解的程度受到CSN突變的影響卻有所不同,表示受質能夠左右CRL接合脢活性受neddylation/deneddylation調節的程度.Ubiquitin-proteasome mediated proteolysis is an essential pathway involved in many cellular processes. Ubiquitin is a small polypeptide. Polymerized ubiquitin chains are first attached to the proteins designated to be degraded by a substrate specific ubiquitin E3 ligase, a process called ubiquitylation. The ubiquitylated protein is then recognized by the 19S proteasome and subsequently degraded by the 20S proteasome. Cullins are scaffold proteins that assemble substrate binding receptors/adaptors, and Roc1 into cullin-RING E3 ligase (CRL) complexes transferring ubiquitin moieties onto many cellular proteins. Nedd8 is an ubiquitin-like polypeptide that can be transferred onto the conserved lysines of cullins, a process called neddylation. Deneddylation, the reverse reaction that removes the Nedd8 moieties from cullins, is carried out by COP9 signalosome (CSN). Neddylation is essential for CRL activities in vivo. How deneddylation affects the activities CRLs hasn’t been fully explored. In this thesis, I first showed that cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable in CSN mutant cells. Moreover, deneddylating activity in CSN is required to preserve Cul1 and Cul3 protein levels. In addition to cullins, Nedd8 and Slimb, one of the substrate receptors in Cul1 organized E3 (SCF), are also unstable in CSN mutant cells. These data indicate that the several CRL components could be protected by CSN from neddylation-induced degradations. The significance of CSN-mediated protection on CRLs is suggested by synergistic interaction between CSN5 mutant and several cullin mutants, Nedd8 mutant, as well as a dominant-negative Slimb mutant. Because neddylation-induced degradation of Slimb depends on SCF activity, rescuing Cul1 and Nedd8 protein levels in CSN mutant cells was found to paradoxically compromise SCFSlimb activity. This result is possibly due to the enhanced neddylation-induced degradation of Slimb. The significance of neddylation-induced degradation of cullins and Nedd8 is to preserve substrate degradation in this context, implying another layer of complexity in regulating CRL activity through neddylation and deneddylation. Finally, I found the protein levels of two SCFSlimb substrates changed differently in CSN mutant cells, implying that substrates may play a role on modulating the effects of neddylation and deneddylation on SCFSlimbACKNOWLEDGEMENTS………………………………………………………v ABSTRACT IN CHINESE…………………………………………………vi ABSTRACT……………………………………………………………viii INTRODUCTION……………………………………………………………1 Ubiquitin proteasome pathway………………………………………1 Hierarchical organization and substrate diversity of cullin-RING ubiquitin ligases ……………………………………4 Neddylation as a way to regulate the activities of CRLs………………………………………………………………………7 CSN-mediated deneddylation of CRLs………………………………9 CSN paradox…………………………………………………………………11 The CAND1-assisted cyclic model of neddylation and deneddylation…………………………………………………………13 MATERIALS AND METHOD Genetics and molecular biology…………………………………………………………………15 Clonal analysis in mutant imaginal discs……………………15 RNA interference and cycloheximide chase……………………17 Immunostaining and immunoblotting………………………………17 RESULTS Cullin protein levels in CSN5null cells and animals………19 Cul1 and Cul3 protein stability controlled by CSN5………20 Cul1 and Cul3 stabilities regulated by CSN deneddylating activity………………………………………………………………20 Degradation of cullins induced by neddylation………………22 Nedd8 protein turnover regulated by CSN and en bloc degradation of the neddylated cullins…………………………24 The effect of deneddylation on the stability of F-box protein Slimb…………………………………………………………25 The significance of Cul1, Cul3, Cul4, and Nedd8 protein levels maintained by CSN deneddylating activity………………………………………………………………26 Degradation mechanism of neddylated cullins…………………28 Slimb shielding Cul1 and Nedd8 from depletion………………29 The effects of deneddylation on SCF substrates, Cubitus interruptus (Ci), Armadillo (Arm), and dMyc…………………30 Delicate regulations on Ci degradation by deneddylation of SCFSlimb………………………………………………………………31 Paradoxical role of neddylation induced Cul1 and Nedd8 degradation in promoting Ci degradation………………………32 DISCUSSION AND PROSPECTIVES Degradation of neddylated cullins in different species…34 The mechanism of neddylation-induced cullin degradation…35 The significance of neddylation-induced Cul1 and Nedd8 degradation……………………………………………………………36 Pro-degradation and anti-degradation of Cul1 and its transition by CSN……………………………………………………37 The possible clinical implications of the hypothetical biphasic CRL activities……………………………………………38 The opposite outcomes of the Ci and Arm in CSN5 mutant clones…………………………………………………………………39 Conclusion……………………………………………………………40 FIGURES Figure 1. A schematic illustration of cullin-RING ligases (CRLs) and neddylation ……………………………………………42 Figure 2. Reduction of Cul1 and Cul3 protein levels in cells harbouring mutations in the CSN complex………………43 Figure 3. The effects of CSN5 on the protein stability of Cul1 and Cul3.………………………………………………………44 Figure 4. Requirement of CSN deneddylating activity to maintain Cul1 and Cul3 protein stability……………………45 Figure 5. CSN regulation of Cul1 and Cul3 protein levels through a neddylation-dependent mechanism……………………46 Figure 6. Degradation of Nedd8 in CSN5null cells…………48 Figure 7. The effects of neddylation and deneddylation on the autocatalytic destruction of Slimb………………………49 Figure 8. Genetic interactions between CSN5 and Cul1……50 Figure 9. Degradation of Cul1 and Cul3 in the absence of proteasome and lysosome……………………………………………51 Figure 10. en bloc degradation of neddylated Cul1in Slimbp1493 cells……………………………………………………53 Figure 11. Degradations of SCF substrates Ci, Arm and dMyc in CSN5null clones…………………………………………………54 Figure 12. The regulatory roles of Neddylated Cul1 and the autocatalytic destruction of Slimb on Ci protein levels in CSN5null clones………………………………………………………55 TABLE……………………………………………………………………56 REFERENCES……………………………………………………………57 APPENDIX………………………………………………………………7

Neddylation and Deneddylation Regulate Cul1 and Cul3 Protein Accumulation

Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities1, 2. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN)3, 4. Here we show that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3 ) proteins are unstable, and that to preserve their normal cellular levels , CSN isopeptidase activity is required. We further show that neddylated Cul1 and Cul3 are unstable — as suggested by the evidence that Nedd8 promotes the instability of both cullins — and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin- conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. We propose that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo

The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence.

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. We found that the COP9 signalosome (CSN) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. We propose a model in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM

Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly

<div><p>In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines <em>in-vivo</em> isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (<em>Drosophila melanogaster</em>). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (¹³C₆-glucose) for 12 h – either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate – possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism.</p> </div

Notation used in this report.

<p>Before the incubations, the flies were entrained to light/dark cycle in the following way: day (light), 9:00 AM–9:00 PM; night (dark), 9:00 PM–9:00 AM.</p

Experimental design and chemical analysis workflow.

<p>(1) pre-conditioning (entrainment) of the culture stock (adaptation to the 12/12-h (light/dark) cycle); (2) incubation of female fruit flies with ¹³C₆-glucose solution; (3) dissection of the anesthetized flies; (4a/4b) preparation of individual eggs for mass spectrometric analysis; (5) mass spectrometry, and (6) data analysis.</p

Histograms showing the distributions of labeling levels within the eggs obtained from the fruit flies incubated with ¹³C₆-glucose (during 12 h) at different illumination conditions and at different times during the day/night cycle.

<p>ML – flies incubated during day (starting from the morning) at light; MD – flies incubated during day (starting from the morning) at dark; EL – flies incubated during night (starting from the evening) at light; ED – flies incubated during night (starting from the evening) at dark.</p

Histograms showing differences in the labeling of eggs among flies from the same treatment.

<p>Flies incubated during night (starting from the evening) at dark (ED): arbitrarily selected examples of five single-fly histograms, ordered according to the increasing level of labeling (Fly #1 to #5: top to bottom). All single-fly histograms from this experimental variant are displayed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050258#pone.0050258.s009" target="_blank">Figure S9</a></b>.</p

Influence of temperature (21 <i>vs.</i> 28°C) and illumination (dark <i>vs.</i> light) on the labeling of glucose moiety in UDP-glucose molecules extracted from individual eggs.

<p>Female flies were incubated with ¹³C₆-glucose solution during 24 h. The default conditions were: white light on, ∼4000 lux (in the study involving the change of temperature); temperature, 28°C (in the study involving the change of illumination).</p