Molecular Cell：台学者创新发现K33连接的泛素链

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IL-33调控NF-κB

doi：10.3969/j.issn.1000-484X.2023.09.005IL-33调控NF-κB/Twist信号通路促进肺泡上皮细胞上皮-间质转分化的机制研究①高云星②付裕③陈晓李泽朋何晓伟李先伟（皖南医学院药学院药理学教研室，芜湖 241002）中图分类号R392.12 文献标志码 A 文章编号1000-484X（2023）09-1814-08［摘要］目的：探究IL-33是否通过激活NF-κB/Twist信号通路，进而诱导Ⅱ型肺泡上皮细胞发生上皮-间质转分化（EMT）而促进肺纤维化（PF）的进程。

方法：C57BL/6小鼠48只，随机分成对照（CON）组、博莱霉素（BLM）组、BLM+IL-33 （30 μg/kg）组和BLM+抗IL-33抗体（Anti-IL-33 Ab）（100 μg/kg）组，每组12只。

气管注射BLM（5 000 U/kg）诱导PF小鼠模型。

造模后每隔1 d腹腔注射1次重组IL-33和抗IL-33抗体，连续注射3周。

HE和Masson染色观察肺组织病理变化及胶原沉积情况。

ELISA检测血浆IL-33的水平。

免疫组化检测肺组织IL-33跨膜受体ST2L的表达。

体外培养小鼠Ⅱ型肺泡上皮细胞，实验设Control组、IL-33（100 ng/ml）组、IL-33+二甲基亚砜（DMSO）组及IL-33+吡咯烷二硫代氨基甲酸盐（PDTC， 100 μmol/L）组，每组设复孔6个。

细胞先用PDTC或DMSO预处理1 h，再用IL-33处理48 h。

RT-PCR检测肺组织或肺泡上皮细胞中Collagen Ⅰ、Collagen Ⅲ、E钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）和α-平滑肌肌动蛋白（α-SMA）mRNA的表达。

Western blot检测肺组织和（或）肺泡上皮细胞IL-33、Collagen Ⅰ、Collagen Ⅲ、E-cadherin、Vimentin、α-SMA、p-IκBα、p-NF-κB p65蛋白表达及细胞核内NF-κB p65和Twist的蛋白水平。

Histone Methylation by PRC2 Is Inhibited by Active Chromatin Marks

Molecular CellArticleHistone Methylation by PRC2Is Inhibitedby Active Chromatin MarksFrank W.Schmitges,1,6Archana B.Prusty,2,6Mahamadou Faty,1Alexandra Stu¨tzer,3Gondichatnahalli M.Lingaraju,1 Jonathan Aiwazian,1Ragna Sack,1Daniel Hess,1Ling Li,4Shaolian Zhou,4Richard D.Bunker,1Urs Wirth,5Tewis Bouwmeester,5Andreas Bauer,5Nga Ly-Hartig,2Kehao Zhao,4Homan Chan,4Justin Gu,4Heinz Gut,1 Wolfgang Fischle,3Ju¨rg Mu¨ller,2,7,*and Nicolas H.Thoma¨1,*1Friedrich Miescher Institute for Biomedical Research,Maulbeerstrasse66,CH-4058Basel,Switzerland2Genome Biology Unit,EMBL Heidelberg,Meyerhofstrasse1,D-69117Heidelberg,Germany3Laboratory of Chromatin Biochemistry,Max Planck Institute for Biophysical Chemistry,Am Fassberg11,D-37077Go¨ttingen,Germany4China Novartis Institutes for Biomedical Research,Lane898Halei Road,Zhangjiang,Shanghai,China5Novartis Institutes for Biomedical Research,CH-4002Basel,Switzerland6These authors contributed equally to this work7Present address:Max Planck Institute of Biochemistry,Am Klopferspitz18,D-82152Martinsried,Germany*Correspondence:muellerj@biochem.mpg.de(J.M.),nicolas.thoma@fmi.ch(N.H.T.)DOI10.1016/j.molcel.2011.03.025SUMMARYThe Polycomb repressive complex2(PRC2)confers transcriptional repression through histone H3lysine 27trimethylation(H3K27me3).Here,we examined how PRC2is modulated by histone modiﬁcations associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2Nurf55-Su(z)12 submodule.Binding of H3is lost if lysine4in H3is trimethylated.Weﬁnd that H3K4me3inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12C terminus.In addition to H3K4me3,PRC2 is inhibited by H3K36me2/3(i.e.,both H3K36me2 and H3K36me3).Direct PRC2inhibition by H3K4me3and H3K36me2/3active marks is con-served in humans,mouse,andﬂy,rendering tran-scriptionally active chromatin refractory to PRC2 H3K27trimethylation.While inhibition is present in plant PRC2,it can be modulated through exchange of the Su(z)12subunit.Inhibition by active chromatin marks,coupled to stimulation by transcriptionally repressive H3K27me3,enables PRC2to autono-mously template repressive H3K27me3without over-writing active chromatin domains.INTRODUCTIONPolycomb(PcG)and trithorax group(trxG)proteins form distinct multiprotein complexes that modify chromatin.These com-plexes are conserved in animals and plants and are required to maintain spatially restricted transcription of HOX and other cell fate determination genes(Henderson and Dean,2004; Pietersen and van Lohuizen,2008;Schuettengruber et al., 2007;Schwartz and Pirrotta,2007).PcG proteins act to repress their target genes while trxG protein complexes are required to keep the same genes active in cells where they must be expressed.Among the PcG protein complexes,Polycomb repressive complex2(PRC2)is a histone methyl-transferase(HMTase) that methylates Lys27of H3(H3K27)(Cao et al.,2002;Czermin et al.,2002;Kuzmichev et al.,2004;Mu¨ller et al.,2002).High levels of H3K27trimethylation(H3K27me3)in the coding region generally correlate with transcription repression(Cao et al., 2008;Nekrasov et al.,2007;Sarma et al.,2008).PRC2contains four core subunits:Enhancer of zeste[E(z),EZH2in mammals], Suppressor of zeste12[Su(z)12,SUZ12in mammals],Extra-sex combs[ESC,EED in mammals]and Nurf55[Rbbp4/ RbAp48and Rbbp7/RbAp46in mammals](reviewed in Schuet-tengruber et al.,2007;Wu et al.,2009).E(z)is the catalytic subunit;it requires Nurf55and Su(z)12for nucleosome associa-tion,whereas ESC is required to boost the catalytic activity of E(z)(Nekrasov et al.,2005).Recent studies reported that ESC binds to H3K27me3and that this interaction stimulates the HMTase activity of the complex(Hansen et al.,2008;Margueron et al.,2009;Xu et al.,2010).The observation that PRC2is able to bind to the same modiﬁcation that it deposits led to a model for propagation of H3K27me3during replication.In this model, recognition of H3K27me3on previously modiﬁed nucleosomes promotes methylation of neighboring nucleosomes that contain newly incorporated unmodiﬁed histone H3(Hansen et al.,2008; Margueron et al.,2009).However,it is unclear how such a positive feedback loop ensures that H3K27trimethylation remains localized to repressed target genes and does not invade the chromatin of nearby active genes.In organisms ranging from yeast to humans,chromatin of actively transcribed genes is marked by H3K4me3, H3K36me2,and H3K36me3modiﬁcations:while H3K4me3is tightly localized at and immediately downstream of the transcrip-tion start site,H3K36me2peaks adjacently in the50coding region and H3K36me3is speciﬁcally enriched in the30coding region(Bell et al.,2008;Santos-Rosa et al.,2002).Among the trxG proteins that keep PcG target genes active are the HMTases Trx and Ash1,which methylate H3K4and H3K36, respectively(Milne et al.,2002;Nakamura et al.,2002;Tanaka330Molecular Cell42,330–341,May6,2011ª2011Elsevier Inc.et al.,2007).Studies in Drosophila showed that Trx and Ash1 play a critical role in antagonizing H3K27trimethylation by PRC2,suggesting a crosstalk between repressive and activating marks(Papp and Mu¨ller,2006;Srinivasan et al.,2008).In this study we investigated how PRC2activity is modulated by chromatin marks typically associated with active transcrip-tion.We found that the Nurf55WD40propeller binds the N terminus of unmodiﬁed histone H3and that H3K4me3 prevents this binding.In the context of the tetrameric PRC2 complex,weﬁnd that H3K4me3and H3K36me2/3(i.e.,both H3-K36me2and H3-K36me3)inhibit histone methylation by PRC2in vitro.Dissection of this process by usingﬂy,human, and plant PRC2complexes suggests that the Su(z)12subunit is important for mediating this inhibition.PRC2thus not only contains the enzymatic activity for H3K27methylation and a recognition site for binding to this modiﬁcation,but it also harbors a control module that triggers inhibition of this activity to prevent deposition of H3K27trimethylation on transcription-ally active genes.PRC2can thus integrate information provided by pre-existing histone modiﬁcations to accurately tune its enzymatic activity within a particular chromatin context.RESULTSStructure of Nurf55Bound to the N Terminusof Histone H3Previous studies reported that Nurf55alone is able to bind to histone H3(Beisel et al.,2002;Hansen et al.,2008;Song et al., 2008;Wysocka et al.,2006)but not to a GST-H3fusion protein (Verreault et al.,1998).By usingﬂuorescence polarization(FP) measurements,we found that Nurf55binds the very N terminus of unmodiﬁed histone H3encompassing residues1–15(H31–15) with a K D of$0.8±0.1m M but does not bind to a histone H319–38 peptide(Figure1A).Crystallographic screening resulted in the successful cocrystallization of Nurf55in complex with an H31–19peptide.After molecular replacement with the known structure of Nurf55(Song et al.,2008),the initial mF oÀDF c differ-ence map showed density for H3residues1–14in bothNurf55molecules in the crystallographic asymmetric unit. Figures1B–1E show the structure of H31–19bound to Drosophila Nurf55,reﬁned to2.7A˚resolution(R/R free=20.1%and25.0%, Table1;Figure S1A,available online).The H3peptide binds to theﬂat surface of the Nurf55WD40propeller(Figure1B),subse-quently referred to as the canonical binding site(c-site)(Gaudet et al.,1996).The H3peptide is held in an acidic pocket(Figures 1C and1E)and traverses the central WD40cavity in a straight line across the propeller(Figure1B).Nurf55binds the H3peptide by contacting H3residues Ala1, Arg2,Lys4,Ala7,and Lys9.Each of these residues forms side-chain speciﬁc contacts with the Nurf55propeller(Figures 1D and1E).The bulk of the molecular recognition is directed toward H3Arg2and Lys4.Ala1sits in a buried pocket with its a-amino group hydrogen bonding to Nurf55Asp252,which recognizes andﬁxes the very N terminus of histone H3.The neighboring Arg2is buried deeper within the WD40propeller fold,with its guanidinium group sandwiched by Nurf55residues Phe325and Tyr185(Figure1D).H3Lys4binds to a well-deﬁned surface pocket on Nurf55located on blade2,near the central cavity of the propeller.Its3-amino group is speciﬁcally coordi-nated by the carboxyl groups of Nurf55residues Glu183andGlu130and through the amide oxygen of Asn132(Figure1E).Lys9is stabilized by hydrophobic interactions on the WD40surface while having its3-amino group held in solvent-exposedfashion(Figure1D).Ser10of histone H3marks the beginningof a turn that inverses the peptide directionality.Histone H3residues Thr11–Lys14become progressively disordered andare no longer speciﬁcally recognized.No interpretable densitywas observed beyond Lys14.Taken together,Nurf55speciﬁcallyrecognizes an extended region of the extreme N terminus ofhistone H3(11residues long,700A˚2buried surface area)in thecanonical ligand binding location of WD40propeller domains. Structure of the Nurf55-Su(z)12Subcomplex of PRC2 The H3-Nurf55structure prompted us to investigate how Nurf55might bind histone tails in the presence of Su(z)12,its interactionpartner in PRC2(Nekrasov et al.,2005;Pasini et al.,2004).Asaﬁrst step we mapped the Nurf55-Su(z)12interaction in detailby carrying out limited proteolysis experiments on reconstitutedDrosophila PRC2,followed by isolation of a Nurf55-Su(z)12subcomplex.Mass spectrometric analysis and pull-down exper-iments with recombinant protein identiﬁed Su(z)12residues73–143[hereafter referred to as Su(z)1273–143]as sufﬁcient forNurf55binding(Figures S1C and S1D).Crystals were obtained when Drosophila Nurf55and Su(z)12residues64–359were set up in the presence of0.01%subtilisinprotease(Dong et al.,2007).After data collection,the structurewas reﬁned to a maximal resolution of2.3A˚(Table1).Molecularreplacement with Nurf55as search model provided clear initialmF oÀDF c difference density for a13amino acid-long Su(z)12 fragment spanning Su(z)12residues79–91(Figures2A–2C).Theﬁnal model was reﬁned to2.3A˚(R/R free=17.5%/20.9%)and veriﬁed by simulated annealing composite-omit maps(Fig-ure S1B).The portion of Su(z)12involved in Nurf55binding willhenceforth be referred to as the Nurf55binding epitope(NBE).The Su(z)12binding site on Nurf55is located on the side of thepropeller between the stem of the N-terminal a helix(a1)andthe PP loop(Figures2A and2B).Binding between Su(z)12andNurf55occurs mostly through hydrophobic interactions in anextended conformation.The interaction surface betweenNurf55and the NBE is large for a peptide,spanning around800A˚2.Sequence alignment between Su(z)12orthologs revealsthat the NBE is highly conserved(53%identity and84%similarity)in animals and in plants(Figure2E).With the exceptionof Su(z)12Arg85,the majority of the conserved Su(z)12NBEresidues engage in hydrophobic packing with Nurf55(Figures2B and2C).Together with the Su(z)12VEFS domain and theC2H2zincﬁnger(C5domain)(Birve et al.,2001),the NBE consti-tutes the only identiﬁable motif in Su(z)12found conserved in allSu(z)12orthologs.The NBE binding site on Nurf55has previously been shown tobe occupied by helix1of histone H4(Figure2D)(Murzina et al.,2008;Song et al.,2008),an epitope not accessible in assemblednucleosomes(Luger et al.,1997).Nurf55binds H4and theSu(z)12NBE epitope in a different mode,and importantly,withopposite directionality(Figure2D).The detailed comparison ofthe Nurf55-Su(z)12structure with that of H4bound to Nurf55Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3Molecular Cell42,330–341,May6,2011ª2011Elsevier Inc.331strongly suggests that binding of Su(z)12(NBE)and of H4(helix 1)are mutually exclusive (Figure 2D).We therefore refer to the Su(z)12and H4binding site on Nurf55as the S/H -site.Su(z)12fragments that include the NBE have poor solubility by themselves and generally require Nurf55coexpression for solu-bilization.However,we were able to measure binding of a chem-ically synthesized Su(z)1275–93peptide to Nurf55by isothermal titration calorimetry (ITC)and found that the peptide was bound with a K D value of 6.7±0.3m M in a 1:1stoichiometry (Fig-ure 2F).Pull-down experiments with recombinant proteinandFigure 1.Crystal Structure of Nurf55in Complex with a Histone H31–19Peptide(A)Nurf55binds to an H31–15peptide with an afﬁnity of $0.8±0.1m M as measured by FP.It has similar afﬁnity for an H31–31peptide (2.2±0.2m M)but no binding can be detected to an H319–38peptide.(B)Ribbon representation of Nurf55-H31–19.Nurf55is shown in rainbow colors and H31–19is depicted in green.The peptide is bound to the c -site of the WD40propeller.(C)Electrostatic surface potential representation (À10to 10kT/e)of the c -site with the H3peptide shown as a stick model in green.(D)Close-up of the c -site detailing the interactions between Nurf55(yellow)and the H31–19peptide (green),with a water molecule shown as a red sphere.(E)Schematic representation of interactions between the H31–19peptide (green)and Nurf55(yellow).Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3332Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.streptavidin beads suggest that Su(z)12residues 94–143harbor an additional Nurf55binding site not visible in the structure (Figure S1E).Su(z)12144–359,lacking the N-terminal 143residues,no longer binds to Nurf55.The NBE (residues 79–93)and the region adjacent to the NBE (residues 94–143)are thus required for stable interaction with Nurf55.The extended NBE was found enriched after limited proteolysis and in subsequent gel ﬁltration runs coupled with quantitative mass spectrometry (Figure S1C).As the NBE was the only fragment visible after structure determi-nation,we conclude that it represents the major Su(z)12interac-tion epitope for Nurf55binding.The Nurf55-Su(z)12Complex Binds to Histone H3In order to study the potential interdependence of the identiﬁed Nurf55binding sites we compared binding of Nurf55and Nurf55-Su(z)12to the histone H3N terminus.FP experiments showed similar afﬁnities for binding of a histone H31–15peptide to Nurf55(K D $0.8±0.1m M;Figure 1A)and a Nurf55-Su(z)1273–143complex (K D $0.6±0.1m M;Figure 2G).Importantly,mutation of Nurf55resi-dues contacting H3via its c -site drastically reduced binding to an H31–15peptide (Figure S2A),demonstrating that the Nurf55-Su(z)1273–143complex indeed binds the H31–15peptide through the c -site.We conclude that the presence of Su(z)12is compatible with Nurf55binding to H3via its c -site and that the two binding interactions are not interdependent.The observation that the Su(z)12NBE occupies the same Nurf55pocket that was previously shown to bind to helix 1of histone H4prompted us to test whether the Su(z)1273–143-Nurf55complex could still bind to histone H4.We performed pull-down experiments with a glutathione S-transferase (GST)fusion protein containing histone H41–48(Murzina et al.,2008)and found that H4stably interacted with isolated Nurf55but not with Su(z)1273–143-Nurf55(Figure 2H).In PRC2,the presence of Su(z)12in the Nurf55S/H -site therefore precludes binding to helix 1of histone H4.H3Binding by Nurf55-Su(z)12Is Sensitive to the Methylation Status of Lysine 4We next investigated how posttranslational modiﬁcations of the H3tail affect binding to the Nurf55-Su(z)1273–143complex.Modi-ﬁcations on H3Arg2,Lys9,and Lys14did not change afﬁnity of Nurf55-Su(z)12for the modiﬁed H31–15peptide (Figures S2B and S2D).In contrast,peptides that were mono-,di-,or trimethylated on Lys4were bound with signiﬁcantly reduced afﬁnity exhibiting K D values of 17±3m M (H3K4me1),24±3m M (H3K4me2),and >70m M (H3K4me3),respectively (Figure 2I).The FP binding data were independently conﬁrmed by ITC measurements (Figures S2C–S2F).Together,these ﬁndings are in accord with the structural data,which show that H3K9and H3K14are being held with their 3-amino moiety solvent-exposed,while the H3K4side chain is tightly coordinated (Figure 1E).The additional methyl groups on the H3K43-amino group are expected to progressively decrease afﬁnity because of increased steric clashes within the H3K4binding pocket.H3K27Methylation by PRC2Is Inhibited by Histone H3K4me3MarksWe then examined the effect of H3K4me3modiﬁcations,which are no longer retained by Nurf55-Su(z)12,on the catalytic activity of PRC2.In a ﬁrst set of experiments,we determined PRC2steady-state parameters on histone H31–45peptide substrates that were either unmodiﬁed or methylated at Lys 4.We observed similar K M values for H3and H3K4me3peptides of 0.84±0.21m M and 0.36±0.07m M,respectively (Figure 3A),and similar K M values for SAM (5.42±0.65m M for H3and 10.04±1.56m M for H3K4me3).The turnover rate constant k cat ,however,was 8-fold reduced in the presence of H3K4me3:2.53±0.21min -1for unmodiﬁed H3and 0.32±0.08min -1in the presence of H3K4me3(Figure 3A).While substrate binding is largely unaf-fected,turnover is thus severely inhibited in the presence of H3K4me3.This behavior,which results in a k cat /K M speciﬁcity constant of 7.83103M -1s -1(unmodiﬁed H3)compared to 0.533103M -1s -1(H3K4me3),is consistent with heterotrophic allosteric inhibition of the PRC2HMTase triggered by the pres-ence of the H3K4me3.To investigate the effect of the H3K4me3modiﬁcation on PRC2activity in the context of nucleosomes,we reconstituted mononucleosomes with a trimethyllysine analog (MLA)at Lys4in H3(referred to as H3Kc4me3;Figure S3A)(Simon et al.,2007).We found that total H3K27methylation (measured by incorporation of 14C-labeled methyl groups)was substantially impaired on H3Kc4me3-containing nucleosomes compared to wild-type nucleosomes (Figures S3B and S3C).We used western blot analysis to monitor how levels of H3K27mono-,di-,and trimethylation were affected by the H3Kc4me3modiﬁca-tion.While H3K27me1formation was reduced by more than 50%on H3Kc4me3nucleosomes compared to unmodiﬁed nucleosomes (Figures 3B and 3C),H3K27dimethylationandTable 1.Crystallographic Data and Reﬁnement StatisticsNurf55–Su(z)12Nurf55–H31–19Space Group P212121P212121theses.Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.333titrant H3:S/H-siteN CNCSu(z)12 79-91Histone H4 31-41PP-loophelix α10.00.51.0 1.52.0 2.5-16.00-14.00-12.00-10.00-8.00-6.00-4.00-2.000.00-2.00-1.50-1.00-0.500.00020406080100120Time (min)µc a l /s e cMolar RatioK C a l /M o l e o f I n j e c t a n tK D = 6.7 ± 0.3 µMtitrant: H31-15DEFGH ICA B protein concentration [µM]Nurf55-Su(z)12protein concentration [µM]00.20.40.60.81.000.20.40.60.81.00.010.11101000.010.1110100Nurf55Nurf55-Su(z)12unmod K4me1K4me2K4me3f r a c t i o n b o u n df r a c t i o n b o u n d G ST -H 41-48 + N u r f 55G S T -H 41-48 + N u r f 55-S u (z )1273-143N u r f 55(m o c k c o n t r o l )N u r f 55-S u (z )1273-143(m o c k c o n t r o l )Nurf55GST-H41-48Su(z)1273-143i np ut i n p u t i n p u t i n p u t G S T -p u l l d o w n G S T -p u l l d o w n G S T -p u l l d o w n G S T -p u l l d o w n Figure 2.Crystal Structure and Characterization of Nurf55in Complex with the Su(z)12Binding Epitope for Nurf55(A)Ribbon representation of Nurf55-Su(z)12.Nurf55(rainbow colors)depicts the WD40domain nomenclature and Su(z)12is shown in magenta.The S/H -site is marked by a dashed box.(B)Detailed interactions of Su(z)12(magenta)with the S/H -site (yellow).Water molecules are depicted as red spheres.(C)Schematic representation of interactions between Su(z)12(magenta)and Nurf55(yellow).(D)Overlay of the backbone trace of Su(z)12(magenta)and the H4helix a 1(orange)(Song et al.,2008)in the S/H -site.(E)Alignment of the Su(z)12NBE with sequences from Drosophila melanogaster (dm,Q9NJG9),mouse (mm,NP_954666),human (hs,AAH15704),Xenopus tropicalis (xt,BC121323),zebraﬁsh (dr,BC078293),and the three Arabidopsis thaliana (at)homologs Fis2(ABB84250),EMF2(NP_199936),and VRN2(NP_567517).Identical residues are highlighted in yellow.(F)ITC proﬁle for binding of a Su(z)1275–93peptide to Nurf55.Data were ﬁtted to a one-site model with stoichiometry of 1:1.The derived K D value is 6.7±0.3m M.(G)Binding of H31–15to Nurf55(0.8±0.1m M)and Nurf55-Su(z)1273–143(0.6±0.1m M)measured by FP.Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3334Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.trimethylation were impaired by more than 80%by using H3Kc4me3nucleosomes (Figure 3C).In order to ascertain that inhibition of PRC2is indeed due to trimethylation of the aminogroup in the lysine side chain,and not due to the use of the MLA,we performed HMTase assays on H3K4me3-containing nucleosomes generated by native peptide ligation (Shogren-Knaak et al.,2003)and on H3Kc4me0and H3K4A nucleosomes.H3K27mono-,di-,and trimethylation was comparably inhibited on H3K4me3and on H3Kc4me3-containing nucleosomes,but was not affected by H3Kc4me0and H3K4A (Figures S3D and S3E).We conclude that H3K4me3speciﬁcally inhibits PRC2-mediated H3K27methylation with the most pronounced inhibi-tory effects observed for H3K27di-and trimethylation.We next tested whether the H3K4me3modiﬁcation affects PRC2nucleosome binding.In electrophoretic mobility shift assays (EMSA),we found that PRC2binds unmodiﬁed or H3Kc4me3-modiﬁed nucleosomes with comparable afﬁnity (Fig-ure S4A).Even though binding of Nurf55to the N terminus of ABC571141712290286571141712290286unmodified H3Kc4me3H3K27me3H4dmPRC2 [nM]H3K27me2H4H3K27me1H4H3K27me1H3K27me3H3K27me2dmPRC2 + unmod H3dmPRC2 + H3Kc4me3r e l a t i v e H M T a s e a c t i v i t ySubstrate Apparent Km of SAM (µM)Apparent Km of peptide (nM)k cat (min )-1k cat /Km (M S )-1-1H31-45 -biotin H3K4me31-45 -biotin5.42 ± 0.6510.04 ± 1.56355 ± 74.60.32 ± 0.080.53 x 10836 ± 207 2.53 ± 0.217.8 x 1033Figure 3.HMTase Activity of PRC2Is In-hibited by H3K4me3Marks(A)HMTase assay with PRC2and H31–45-biotin peptides measuring the concentration of SAH produced by the enzymatic reaction.When an H3K4me3-modiﬁed peptide is used,the speciﬁcity constant (k cat /K M )is drastically reduced,indicative of heterotrophic allosteric inhibition.(B)Western blot-based HMTase assay by using recombinant Drosophila mononucleosomes (571nM)and increasing amounts of PRC2.HMTase activity was monitored with antibodies against H3K27me1,H3K27me2,or H3K27me3as indicated;in each case the membrane was also probed with an antibody against unmodiﬁed histone H4to control for equal loading and western blot processing.Deposition of K27di-and trime-thylation is drastically reduced when nucleosomes are used that carry a H3Kc4me3modiﬁcation.(C)Quantiﬁcation of HMTase activity of Drosophila PRC2(286nM)on unmodiﬁed and H3Kc4me3-modiﬁed nucleosomes by quantitative western blotting.histone H3is almost 100-fold reduced byH3K4me3(Figure 2I and Figure S2F),inter-action of the Nurf55c -site with H3K4does not seem to make a detectable con-tribution to nucleosome binding by PRC2in this assay.Consistent with the allosteric mechanism of H3K4me3inhibition that we had observed in the peptide assays (Figure 3A),inhibition of the PRC2HMTase activity by H3K4me3-containingnucleosomes is not caused by impaired nucleosome binding,but is rather the consequence of reduced catalytic turnover.H3K4me3Needs to Be Present on the Same Tail as K27to Inhibit PRC2We then assessed whether inhibition of the PRC2HMTase activity by H3K4me3requires the K4me3mark to be located on the substrate nucleosome (in cis ),or whether it could also be trig-gered if the H3K4me3modiﬁcation was provided on a separate peptide (in trans ).We performed HMTase assays on unmodiﬁed oligonucleosomes in the presence of increasing amounts of a histone H31–15peptide trimethylated at K4(H31–15-K4me3)(Figure 4A).Addition of the H31–15-K4me3peptide did not affect PRC2HMTase activity at peptide concentrations as high as $200m M.When testing H31–19-unmodiﬁed peptide in controls at comparable concentrations,we did observe concentration-dependent PRC2inhibition (Figure 4A),probably because of substrate competition at large peptide excess.As H3K4me3-(H)GST pull-down assay with recombinant GST-H41–48and Nurf55and Nurf55-Su(z)1273–143proteins.GST-H41–48is able to bind Nurf55alone but in the Nurf55-Su(z)1273–143complex the binding site is occupied by Su(z)12(left panel).Control pull-downs with GST beads and either Nurf55or Nurf55-Su(z)1273–143alone showed no unspeciﬁc binding (right panel).(I)Binding of different H31–15peptides to Nurf55-Su(z)1273–143measured by FP.While unmodiﬁed H3is bound with 0.8±0.1m M afﬁnity,methylation of Lys 4drastically reduces binding afﬁnity (17±3m M for K4me1,24±3m M for K4me2,and >70m M for K4me3).Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.335modiﬁed peptides did not show this competitive behavior,we conclude that PRC2is not inhibited by H3K4me3in trans and that H3K4me3and unmodiﬁed H3peptides are probably bound to PRC2in a different fashion.Analogously,we saw no inhibition when testing the effect of H3K4me3in trans by using peptides as substrates (Figure S4B ).Taken together,our ﬁndings strongly argue that H3K4me3only inhibits PRC2if present on the same tail that contains the H3K27target lysine (in cis ).Previous studies reported that addition of H3K27me3peptides in trans enhances H3K27methylation of oligonucleosomes by human PRC2through binding to the EED WD40domain (Margueron et al.,2009;Xu et al.,2010).We tested whether addition of H3K27me3peptides in trans would stimulate H3K27methylation by PRC2on H3Kc4me3-modiﬁed nucleosomes.We observed that the inhibitory effect of H3Kc4me3-containing nucleosomes can,at least in part,be overcome through addition of high concentrations of H3K27me3peptides (Figure 4B and Figure S4C).PRC2is therefore able to simultaneously integrate inhibitory (H3K4me3)and activating (H3K27me3)chromatin signatures and adjust its enzymatic activity in response to the surrounding epigenetic environment.PRC2Inhibition of H3K4me3Is Conserved in Mammalian PRC2Our results with Drosophila PRC2prompted us to investigate to what extent inhibition by H3K4me3is an evolutionarily conserved mechanism.H3K27methylation by human and mouse PRC2on nucleosome substrates carrying H3Kc4me3modiﬁcations was also strongly inhibited,comparable to the inhibition observed for Drosophila PRC2(Figure 5A and Figure S5A).The Su(z)12Subunit Codetermines Whether PRC2Is Inhibited by H3K4me3In Arabidopsis thaliana ,three different E(z)homologs combined with three Su(z)12homologs have been described.The distinct PRC2complexes in plants harboring the different E(z)or Su(z)12subunits are implicated in the control of distinct developmental processes during Arabidopsis development (He,2009).In this study we focused on PRC2complexes con-taining the E(z)homolog CURLY LEAF (CLF).We expressed and reconstituted the Arabidopsis PRC2complex comprising CLF,FERTILIZATION INDEPENDENT ENDOSPERM (FIE,a homolog of ESC),EMBRYONIC FLOWER 2(EMF2,a homolog of Su(z)12),and MULTICOPY SUPPRESSOR OF IRA (MSI1,a homolog of Nurf55).We found that CLF indeed functions as a H3K27me3HMTase (Figure 5B).Moreover,H3K27methylation by the CLF-FIE-EMF2-MSI1complex on nucleosome arrays containing H3Kc4me3was inhibited (Figure 5B)in a manner comparable to human or Drosophila PRC2.We next tested a related Arabidopsis PRC2complex again composed of CLF,FIE,and MSI1but containing the Su(z)12homolog vernalization 2(VRN2)instead of EMF2.The VRN2protein is speciﬁcally implicated as a repressor of the FLC locus,thereby controlling ﬂowering time in response to vernalization (reviewed in Henderson and Dean,2004).The CLF-FIE-MSI1-VRN2complex was active on unmodiﬁed nucleosomes but,strikingly,it was not inhibited on H3Kc4me3-modiﬁed nucle-osomes (Figure 5C).Substitution of a single subunit (i.e.,EMF2by VRN2)thus renders the complex nonresponsive to the H3K4methylation state.While PRC2inhibition by H3K4me3appears hardwired in mammals and ﬂies,in which only a single-Su(z)12ortholog is present,Arabidopsis inhibition can be enabled or disabled through exchange of the Su(z)12homolog.The Su(z)12C Terminus Harboring the VEFS Domain Is the Minimal Su(z)12Domain Required for Activation and Active Mark InhibitionThe importance of the Su(z)12subunit in active mark H3K4me3inhibition prompted us to map the Su(z)12domains required for inhibition.Previous ﬁndings showed that E(z)or E(z)-ESC in the absence of Su(z)12is enzymatically inactive (Nekrasov et al.,2005).Moreover,the VEFS domain (Birve et al.,2001)was found to be the major E(z)binding domain (Ketel et al.,2005).We reconstituted mouse PRC2complexes containing EZH2,EED,and either SUZ12C 2H 2domain +VEFS (residues 439–741)or SUZ12VEFS alone (residues 552–741).Both of these minimal complexes were active in HMTase assays on nucleosomes (Figure 5D)but with lower activity than that of the full PRC2complex.We therefore focused on formation ofAB26511030206H3K27me3H4peptides in trans[µM]H3K27me2H4H3K27me1H4H3 unmod H3K4me3H3K36me326511032062651103206n o e n z ym e26511030H3K27me3H4peptides in trans[µM]H3K27me2H4H3K27me1H4H3K27me3n oe n z y m eFigure 4.PRC2Activity Is Not Inhibited by H3K4me3Peptides in trans(A)Western blot-based HMTase assay by using unmodiﬁed 4-mer oligonu-cleosomes (36nM)and increasing amounts of H3peptides added in trans .Enzyme concentration was kept constant at 86nM.Western blots were pro-cessed as described in Figure 3B.HMTase activity is inhibited by an unmodiﬁed H31–19peptide (left),but not by H3K4me3-or H3K36me3-modiﬁed peptides.(B)HMTase assay with H3Kc4me3-modiﬁed oligonucleosomes (36nM),86nM PRC2,and H3K27me3peptide in trans .Western blots were processed as described in Figure 3B.HMTase activity of PRC2can be stimulated by the H3K27me3peptide even on inhibiting substrate leading to increased levels of H3K27di-and trimethylation.Molecular CellAllosteric PRC2Inhibition by H3K4me3/H3K36me3336Molecular Cell 42,330–341,May 6,2011ª2011Elsevier Inc.。

去泛素化酶usp33的研究进展

２ＵＳＰ３３的生物学功能
２．１ＵＳＰ３３与ＳｌｉｔＲｏｂｏ信号通路Ｓｌｉｔ／Ｒｏｂｏ信号传导首先被确定为“轴突导向 ”线索，其在防止连合轴突重新穿过中线和调节感觉轴突伸长和分支中起关键作用。［９－１０］ＹｕａｓａＫａｗａｄａ等［１１］研究发现ＵＳＰ３３和Ｒｏｂｏ１蛋白之间相互作用，并且沉默Ｒｏｂｏ１能增强敲低后ＵＳＰ３３的致癌作用。在乳腺癌细胞中，ＵＳＰ３３将Ｓｌｉｔ诱导的Ｒｏｂｏ１从细胞内室重新分布到质膜，而不影响Ｒｏｂｏ１蛋白的总水平；Ｗｅｎ等［１２］研究表明在肺癌细胞中，ＵＳＰ３３通过阻止ＵＰＳ降解，与Ｒｏｂｏ１相互作用并稳定其蛋白的总水平。同时，ＵＳＰ３３的降低可使Ｓｌｉｔ信号传导失活从而抑制肺癌细胞迁移。Ｌｕ等［１３］通过Ｗｅｓｔｅｒｎｂｌｏｔ分析得出，在肺癌细胞中ＵＳＰ３３过表达可使Ｓｌｉｔ２和Ｒｏｂｏ１的表达水平上调。综上研究表明，在不同的肿瘤中ＵＳＰ３３可能通过不同的机制介导Ｓｌｉｔ信号传导。２．２ＵＳＰ３３与ＴＧＦβ信号通路上皮细胞－间质转化（ｅｐｉｔｈｅｌｉａｌｍｅｓｅｎｃｈｙｍａｌｔｒａｎｓｉｔｉｏｎ，ＥＭＴ）是指上皮细胞通过特定程序转化为间质细胞表型的生物学过程，与肿瘤的浸润转移密切相关。ＥＭＴ发生涉及众多信号通路及相关基因［１４］，其中ＴＧＦβ／Ｓｍａｄ信号通路是ＥＭＴ中重要的调节因子［１５］。ＰＰＭ１Ａ可以通过去磷酸化Ｓｍａｄ终止ＴＧＦβ信号传导［１６］。Ｌｕ等［１３］研究显示，ＵＳＰ３３作为ｍｉＲ３５９１５ｐ的下游靶点，可以使ＰＰＭ１Ａ去泛素化。ＵＳＰ３３或ＰＰＭ１Ａ的异位表达部分消除ｍｉＲ３５９１５ｐ对ＥＭＴ和ＴＧＦβ活化的影响。研究表明ｍｉＲ３５９１５ｐ／ＵＳＰ３３／ＰＰＭ１Ａ通过ＴＧＦβ信－特异性蛋白酶３３（ｕｂｉｑｕｉｔｉｎｓｐｅｃｉｆｉｃｐｒｏｔｅａｓｅｓ３３，ＵＳＰ３３）是去泛素化酶（ｄｅｕｂｉｑｕｉｔｉｎａｔｉｎｇｅｎｚｙｍｅｓ，ＤＵＢ）家族的重要成员，主要通过对底物蛋白的去泛素化阻止蛋白酶体降解，进而调节细胞内多种生命活动。同时，ＵＳＰ３３在不同肿瘤中的特异性可为肿瘤的预防、治疗及预后提供新方向。该文现就ＵＳＰ３３在信号通路、自噬、中心体扩增、肿瘤发生、发展中的研究进展进行回顾和综述。关键词：肿瘤；ＵＳＰ３３；去泛素化酶；文献综述中图分类号：Ｒ７３０２文献标志码：Ａ文章编号：１００１－７３９９（２０１９）１０－１２０７－０３ｄｏｉ：１０．１３３１５／ｊ．ｃｎｋｉ．ｃｊｃｅｐ．２０１９．１０．０１６

AD治疗

香港研究团队发现减缓阿尔兹海默症病变的蛋白IL-33作者：佚名来源：生物谷日期：2016-06-14一个由香港科学与工艺大学（HKUST）领军的研究团队发现一个在人体内的蛋白能被开发成有效治疗阿尔兹海默症（AD）。

关键字：|阿尔兹海默症|病变的蛋白IL-33一个由香港科学与工艺大学(HKUST)领军的研究团队发现一个在人体内的蛋白能被开发成有效治疗阿尔兹海默症(AD)。

AD是一个渐进式和使人弱化的脑部疾病，其在脑部以β淀粉样蛋白斑块(Aβ)和神经纤维缠结形式出现。

在全球，AD影响46.8 百万人，主要是年龄超过65岁者，而病例预测在2050年将达到131.5 百万。

病患有认知缺陷比如受损的记忆，推理，判断和运动能力。

此病目前不可逆转和不能治愈。

该团队，由Nancy Ip 教授，HKUST的科学院长领导，发现白介素-33(IL-33)能减缓认知退化和减少脑部Aβ斑块。

成果刊载在《美国国家科学院期刊》。

白介素-33是一个由人体制造来调节免疫功能的蛋白。

研究团队强调IL-33，由于其在有轻微认知损害和有高风险患上AD的人中有受损的功能。

他们发现在改变基因以显示AD症状的老鼠中注射IL-33，能取得认知功能快速复原的结果。

在一周内，研究员观察到神经通讯缺陷和记忆衰退在老鼠中逆转。

显着的，该团队发现连续两天注射IL-33足以减少Aβ蛋白，依次减少淀粉样蛋白在这些老鼠脑中的存量。

去除脑中Aβ蛋白的缺陷是AD形成的主因。

研究员也显示IL-33的存在使脑部免疫细胞(小神经胶质细胞)移动至淀粉样斑块，促使Aβ蛋白的去除。

IL-33也引发小神经胶质细胞的改变，减低整体炎症，后者促使疾病在脑部的病变。

“这个令人振奋的成果让我们在理解这个复杂，多因素疾病的病理过程中更进一步，在开发AD治疗方面提供了新路，”Ip说。

血管因素亦可影响阿尔茨海默病进程？作者：伊文来源：医脉通日期：2015-10-21通常认为，痴呆分为神经变性性痴呆及血管性痴呆。

藏红花素介导DKK3调控GSK-3β

◇基础研究◇摘要目的：探讨藏红花素（crocin ）对阿尔兹海默症（Alzheimer's disease ，AD ）小鼠认知能力的改善作用及机制。

方法：SD 大鼠海马区注射A β25-35建立AD 模型，随机分为AD 组、AD+L 、M 、H-crocin 组（10、20、40mg/kg ）和AD+donepezil 组（1mg/kg 盐酸多奈哌齐），腹腔注射治疗4周，另设置Sham 组。

采用避暗实验、水迷宫实验评估大鼠学习、记忆能力，ELISA 测定大鼠血清A β含量，HE 染色和Tunel 染色确定大鼠海马区内病理改变及神经元细胞凋亡，免疫组化测定大鼠海马区Brdu 、Dcx 、NeuN 表达，Western blot 测定大鼠脑组织A β、DKK3、β-catenin 、p-GSK-3β/GSK-3β、Caspase-3、Bax 、Bcl-2蛋白表达。

结果：与Sham 组相比，AD 组大鼠的学习、记忆能力下降，血清A β含量升高，且海马区的病理改变严重，神经元细胞凋亡增加，Brdu 、Dcx 、NeuN 含量降低，A β、DKK3、p-GSK-3β/GSK-3β、Caspase-3、Bax 蛋白表达升高，β-catenin 、Bcl-2蛋白表达降低（P <0.01）。

与AD 组相比，给予不同剂量crocin 和donepezil 治疗后，AD 大鼠学习、记忆能力提高，血清A β含量降低，海马区的病理改变减轻，神经元细胞凋亡减少，Brdu 、Dcx 、NeuN 含量升高，A β、DKK3、p-GSK-3β/GSK -3β、Caspase-3、Bax 蛋白表达升高，β-catenin 、Bcl-2蛋白表达降低（P <0.05），crocin 的剂量依赖效应显著。

结论：crocin 通过减少神经元细胞凋亡，介导DKK3调控GSK-3β/β-catenin 通路来改善AD 大鼠认知损伤。

多聚泛素化链类型

多聚泛素化链类型
多聚泛素化链是泛素分子通过共价键连接而形成的链状结构，其中每个泛素分子通过赖氨酸残基与下一个泛素分子连接。

目前已知的多聚泛素化链类型主要包括以下几种：
1. K63多聚泛素化链：由63个泛素分子组成，每个泛素分子通过赖氨酸残基与下一个泛素分子连接，形成一条线性链。

2. K48多聚泛素化链：由48个泛素分子组成，每个泛素分子通过赖氨酸残基与下一个泛素分子连接，形成一条线性链。

3. K27/K33多聚泛素化链：由27个泛素分子组成，其中25个泛素分子通过赖氨酸残基与下一个泛素分子连接，另外2个泛素分子通过甲硫氨酸残基与下一个泛素分子连接，形成一条分支链。

4. K29/K33多聚泛素化链：由29个泛素分子组成，其中27个泛素分子通过赖氨酸残基与下一个泛素分子连接，另外2个泛素分子通过甲硫氨酸残基与下一个泛素分子连接，形成一条分支链。

5. K11/K33多聚泛素化链：由11个泛素分子组成，其中10个泛素分子通过赖氨酸残基与下一个泛素分子连接，另外1个泛素分子通过甲硫氨酸残基与下一个泛素分子连接，形
成一条分支链。

6. K15/K33多聚泛素化链：由15个泛素分子组成，其中14个泛素分子通过赖氨酸残基与下一个泛素分子连接，另外1个泛素分子通过甲硫氨酸残基与下一个泛素分子连接，形成一条分支链。

这些多聚泛素化链类型在细胞内具有不同的生物学功能，例如调节蛋白质的降解、转录调控等。

植物耐铝的生物化学与分子机理

摘要：某些耐铝植物在铝胁迫下分泌有机酸被认为是一个重要的抗性机制。从根系分泌出来的有机酸能与根际的 >-$ ? 结合，形成无毒性的螯合物，从而减轻了铝对根系的毒害。但是，铝诱导有机酸分泌的中间环节及调节机制至今仍不清楚。一些证据表明，铝能激活根尖细胞质膜内的阴离子通道，因而可以调节有机酸的分泌。近年来，人们开始注意一些信号分子如蛋白激酶、水杨酸等介导铝诱导有机酸的分泌，已经获得一些成果。同时，铝胁迫基因的分离和鉴定也为人们从分子水平上研究和认识铝胁迫下植物的抗性机制奠定了基础。关键词：铝；有机酸；蛋白激酶；水杨酸；铝诱导基因中图分类号：@=&<
G<0
植物生理与分子生物学学报\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ 0* 卷
时，我们也测定了柠檬酸的含量，结果显示铝处理的根尖柠檬酸的含量高出对照 ! 倍左右。铝刺激有机酸相关合成酶活性提高的这种机制得到转基因实验的支持。有人将大肠杆菌柠檬酸合酶基因转入到烟草幼苗，结果显示转基因植株具有较高的柠檬酸合酶活性，柠檬酸分泌量增加，植株表现出较强的抗性（ "# $% &’#()# 等 !**+ ）。之后， ,#-.%/# 等（ 011! ）用豆科植物根瘤促进型（ (2"’$#3#(4%(5#" .267-）的 89: 基因导入苜蓿后，发现转基因植株 89: 活性提高 !; < 倍，根尖内苹果酸含量提高了 =; 0 倍，有机酸的分泌提高了 +; ! 倍。但也有一些结果不支持铝诱导相关酶活性提高的有机酸分泌机制。例如，在以铝诱导苹果酸分泌为主的抗性小麦上，观察不到 89: 活性的提高，表明铝诱导苹果酸的分泌并不一定与根系内相应酶活性的提高有直接关系（ >/%( 等 !**? ）。

白介素-33在阿尔茨海默病中的研究进展

白介素-33在阿尔茨海默病中的研究进展
戴莉莉;周涛;哈斯也提·依不来音
【期刊名称】《中国实用神经疾病杂志》
【年(卷),期】2024(27)2
【摘要】随着老龄化社会的到来,阿尔茨海默病(AD)的发病率越来越高,给社会带来了巨大的负担。

该疾病目前影响着全球5000多万人,预计到2050年将增加到1.52亿人。

尽管其流行率高,但该病的致病机制尚未完全阐明,不能有效地预防和治疗,因此有必要寻找针对阿尔茨海默的新治疗靶点。

白介素-33(IL-33)是一种重要的神经炎症因子,在哺乳动物大脑的各种组织和细胞中高度表达。

在阿尔茨海默病中,IL-33通过促进淀粉样斑块清除,减少tau蛋白的过度磷酸化,增强小胶质细胞的吞噬活性,并影响突触可塑性来发挥保护作用,本文就IL-33在AD中的作用做一综述,为AD的治疗提供一些新思路。

【总页数】6页(P259-264)
【作者】戴莉莉;周涛;哈斯也提·依不来音
【作者单位】新疆医科大学第二附属医院
【正文语种】中文
【中图分类】R749.16
【相关文献】
1.白介素33在类风湿关节炎中的研究进展
2.白介素-33在心血管系统中的研究进展
3.白介素-17、白介素-33与支气管哮喘关系的研究进展
4.白介素-33在妇科肿瘤中的研究进展
5.白介素-33在急性呼吸窘迫综合征中的研究进展
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细胞真题名词解释

2017一、名词解释1.胞质溶胶Lipid raft 自噬溶酶体亚线粒体小泡染色体骨架联会复合体原初反应Cotransport 信号斑多能干细胞与单能干细胞2016一、名词解释胚胎诱导端粒酶Tight junction 核纤层蛋白Cyclin 抑癌基因信号识别颗粒氧化磷酸化核纤层Cell communication2015一、名词解释1.细胞学说2.核孔复合体3.多线染色体4.化学渗透学说：解释氧化磷酸化过程中电子传递与磷酸化之间偶联机制的一种学说。

其主要要点为电子传递链不对称分布，起着质子泵的作用，在电子传递莱奶过程中所释放的能量转化成跨膜的PH梯度和电位梯度，由于内膜具有完整性，因此在将质子从内室泵至外室时，质子只能从ATP合成酶返回基质，该酶便用其能量合成ATP。

5.端粒6.信号转导7.限制点：是细胞周期监控点之一。

8.肿瘤抑制基因I（抑癌基因）9.细胞周期10微管组织中心2014一、名词解释1.肌质网2.异噬溶酶体中心体分子伴侣重组小节成帽反应极细胞核定位信号细胞外被肌球蛋白2013一、名词解释胚胎干细胞胚胎诱导细胞拆合联会复合体生殖质程序性细胞死亡嵌合体多线染色体收缩环随体2012一、名词解释1.细胞学说2细胞识别3.细胞拆合4.原生质：原生质是细胞内生命物质的总称。

它的主要成分是糖类、蛋白质、核酸、脂质等。

原生质分化产生细胞膜、细胞质和细胞核，构建成具有特定结构体系的原生质体，即细胞。

一个动物细胞就是一个原生质体。

植物细胞由原生质体和细胞壁组成。

5.重组小节6.细胞外被7.核小体8.多核糖体：在蛋白质合成过程中,同一条mRNA分子能够同多个核糖体结合,同时合成若干条蛋白质多肽链,结合在同一条mRNA上的核糖体就称为多聚核糖体(polysome 或polyribosomes).在电镜下观察呈现各种各样的结构。

蛋白质合成时多聚核糖体的形成对生命活动的意义在于:节省了遗传信息量,减轻了核的负担.原9.癌基因点突变：这是原癌基因激活的途径之一，有的癌细胞基因激活是由于原癌基因本身一定部位的核苷酸序列发生了变化，合成了异常的蛋白质产物，从而使细胞出现转化表型，所谓的点突变就是基因中只有一对碱基发生了突变。

k29连接的泛素化作用

k29连接的泛素化作用
K29连接的泛素化作用目前还不明晰，泛素化功能在不同位点泛素化的蛋白有不同的功能，其中通过K48连接的泛素链是最典型的聚泛素链，占了所有链型的50%以上，是底物被26S蛋白酶体降解的信号；K63泛素链则介导信号传导；M1泛素链是NF-κB信号的重要正调节因子；K6泛素链与调节UV引起的DNA损伤应答及线粒体的体内稳态有关；K11泛素链是细胞周期调控中另一种蛋白酶体降解信号；K27泛素链与线粒体损伤等相关；K29泛素链与蛋白酶体降解和表观遗传调控有关；K33泛素链作用于高尔基体膜上的蛋白交换。

载DOX纳米粒子在SKOV3细胞内累积的研究

载DOX纳米粒子在SKOV3细胞内累积的研究DOX纳米粒子是一种将阿霉素（Doxorubicin, DOX）包裹在纳米粒子中的药物载体。

阿霉素是一种常用的抗癌药物，但其应用受到很大限制，因为其会对健康细胞造成严重的毒副作用。

纳米粒子作为药物的载体具有很多优势，例如可以提高药物的稳定性，改善药物的溶解度，降低药物在体内的代谢和排泄速度，同时还可以通过改变纳米粒子的性质来实现药物的靶向输送。

在这项研究中，研究人员将DOX纳米粒子加入到SKOV3细胞培养基中。

SKOV3细胞是一种常用的卵巢癌细胞系，对药物敏感。

研究人员首先通过扫描电子显微镜（SEM）观察纳米粒子的形貌和粒径分布。

结果显示，DOX纳米粒子呈现规则的球形结构，粒径分布较为均匀，平均粒径约为100 nm。

接下来，研究人员通过MTT实验评估DOX纳米粒子对SKOV3细胞的毒性。

MTT实验是通过测量细胞色素c还原酶的活性来评估细胞的活力。

结果显示，DOX纳米粒子显示出显著的抗癌活性，并且其对SKOV3细胞的毒性明显高于自由DOX药物。

研究人员进一步采用荧光显微镜观察DOX纳米粒子在SKOV3细胞内的累积情况。

荧光显微镜通过DOX纳米粒子的自身荧光特性来观察其在细胞内的分布情况。

结果显示，DOX纳米粒子能够迅速穿过细胞膜，进入细胞核内，并在细胞核中积累。

与自由DOX药物相比，DOX纳米粒子在细胞内的累积量更多，说明其在SKOV3细胞内的靶向输送效果更好。

研究人员通过Western blot实验检测DOX纳米粒子对SKOV3细胞的作用机制。

通过检测细胞凋亡相关蛋白Bcl-2和Bax的表达水平，结果显示DOX纳米粒子可以诱导SKOV3细胞凋亡，同时还能够抑制细胞增殖和促进细胞凋亡的相关蛋白如Cyclin D1和Caspase-3的表达。

这项研究证明了DOX纳米粒子具有较好的抗癌活性，并且可以在SKOV3细胞内实现靶向输送和积累，从而提高药物的疗效。

这为纳米药物的设计和应用提供了有力的参考。

k63位泛素化作用_概述及解释说明

k63位泛素化作用概述及解释说明1. 引言1.1 概述在细胞内，泛素化作为一种重要的蛋白质修饰机制，参与了许多生物学过程的调控。

其中，k63位泛素化作用是一种特殊的泛素化方式，以k63位点上的链型连接方式来修饰目标蛋白。

近年来，对于k63位泛素化作用的研究不断深入，揭示了它在细胞信号转导、DNA损伤修复、免疫应答等方面具有重要作用。

1.2 文章结构本文将从以下几个方面对k63位泛素化作用进行概述和解释说明。

首先，在第2部分将详细介绍k63位泛素化的定义和其特征。

接着，在第3部分将阐述k63位泛素化作用的具体作用机制，并详细讨论其在生物学功能中的重要性。

最后，在第4部分将总结主要观点，并展望未来研究方向。

1.3 目的本文旨在系统地介绍和解释k63位泛素化作用，以增加读者对该功能的理解。

通过对近年来相关研究进展、相关疾病和治疗潜力、技术方法和应用前景的探讨，希望能够为进一步研究和应用k63位泛素化作用提供有益的指导和启示。

2. k63位泛素化作用2.1 定义和特征：k63位泛素化（K63-linked ubiquitination）是一种特殊类型的泛素化修饰过程，它通过将一条或多条含有29号赖氨酸（Lysine 63，K63）残基的泛素蛋白共价连接到靶蛋白上来进行调控。

与其他类型的泛素化修饰相比，k63位泛素化具有独特的生物学特征和功能。

2.2 作用机制：k63位泛素化主要通过E1激活酶、E2结合酶和E3连接酶等多个酶参与的级联反应来进行。

首先，E1激活酶将游离态的泛素蛋白与ATP结合并激活。

然后，激活后的泛素通过E2结合酶转移给E3连接酶。

最后，在E3连接酶的催化下，k63位泛素被共价连接到靶蛋白上的K63位点上。

2.3 生物学功能：k63位泛素化在细胞内发挥着多种重要的生物学功能。

首先，它参与了细胞质相互作用、信号通路传导和蛋白质降解等多种细胞过程。

其次，k63位泛素化在DNA损伤修复、免疫应答和炎症调节等生物学过程中发挥关键作用。

泛素化 sh3结构域

泛素化 sh3结构域
泛素化是一种生物学过程，它涉及到泛素蛋白与其他蛋白质结
合的化学修饰。

泛素是一种小的蛋白质，它可以与其他蛋白质发生
共价结合，这个过程被称为泛素化。

泛素化通常发生在蛋白质的赖
氨酸残基上，它可以影响蛋白质的稳定性、活性、亚细胞定位以及
与其他蛋白质的相互作用。

SH3结构域是一种常见的蛋白质结构域，它通常包含50-60个
氨基酸残基，具有β-折叠结构。

SH3结构域在细胞信号传导、细胞
骨架的重组以及蛋白质的互相作用中发挥着重要作用。

SH3结构域
通常与多肽序列特定的蛋白质结合，从而调控细胞内的信号传导通路。

关于泛素化和SH3结构域的关系，研究表明泛素化可以影响与
SH3结构域相关的蛋白质相互作用。

泛素化修饰可以改变蛋白质的
构象和亲和性，从而影响SH3结构域与其结合的蛋白质的相互作用。

这种影响可能对细胞信号传导通路和细胞功能产生重要影响。

总的来说，泛素化和SH3结构域在细胞生物学中都扮演着重要
的角色。

它们之间的相互作用和影响关系对于我们理解细胞内信号
传导和蛋白质相互作用的机制具有重要意义。

希望这个回答能够满足你的要求。

白杨素通过PI3K／AKT信号通路抑制LPS诱导的软骨细胞自噬

网络出版时间：２０２１－４－２２１７：０７：００　网络出版地址：ｈｔｔｐｓ：／／ｋｎｓ．ｃｎｋｉ．ｎｅｔ／ｋｃｍｓ／ｄｅｔａｉｌ／３４．１０８６．Ｒ．２０２１０４２２．１４１２．０２６．ｈｔｍｌ白杨素通过ＰＩ３Ｋ／ＡＫＴ信号通路抑制ＬＰＳ诱导的软骨细胞自噬杨　阳１，何　宇２，史于传２，舒见威３，虞　乐３，胡　伟１（安徽医科大学第二附属医院１．药物临床试验研究中心、２．妇产科，３．麻醉与围术期医学安徽省普通高校重点实验室，安徽合肥　２３０６０１）ｄｏｉ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－１９７８．２０２１．０５．０１３文献标志码：Ａ文章编号：１００１－１９７８（２０２１）０５－０６６２－０７中国图书分类号：Ｒ３３２；Ｒ２８５５；Ｒ３２９２４；Ｒ３９２；Ｒ６８４３；Ｒ９７７６摘要：目的　探索白杨素对脂多糖诱导的大鼠软骨细胞骨关节炎模型中对软骨细胞自噬的作用及其机制。

方法　提取１０只ＳＰＦ级ＳＤ大鼠关节软骨正常细胞进行体外分离培养，通过ＬＰＳ诱导大鼠软骨细胞自噬。

实验分为空白对照组、ＬＰＳ组、白杨素（ＣＨＲ）组和ＬＰＳ＋ＣＨＲ组；ＣＣＫ８法检测各分组细胞的活性、Ｒｈｏｄａｍｉｎｅ１２３检测各组细胞中线粒体膜电位、ＤＣＦＨＤＡ检测各组细胞的活性氧，Ｗｅｓｔｅｒｎｂｌｏｔ法检测各组细胞中ＰＩ３Ｋ、ＡＫＴ、ｐＰＩ３Ｋ、ｐＡＫＴ、Ｂｅｃｌｉｎ１及ＬＣ３Ⅱ的蛋白表达。

结果　白杨素对ＬＰＳ诱导的软骨细胞自噬具有抑制作用，且当白杨素浓度为１０ｍｍｏｌ·Ｌ－１时抑制自噬作用最为显著；白杨素能够抑制ＬＰＳ所致ＬＰＳ诱导的软骨细胞的活性氧（ＲＯＳ）的增加；白杨素抑制了细胞受损后线粒体膜电位的降低；同时白杨素抑制Ｂｅｃｌｉｎ１、ＬＣ３Ⅱ蛋白的表达，抑制ＰＩ３Ｋ和ＡＫＴ的磷酸化。

结论　白杨素可能通过抑制ＰＩ３Ｋ／ＡＫＴ信号通路，下调自噬蛋白Ｂｅｃｌｉｎ１和ＬＣ３Ⅱ的表达抑制自噬，进而保护软骨细胞。

链霉菌挥发性化合物提高植物抗病能力的研究进展

不合理使用化学农药会破坏生态环境，威胁人类健康。

植物根际促生菌（plant growth promoting rhi-zobacteria ，PGPR ）为一种生防剂，具有安全、环保、高效的特点，逐渐成为农业绿色高质量发展关注的热点。

链霉菌是PGPR 的一类，广泛分布于土壤、水体等生态系统中，大多具有抗菌活性和能产生天然次级代谢产物的能力。

链霉菌不但可以通过溶磷、固氮、产铁和调节植物激素等方式[1，2]促进植物生长，而且其产生的挥发性化合物（volatile organic compounds ，VOCs ）也具有较高的抗菌活性，对细菌和真菌引起的各种植物病害具有防治作用。

对链霉菌VOCs 的分类、VOCs 常用分离鉴定技术、VOCs 对常见植物病原菌的抑制作用以及作用机制研究进展等进行总结，旨为链霉菌VOCs 的深入研究和产品定向开发提供参考。

1微生物的VOCsVOCs 是一类小分子、易挥发的化合物，主要包括烯烃、烷烃、醇、酮、萜类、苯类、醛、吡嗪、酸、酯和含硫化合物[3]。

VOCs 检测在生物学、环境科学、医学、食品工业等领域应用广泛。

微生物产生的VOCs 具有调节植物生长、刺激植物产生激素、诱导植物系统免疫抗性等功能。

VOCs 通过与细胞之间的相互作用[4]，参与调节植物生长发育、抑制病原菌侵染的过程。

合理利用微生物产生的VOCs ，在未来具有很大的应用潜力。

不同细菌的代谢途径不完全一摘要：研究微生物代谢相关新技术的迅猛发展，使研究者们从微生物次生代谢产物中发现了更多新的挥发性物质，这些挥发性物质在植物生长发育过程中起到多种重要作用。

在众多微生物中，链霉菌产生的次生代谢产物种类最多、功能最复杂。

以链霉菌为例，综述了其产生的挥发性化合物（VOCs ）的类型、VOCs 常用分离鉴定方法、VOCs 对几种常见植物病原菌的抑制作用以及作用机制，旨为链霉菌VOCs 的深入研究和产品开发提供参考。

关键词：链霉菌；挥发性化合物；植物病害；ISR 中图分类号：Q939文献标识码：A 文章编号：1008-1631（2022）03-0053-06收稿日期：2022-03-04基金项目：国家重点研发计划项目（2021YFD1901004-4）；河北省农林科学院创新工程专项（2022KJCXZX-ZHS-3）；河北省重点研发计划项目（19222902D ）作者简介：郭纹余（1996-），女，壮族，广西桂林人，硕士研究生在读，研究方向为资源利用与植物保护。

PI3K抑制剂在乳腺癌治疗中的应用现状

及评估乳腺癌患者预后和诊断价值的重要研究对
象［４］。本文将总结ＰＡＭ信号通路作用机制以及
ＰＩ３Ｋ抑制剂在不同亚型乳腺癌治疗中的应用现状，
以期为不同亚型乳腺癌提供靶向治疗方案。
· ２５２·
中国肿瘤外科杂志２０２１年６月第１３卷第３期ＣｈｉｎＪＳｕｒｇＯｎｃｏｌ，Ｊｕｎｅ２０２１，Ｖｏｌ．１３，Ｎｏ．３
ＥＲ＋ＭＣＦ⁃７细胞系）的生长来发挥协同作用［１３］。
ＢＥＺ⁃２３５与自噬抑制剂的结合，这些组合导致乳腺癌
细胞增殖抑制和凋亡增加［１４］。
族酶，ⅠＡ类ＰＩ３Ｋ是一组由催化亚基ｐ１１０（ｐ１１０α、
３ＰＩ３Ｋ抑制剂与乳腺癌
ｐ８５β、ｐ８５γ、ｐ５０α 和ｐ５５α）组成的异二聚体，其中催
２ＰＩ３Ｋ抑制剂
鉴于ＰＩ３Ｋ通路的过度激活是乳腺癌最常见的信
雌激素受体（ｅｓｔｒｏｇｅｎｒｅｃｅｐｔｏｒ，ＥＲ）信号传导是乳
乳腺癌病例为激素受体阳性（ｈｏｒｍｏｎｅｒｅｃｅｐｔｏｒ⁃
ｐｏｓｉｔｉｖｅ，ＨＲ）［即雌激素受体和／或孕酮受体（ｐｒｏｇｅｓ⁃
ｔｅｒｏｎｅｒｅｃｅｐｔｏｒ，ＰＲ）］。靶向雌激素作用的内分泌疗
法（ｅｎｄｏｃｒｉｎｅｔｈｅｒａｐｉｅｓ，ＥＴｓ）大大降低了乳腺癌的死
亡率。然而，它们的功效受到天然和获得性内分泌抗
性的限制［１５］。例如，使用他莫昔芬的患者中有１／３
最终将在１５年内因内分泌耐药性晚期肿瘤复发；在
ｃｏｍｍｏｎｉｎｂｒｅａｓｔｃａｎｃｅｒ，ｗｈｉｃｈｍａｋｅｓｔｈｉｓｐａｔｈｗａｙａｎｅｗｔｈｅｒａｐｅｕｔｉｃｔａｒｇｅｔｆｏｒｂｒｅａｓｔｃａｎｃｅｒ．Ａｖａｒｉｅｔｙｏｆ

维生素K3电化学反应机理的紫外光谱电化学研究

维生素K3电化学反应机理的紫外光谱电化学研究
朱世民;马永钧
【期刊名称】《分析化学》
【年(卷),期】1998(026)002
【摘要】用薄层池循环伏安法和现场薄层池紫外光谱电化学法研究了维生素Ｋ３（ＶＫ３）在铂电极上的电化学反应机理。

薄层池循环伏安实验结果表明：ＶＫ３的电化学反应为二步１ｅ准可逆过程，现场薄层池紫外光谱电化学的实验结果和Ｎｅｒｎｓｔ图解分析表明：电解还原反应的最后产物为２－甲基－１，４－萘酚。

该反应偶合有前行化学反应；还原产物经电解氧化的产物为２－甲基－１，４－萘酚。

该反应偶合有前行化学反应；还原产物经电解氧化的
【总页数】4页(P184-187)
【作者】朱世民;马永钧
【作者单位】南京大学化学系;南京大学化学系
【正文语种】中文
【中图分类】Q568.02
【相关文献】
1.聚电解质中电活性物质氧化还原反应机理的现场显微红外光谱电化学研究 [J], 郭黎平;林祥钦
2.间苯二胺的电化学及紫外-可见薄层光谱电化学研究 [J], 焦奎;吕刚;杨涛;吴俊峰
3.肾上腺素电化学氧化生成肾上腺素红反应机理的薄层光谱电化学研究 [J], 刘学军;黎桂辉;郁章玉;翟翠萍
4.辅酶Ⅰ电化学配位反应机理的紫外光谱电化学研究 [J], 马永钧;李根喜
5.维生素K_3电化学反应机理的红外光谱电化学研究 [J], 郑建明;马永钧;朱世民因版权原因，仅展示原文概要，查看原文内容请购买。

Keap1

非小细胞肺癌(non-small cell lung cancer，NSCLC)发病率占据肺癌的75%~80%。

肿瘤细胞进展快且易扩散转移，临床常采用手术、放化疗等进行治疗，但5年生存率低于60%[1-2]。

氧化应激是由活性氧(ROS)生成量增加所致，ROS积累可诱导肺癌细胞凋亡，清除ROS 可阻止癌细胞凋亡，即肺癌细胞存活依赖于癌细胞自身抗氧化能力[3]。

Kelch样环氧氯丙烷相关蛋白-1 (kelch-like epichlorohydrin-associated protein-1，Keap1)/核因子E2相关因子2(nuclear factor E2related factor 2，Nrf2)信号通路在癌症中发挥重要调控作用，氧化应激可激活Keap1，促使Keap1-Nrf2复合物裂解，Nrf2转移至细胞核内，可激活下游靶基因表达，参与肺癌发生发展过程[4]。

Nrf2可维持氧化还原稳态，ROS侵袭细胞时，Nrf2可进入细胞核，结合抗氧化反应元件(ARE)转录编码各种抗氧化蛋白、代谢酶基因，抑制氧化应激反应[5-6]。

目前氧化应激、Keap1/Nrf2信号通路在NSCLC发生过程中的机制尚未明确。

基于此，本研究尝试分析Keap1/Nrf2信号通路与临床病理参数、氧化应激指标的相关性，探讨其在NSCLC氧化应激机制中的作用，为临床研制新药提供参考依据。

1资料与方法1.1一般资料选取2017年4月至2020年4月郑州市第三人民医院收治的100例NSCLC患者为研究对象。

纳入标准：符合NSCLC诊断标准[7]；术前未接受放化疗、免疫治疗者；预计生存期≥6个月；符合手术适应证、禁忌证；Karnofsky功能状态评分≥70分；签署知情同意书。

排除标准：合并凝血功能障碍、肝肾功能障碍、其他恶性肿瘤者；伴有急/慢性感染者；伴有精神疾病者；既往腹部相关外科手术史者。

所有患者均行肺癌根治性切除术，术中收集癌组织、癌旁组织(距离癌组织5cm范围内正常组织)，其中男性63例，女性37例；年龄46~67岁，平均(56.32±3.16)岁；体质量指数(BMI)17~30kg/m2，平均(23.16±2.03)kg/m2；病理类型：鳞癌58例、腺癌42例；病理分级[8]：Ⅰ~Ⅱ级51例、Ⅲ级49例；T分期[9]：T1~T253例、T3~T447例；N分期：N055例、N1~N245例。

keap1 蛋白质域

keap1 蛋白质域Keap1蛋白质是一种重要的细胞调控蛋白质，参与多种细胞信号转导和疾病发生发展的过程。

本文将从Keap1蛋白质的结构与功能、调控机制以及其在疾病中的作用等方面进行介绍。

一、Keap1蛋白质的结构与功能Keap1（Kelch-like ECH-associated protein 1）蛋白质是一种富含Cys和Lys残基的蛋白质，分子量大约为69kDa。

其主要存在于细胞质中，并通过N末端的BTB/POZ结构域与Cul3蛋白相互作用，形成Cullin3-Rbx1-E3泛素连接酶复合物，参与蛋白质的泛素化降解。

Keap1蛋白质的主要功能是通过调控抗氧化应激反应，维持细胞内氧化还原平衡。

Keap1蛋白质与转录因子Nrf2（核因子E2相关因子2）结合，将其定位在细胞质中，并通过泛素连接酶的作用将其泛素化降解。

当细胞受到氧化应激或电子供体的刺激时，Keap1蛋白质的结构发生改变，导致Nrf2脱离Keap1蛋白质的限制，进入细胞核并结合到抗氧化应激反应元件ARE（Antioxidant Response Element）上，激活一系列抗氧化应激反应基因的转录，从而提高细胞的抗氧化能力。

二、Keap1蛋白质的调控机制Keap1蛋白质的功能调控主要涉及氧化应激和电子供体等多种信号通路。

在正常情况下，Keap1蛋白质通过其N末端的Kelch结构域与Nrf2结合，并将其定位在细胞质中，使其泛素化降解。

然而，当细胞受到氧化应激或电子供体的刺激时，Keap1蛋白质的氧化修饰发生改变，导致其结构发生变化，无法有效与Nrf2结合，从而使Nrf2得以稳定并进入细胞核发挥其转录调控功能。

Keap1蛋白质的泛素连接酶活性也受到其他蛋白质的调控。

研究发现，一些蛋白质如p62、WDR23等可以与Keap1蛋白质相互作用，干扰其与Nrf2的结合，从而抑制Keap1蛋白质的泛素连接酶活性，增强Nrf2的稳定性。

三、Keap1蛋白质在疾病中的作用Keap1蛋白质在多种疾病的发生发展中发挥着重要的作用。

单泛素化k53位点

单泛素化k53位点
（原创版）
目录
1.单泛素化 k53 位点的定义与作用
2.单泛素化 k53 位点的研究进展
3.单泛素化 k53 位点的应用前景
正文
单泛素化 k53 位点是一种蛋白质修饰方式，泛素是一种小型蛋白质，可以与其他蛋白质结合，从而影响其功能和活性。

在泛素的修饰过程中，有一种特定的修饰方式称为单泛素化，即泛素分子只与目标蛋白质的一个赖氨酸残基结合。

在单泛素化的过程中，k53 位点是指泛素分子与目标蛋白质结合的位置。

近年来，单泛素化 k53 位点的研究取得了重要进展。

科学家们已经发现了许多可以发生单泛素化 k53 位点修饰的蛋白质，这些蛋白质涉及到多种生物学过程，如细胞生长、凋亡、免疫反应等。

通过对这些蛋白质的单泛素化 k53 位点修饰进行研究，科学家们揭示了这些蛋白质在生物体内的功能和调控机制。

此外，单泛素化 k53 位点在疾病发生发展中也发挥着重要作用。

例如，在某些类型的癌症中，某些蛋白质的单泛素化 k53 位点修饰异常，导致细胞生长和凋亡失控。

因此，研究单泛素化 k53 位点可以为诊断、治疗相关疾病提供新的思路和靶点。

在未来，单泛素化 k53 位点的研究将继续深入，有望为生物医学领域带来新的突破。

在应用方面，单泛素化 k53 位点的研究成果可能有助于开发新型药物，通过调控蛋白质的单泛素化 k53 位点修饰，实现对疾病的治疗。

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