Immunoproteasomes Preserve
Protein Homeostasis upon
Interferon-Induced Oxidative Stress
Ulrike Seifert,1Lukasz P.Bialy,1,4Fre′de′ric Ebstein,1Dawadschargal Bech-Otschir,1Antje Voigt,2Friederike Schro¨ter,1 Timour Prozorovski,3Nicole Lange,1Janos Steffen,1Melanie Rieger,1Ulrike Kuckelkorn,1Orhan Aktas,3
Peter-M.Kloetzel,1,*and Elke Kru¨ger1,*
1Institut fu¨r Biochemie CC2,Charite′-Universita¨tsmedizin Berlin,Oudenarder Strasse16,D-13347Berlin,Germany
2Medizinische Klinik fu¨r Kardiologie und Angiologie CC13,Charite′-Universita¨tsmedizin Berlin,Charite′platz1,D-10117Berlin,Germany 3Department of Neurology,Medical Faculty,Heinrich-Heine-University Dusseldorf,Moorenstrasse5,D-40225Du¨sseldorf,Germany
4Present address:Department of Histology and Embryology,Center for Biostructure Research,Medical University of Warsaw, Chalubinskiego5,02004Warsaw,Poland
*Correspondence:p-m.kloetzel@charite.de(P.-M.K.),elke.krueger@charite.de(E.K.)
DOI10.1016/j.cell.2010.07.036
SUMMARY
Interferon(IFN)-induced immunoproteasomes(i-pro-teasomes)have been associated with improved pro-cessing of major histocompatibility complex(MHC) class I antigens.Here,we show that i-proteasomes function to protect cell viability under conditions of IFN-induced oxidative stress.IFNs trigger the pro-duction of reactive oxygen species,which induce protein oxidation and the formation of nascent, oxidant-damaged proteins.We?nd that the ubiquity-lation machinery is concomitantly upregulated in response to IFNs,functioning to target defective ribosomal products(DRiPs)for degradation by i-pro-teasomes.i-proteasome-de?ciency in cells and in murine in?ammation models results in the formation of aggresome-like induced structures and increased sensitivity to apoptosis.Ef?cient clearance of these aggregates by the enhanced proteolytic activity of the i-proteasome is important for the preservation of cell viability upon IFN-induced oxidative stress. Our?ndings suggest that rather than having a speci?c role in the production of class I antigens, i-proteasomes increase the peptide supply for antigen presentation as part of a more general role in the maintenance of protein homeostasis. INTRODUCTION
The ubiquitin proteasome system(UPS)represents the major ATP-dependent degradation system in eukaryotes that is involved in the maintenance of cellular protein homeostasis. The central task of the26S proteasome is the degradation of poly-ubiquitylated proteins formed by a cascade of E1,E2,and E3 enzymes,which activate,conjugate,and transfer multiple ubiquitin(ub)moieties to protein substrates,targeting these for degradation.Substrates of proteasomes are short-lived regula-tory proteins involved in cell differentiation,cell-cycle regulation, transcriptional regulation,or apoptosis(Kloetzel,2001;Liu et al., 2005).The rapid elimination of proteins by the UPS is also of particular importance under stress conditions,such as heat shock or oxidative stress,that cause the production of misfolded or partially denatured proteins(Goldberg,2003).
The20S proteasome,with its active site b subunits b1,b2, and b5,represents the central catalytic unit of the UPS and the catalytic core of the proteasome.The20S catalytic core associ-ates with19S regulator complexes to form the26S proteasome. In addition,the proteasome activator28(PA28)can interact with the20S complex,forming20S-PA28or hybrid proteasomes (19S-20S-PA28)(Tanahashi et al.,2000).In vertebrates,speci?c catalytically active b subunits,i.e.,b1i/LMP2,b2i/MECL1,and b5i/LMP7,are incorporated into nascent proteasome complexes upon interferon(IFN)-mediated induction to form an alternative proteasome composition(Kloetzel,2001).Two of these subunits (b1i/LMP2,b5i/LMP7)are encoded within the major histocom-patibility complex(MHC)class II region,which led to the term immunosubunits(i-subunits)and immunoproteasome(i-protea-some)(Aki et al.,1994).Together,these?ndings connected i-proteasome function with antigen presentation.Proteasomes are responsible for the generation of peptides derived from path-ogens or cellular proteins that are presented by MHC class I molecules on the cell surface to cytotoxic T cells(CTLs) (Yewdell,2005).
i-proteasomes exhibit altered peptidase activities and cleavage site preferences that result in more ef?cient liberation of many MHC class I epitopes(Kloetzel and Ossendorp,2004; Kru¨ger et al.,2003).The increase in peptide supply by i-protea-somes has a positive effect on ef?cient MHC class I antigen presentation and seems to be important for triggering an effec-tive early CTL response(Chen et al.,2001;Deol et al.,2007; Toes et al.,2001;Van Kaer et al.,1994).Newly translated poly-peptides,a large pool of which are ubiquitylated,represent an important source for MHC class I epitopes(Reits et al.,2000;
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Schubert et al.,2000).It thus was proposed that this pool is mostly composed of defective proteins,and hence these proteins were called defective ribosomal products (DRiPs).The DRiP hypothesis,linking antigen processing to translation,would guarantee that the cellular immune system has access to its substrates before they reach their ?nal destination within or outside the cell (Lelouard et al.,2004;Reits et al.,2000;Schu-bert et al.,2000;Yewdell,2005).However,the idea that a large amount of newly translated proteins should be misfolded or harbor translational errors raised some doubts with regard to the validity of the hypothesis (Vabulas and Hartl,2005;Yewdell and Nicchitta,2006).
Cells rapidly shift to i-proteasome formation in response to proin?ammatory cytokines produced by the innate immune system early upon infection.i-proteasome formation is a strongly accelerated and transient response (Heink et al.,2005),implying an urgent and timely need for their function.However,the absence of fully functional i-proteasomes in mice did not gener-ally reveal effects on T cell priming or on the hierarchy of known T cell epitopes (Nussbaum et al.,2005).Moreover,in pathogen-challenged tissues,noninfected cells also induce i-proteasome formation in response to cytokines without the need for improved antigen processing (Yewdell,2005).It was therefore suggested that i-proteasomes must have important non-antigen-process-ing functions (Nussbaum et al.,2005;Yewdell,2005).
i-proteasome function has been mainly associated with antigen processing and MHC class I antigen presentation.Here,we show that a major function of the i-proteasome is the maintenance of protein homeostasis and the preservation of cell viability under cytokine-induced oxidative stress.
RESULTS
IFNs Induce a Transient Accumulation of K48-Linked Ub Conjugates
Because IFNs enhance proteasome-dependent antigen presen-tation ef?ciency (Kloetzel,2001),we reasoned that IFNs may also enhance the pool of poly-ub conjugates.To test this assumption,we stimulated cells with IFN-g (Figure 1A and Figure S1C available online),-a ,or -b (Figures S1A and S1B).Monitoring of the total amount of poly-ub conjugates over time revealed a signi?cant enrichment of poly-ub conjugates between 4and 12hr after IFN stimulation (Figure 1B and Figure S1).This enrich-ment appeared to be a transient response because the levels of detectable poly-ub conjugates initially increased and the amount of poly-ub conjugates returned to reduced levels between 24and 48hr of IFN stimulation.The accumulation of poly-ub conjugates was independent of the cell type and the type of IFN used and was detectable in both mouse and human cells (Figures 1A and 1B and Figure S1).Concomitant with the induc-tion of i-proteasomes,as determined by increased b 5i/LMP7levels,the amount of poly-ub conjugates gradually declined (Figures 1A and 1B and Figure S1).The strong decrease in poly-ub conjugates after 48hr was due to enhanced protea-some-dependent protein turnover because the poly-ub conju-gates were stabilized by the proteasome inhibitor epoxomicin (Figure 1C and Figure S1E).Previously,it was shown that the rate-limiting pool of free ub can be compensated by depletion of ubiquitylated histone H2A (ubH2A)(Dantuma et al.,2006).Indeed,at time points displaying strongly enriched levels of ub conjugates in response to IFN-g ,we observed decreased levels
C 8 24 48 8 24 48 IFN-γ(h)-
--+ + +epoxomicin K48
K630 2 12 0 2 12 0 2 12 0 2 12 IFN-γ(h)free ub
GAPDH K63 to R poly-ub
0 1 2 3 4 6 8 12 24 48 IFN-γ(h)
A
murine B8fibroblasts
D 0 2 4 6 CHX (h)
-+-+-+-+ 12h IFN-γIP poly-ub
GAPDH β5i/LMP7
GAPDH
poly-ub long
1
2
γ(h)E f o l d i n c r e a s e u b -s t a i n
poly-ub short
K48 to R free ub
ubH2A
poly-ub long poly-ub short
Figure 1.Accumulation of K48Poly-Ub Conjugates Is Caused by IFN-g
(A)Murine ?broblasts were stimulated with IFN-g for up to 48hr.Accumulation of poly-ub conju-gates was visualized by immunoblotting (long and short exposure).Transiently decreased levels
of ubH2A and free ub were obtained with the same experimental setup.Increase in LMP7levels served as controls for IFN-induced i-proteasome
formation and GAPDH as a loading control.(B)A signi?cant accumulation of poly-ub conju-gates was visible at 8hr compared to 48hr of IFN stimulation (densitometric evaluation of four
independent series,±standard deviation [SD];p values from t test).(C)Murine ?broblasts were exposed to IFN-g for 8,
24,and 48hr and were either treated with protea-some inhibitor epoxomicin for the last 4hr of the
incubation period or left untreated.Proteasome
inhibition resulted in a signi?cant accumulation of
poly-ub conjugates.
(D)In vitro ub conjugation was demonstrated after IFN-g treatment of murine ?broblasts with modi?ed
forms of His-tagged ub as indicated.Poly-ub conju-gates stimulated by IFN-g are K48linked as shown in
the left panel (using only K48-linked ub chain)and in the right panel (using K63ub modi?ed to arginine at position 63but containing K48-linked ub chain).
(E)Ubiquitylated proteins were immunoprecipitated with FK2ub antibody from pulse-labeled and IFN-g -stimulated murine ?broblasts (lane 0:cycloheximide [CHX]).CHX chases demonstrated the dependency of poly-ub protein levels on translation.See also Figure S1.
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of ubH2A,and slightly decreased free ub levels (Figure 1A and Figure S1C).The functional role of ubiquitylation and thus the fate of the modi?ed proteins largely depend on the type of linkage between the ub moieties within a poly-ub chain (Mukho-padhyay and Riezman,2007).To study which type of poly-ub conjugates was induced in IFN-g -treated cells,we performed in vitro ub conjugation assays using modi?ed forms of epitope-tagged ub.Substitution of the K48residue by arginine (R)almost completely abrogated the formation of poly-ub conjugates,demonstrating that IFN-g preferentially stimulated the synthesis of poly-ub chains with K48linkages (Figure 1D),targeting these ub conjugates for degradation.The inhibitory effect of the K48R ub mutant on IFN-g stimulated ub conjugation was observed despite the presence of other functional K residues.Thus,other K residues within the ub molecule do not signi?cantly contribute to IFN-g -induced polyubiquitylation.A slight stimulation of K63poly-ub chains by IFN-g is negligible,because the IFN-induced ub conjugation can be completely restored using the K63R ub mutant containing a functional K48residue (Figure 1D).
As expected,IFN-g treatment induced translation via the mTOR pathway (Kaur et al.,2008).After 2hr of IFN stimulation,we detected increased levels of the phosphorylated ribosomal S6subunit,a marker for increased translational activity (Fig-ure S1F).To determine whether the accumulated poly-ub conjugates derive from newly synthesized proteins,we pulse-labeled IFN-g -treated cells and immunoprecipitated ub-modi-?ed proteins.IFN-g induced a strong increase in the amount of poly-ub conjugates (Figure 1E,‘‘0CHX’’)suggesting that primarily newly translated proteins shape the pool of poly-ub substrates in response to IFN-g .In support,cycloheximide
0 1 3 48 IFN-γ(h)
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400006000080000100000120000140000048141825324248IFN-γ(h)U B E 2L 6 (c o p i e s /μl )
free Ub 0
20406080γ
%u b -s t a i n
GAPDH
poly-ub
GAPDH
UBE2L6RT-PCR C
--+ + 8h IFN-γco si co si
UBE2L6170100
230Figure 2.Accumulation of Poly-Ub Conju-gates Is a Result of Increased Ub Conjuga-tion Activity in IFN-Stimulated Cells
(A)In vitro polyubiquitylation activity was deter-mined in cellular lysates after treatment with IFN-g for up to 48hr with His-tagged ub.Increased polyubiquitylation was detected at early time points after IFN-g induction (1hr)and stable for up to 48hr.
(B)Real-time RT-PCR analysis and immunoblot illustrating UBE2L6expression in HeLa cells induced by IFN-g (two independent series ±SD).(C)siRNA knockdown of E2enzyme UBE2L6was performed in HeLa cells treated for 8hr with IFN-g or left untreated.Silencing of UBE2L6(UBE2L6si versus control si)impairs IFN-g -induced upregula-tion of poly-ub conjugates as shown (right panel).UBE2L6knockdown was con?rmed by RT-PCR and immunoblotting (GAPDH,loading control;lower panel,densitomeric evaluation of three independent series ±SD).
See also Figure S2and Table S1.
chase experiments demonstrated that the increase in poly-ub protein levels was strongly dependent on translation (Figure 1E).Thus,the enhanced polyubi-quitylation of newly synthesized proteins in response to IFN-g is accompanied by
augmented de novo protein synthesis.Furthermore,the same experiments also revealed that poly-ub proteins are degraded signi?cantly faster in IFN-g -treated than in untreated cells (see also Figure 4).
The Ub Conjugation Machinery Is Remodeled in Response to IFNs
To determine whether the accumulation of poly-ub conjugates is the result of IFN-induced enhanced polyubiquitylation activity,we performed in vitro ubiquitylation experiments with cellular lysates of IFN-g -stimulated cells.Enhanced polyubiquitylation activity was detected already at early time points and did not signi?cantly decrease over the time period analyzed (Figure 2A).This prompted us to study whether IFNs also exert a more general effect on the ubiquitin-proteasome system (UPS).There-fore,we investigated the expression of UPS-related messenger RNAs (mRNAs)in response to IFN-g in a focused microarray approach.IFNs speci?cally induced 20E3ub ligases.Of the E2ub-conjugating enzymes,only UBE2L6was strongly induced (Table S1),and its mRNA was upregulated more than 50-fold in response to 4hr of IFN treatment (Figure 2B and Figure S2).To study the role of UBE2L6in IFN-induced ub conjugation,we silenced expression of UBE2L6by small interfering RNA (siRNA)to interfere with the thioester cascade.Depletion of UBE2L6signi?cantly abolished the increase in total poly-ub conjugates 8hr after IFN-g stimulation,whereas no differences could be detected in untreated cells (Figure 2C).Thus,UBE2L6-dependent ub conjugation activity plays an important role in accumulation of ub conjugates during the early IFN response.Together,our experiments demonstrate that increased levels
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of polyubiquitylated proteins in response to IFNs are at least in part the result of augmented ub conjugation activity.
Proteasome Activity Is Transiently Decreased in Response to IFNs
In addition to enhanced ubiquitylation activity,a cytokine-dependent partial impairment of proteasomal degradation capacity may also explain the transient increase in cellular levels of poly-ub conjugates.Therefore,we assayed the proteasomal chymotrypsin-like (CT-L)peptide-hydrolyzing activity in total cell lysates of IFN-g -treated HeLa cells (Figure 3A).Indeed,IFN stimulation resulted in a transient decrease of proteasomal CT-L activity 4–12hr after induction,which was restored and even augmented after 24–48hr.This result was con?rmed by measurement of in-gel CT-L activity in native polyacrylamide gel electrophoresis (PAGE)and could be assigned to 26S and to PA28-associated proteasome pools (Figures 3B–3D and Figures S3D and S3E).
Concomitant with the decreased proteolytic activity between 8and 12hr,we detected transiently lower 26S proteasome levels in response to IFN-g (Figure 3C).To de?ne this phenom-enon in more detail,we immunoprecipitated proteasomes from glycerol gradient fractions enriched for 26S complexes,and immunodetection of the 19S regulator subunit Rpn2fol-lowed.In agreement with the measured decrease in proteolytic activity,we observed a transiently reduced association of 19S regulators with 20S complexes (Figure 3E).Total amounts of 20S complexes,as monitored by the a 4subunit levels,remained unchanged,suggesting that IFN-g induces a transient dissocia-tion of the 20S complexes from the 19S regulator.
Next,we studied the kinetics of 26S i-proteasome formation by incorporation of the b 1i/LMP2subunit into the complexes by using native PAGE (Figure 3F).In agreement with the increase in proteasomal activity after 24hr of IFN-g exposure,i26S proteasome levels were strongly enhanced at this time point.Reorganization of proteasomes upon IFN exposure was also shown by immunoprecipitation experiments or two-dimensional separation of puri?ed 20S complexes (Figures S3A–S3C).Impor-tantly,the time frames exhibiting the highest accumulation of poly-ub conjugates corresponded exactly with the decline of proteasomal peptidase activity and coincided with the time period required for the formation of i26S proteasomes.
turnover of Suc-LLVY-AMC R F U
A
0 8 12 24 48 IFN-γ(h)
B
IP:β7 Ab from 26S gradient fractions
0 4 8 12 24 IFN-γ(h)
α4
Rpn2C
α6A b
β1i /L M P 2A b
D
E
0 8 12 24 48 IFN-γ(h)
γ(h)
substrate overlay
0 812 24 48 IFN-γ(h)
immunoblot
26S 20S +PA28
20S
20S + PA2826S
26S
20S
20S + PA28F
26S R p n 2A b
P A 28βA b
hybrid 20S + PA28
0 8 12 24 48 IFN-γ(h)
Figure 3.Altered Proteasome Activity and Composition in Response to IFNs
(A)Total 26S chymotryptic peptide-hydrolyzing activity was analyzed in HeLa cells exposed to IFN-g .After a transient decrease in activity between 4and 8hr of IFN-g treatment,chymotryptic activity increases again and peaks at 48hr of IFN-induction (three independent series ±SD).
(B–D)Cellular proteasome pools were separated on native PAGE,proteasome population was analyzed by in-gel chymotryptic activity (B)and by immunoblot with a 6,Rpn2(C),and PA28b antibodies (D).
(E)26S proteasomes were immunoprecipitated from glycerol gradient fractions with a b 7antibody.Association of 19S subunit Rpn2to 20S proteasome is transiently decreased between 4and 8hr of IFN-g induction.Levels of the constitutive subunit a 4were determined as control.(F)Analysis of 26S i-proteasome amount after IFN-g stimulation on native PAGE with b 1i/LMP2antibody.See also Figure S3.
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i-Proteasomes Are Required for Ef?cient Degradation of Polyubiquitylated Proteins
The above experiments suggest that i-proteasomes may be more ef?cient in degrading poly-ub substrates than standard proteasomes (s-proteasomes).We therefore examined the removal of poly-ub conjugates in mouse embryonic ?broblasts (MEFs)and human cell lines (T1,T2)with either a fully functional i-proteasome (WT,T1)or cells de?cient for one or two of the i-proteasomes subunits (LMP7à/à,T2)after IFN challenge (Figures 4A and 4B and Figure S4A).Furthermore,to study the requirement of i-proteasomes for the degradation of IFN-g induced poly-ub conjugates in vivo,we induced in?ammation in WT mice and in i-proteasome-de?cient mice by using lipopo-lysaccaride (LPS)and analyzed i-proteasome function in experimental autoimmune encephalomyelitis (EAE)(Figures 4C and 4D).
In WT MEFs as well as in LPS-induced acute in?ammation of liver tissue in mice (Figures 4A–4C)or in human T1cells (Fig-ure S4A),we observed a transient accumulation of poly-ub conjugates that returned to basal levels at 48hr after the challenge (see also Figure 1).
In contrast,in LMP7à/àMEFs,that are impaired in i-proteasome formation (Grif?n et al.,1998)(Figures 4A and 4B)or in human T2cells that are devoid of i-proteasomes (Figure S4A),the accumu-lation of poly-ub conjugates persisted over the entire period without returning to basal levels.Most strikingly,in?ammation-challenged liver (LPS)or brain (EAE)displayed a signi?cantly increased accumulation of poly-ub conjugates in LMP7-de?cient
A
B
0 4 8 12 24 480 4 8 12 24 48 IFN-γ(h)
wt MEFs
LMP7-/-MEFs
β5i/LMP70 30 60 120 min
s26S
i26S
D
Ub 5
LMP7-/-wt brain:
EAE diseased poly-ub
GAPDH
β5i/LMP7
LMP7-/-wt liver:LPS 24h C57BL/6liver:
0 24 48 LPS (h)
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GAPDH
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48h IFN-γ
p i x e l d e n s i t y u b -s t a i n
-/-
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GAPDH β5i/LMP7
* p< 0.005
* p< 0.005
GAPDH
E
LPS EAE E n z y m e a c t i v i t y
0h
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6h CHX chase
%r a d i o a c t i v i t y o f u b -c o n j u g a t e s
100 61 35 16 %
100 35 19 8 %
poly-ub short exp.F
Figure 4.Poly-Ub Conjugates Are Degraded by i-Proteasomes
(A and C)Comparison of C57BL/6WT MEFs and LMP7à/àMEFs treated with IFN-g up to 48hr (long and short exposure)(A)or C57BL/6mice treated with LPS or
left naive (C)were sacri?ced after 24or 48hr and processed for ub conjugates in livers by immunoblotting.Representative results out of four independent series are shown (GAPDH,loading control;b 5iLMP7,IFN and knockout control).
(B)In samples expressing i-proteasomes (WT)accumulation of ub conjugates returns to basal levels after 48hr of IFN-g treatment.Statistic evaluation of ub staining out of four independent series (±SD;p values from t test).
(D)In?ammation-challenged liver (left)or brain tissue (right)of C57BL/6WT or LMP7à/àmice stained for poly-ub conjugates by anti-ub antibody in immunoblots.Ub conjugate formation is higher in cells lacking LMP7(LMP7à/à).Representative results are shown (GAPDH,loading control;b 5iLMP7,IFN and knockout control;statistic evaluation of ub staining of four animals ±SD;p values from t test).
(E)In vitro degradation by s26S and i26S proteasomes was analyzed with a Ub 5-Muc 8model substrate and immunodetection with the mucin antibody.Quantitative evaluation of Ub 5-Muc 8digestion by i26S proteasomes showed a 2-fold increase in substrate turnover compared to s26S proteasomes (enzyme activity in ng/min $m g proteasome ±SD,n =5).
(F)Quantitative evaluation of the pulse chase experiment in Figure 1E (±SD,n =2)showing that i-proteasomes degrade poly-ub conjugates faster than do s-proteasomes.See also Figure S4.
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mice compared to the WT(Figure4D).In agreement with these data,the prevention of i-proteasome formation by knockdown of the proteasome maturation protein POMP(Heink et al.,2005) resulted in a considerably slower turnover of poly-ub conjugates (Figure S4B).Together,these experiments show that i-protea-some-de?cient cells are unable to ef?ciently eliminate the accumulating poly-ub conjugates in response to IFN-g,both in cell culture studies and in in vivo in?ammation models.
The observation of an accelerated degradation of poly-ub conjugates dependent on i-proteasomes suggests that i-protea-somes may possess enhanced poly-ub substrate turnover activity.To investigate this in more detail,we performed in vitro degradation assays with af?nity-puri?ed s-and i-proteasomes by using the established poly-ub model substrate Ub5-Muc8 (Bech-Otschir et al.,2009).Indeed,in vitro degradation of Ub5-Muc8revealed an approximately2-fold higher substrate turnover with i-proteasomes than in experiments using s-protea-somes(Figure4E and Figures S4C–S4E),thus explaining at least in part the observed accelerated degradation of accumulated poly-ub conjugates upon IFN-g stimulation.Moreover,the turn-over of i k B a as a known proteasomal substrate in response to TNF-a signaling was faster in cells stably expressing i-protea-somes than in untransfected cells(Figures S4F and S4G).In support of these experiments,the turnover of radiolabeled poly-ub conjugates occurs approximately3-fold faster in response to IFN-g(Figures1E and4F).
Impaired i-Proteasome Function Results
in the Formation of Protein Aggregates
To avoid potentially toxic effects of accumulated poly-ub conju-gates,cells attempt to form aggresomes or aggresome-like induced structures(ALISs).Such ub-rich cytoplasmic inclusions are formed in response to cellular stresses such as heat shock, proteasome inhibition,oxidative stress,transfection,or starva-tion(Kopito,2000;Lelouard et al.,2004;Szeto et al.,2006).To determine whether ALISs are formed in response to IFN,we visualized poly-ub conjugates in different cell types by immuno-cytochemistry.Interestingly,coinciding with the strongest accumulation of poly-ub conjugates and independent of the cell type analyzed,ALISs were transiently formed in response to IFN-g(Figure5A and Figure S5A).In striking contrast to the WT,but in agreement with the inability of LMP7à/àMEFs to remove poly-ub conjugates in response to IFNs(Figure5B),we detected ALISs throughout the IFN-g induction time course (Figures5B and5C and Figure S5B).Thus,we observed the highest accumulation of ALISs in i-proteasome-de?cient cells at time points at which WT cells were already cleared from ALISs (Figure5and Figure S5B).In support,LMP7-de?cient cells dis-played a continuous depletion of ubH2A from nuclei,because ub pools were occupied by ALIS formation,whereas in WT MEFs ubH2A was only transiently depleted from nuclei as long as ALISs were present(Figure S6).
Supporting our data obtained in cell culture studies,in?amma-tion-challenged liver(LPS)or brain(EAE)tissue revealed pro-nounced higher staining of poly-ub in LMP7à/àmice compared to the WT(Figure5D),demonstrating that the observation made in cultured cells can be also applied for the in vivo situa-tion.Thus,at least in part because of their higher substrate-turn-over capacity,i-proteasomes ef?ciently remove poly-ub conju-gates and either remove ALISs or prevent their formation. Under conditions of IFN challenge,such as during an in?amma-tory response,cells that are impaired in i-proteasome formation fail to cope with the increased formation of poly-ub conjugates and consequently produce ALISs.
i-Proteasomes Protect Cells against IFN-Induced Oxidative Stress
IFNs are important triggers for the formation of reactive oxygen species(ROSs)that cause oxidative stress(Pearl-Yafe et al., 2003;Sasaki et al.,2008;Watanabe et al.,2003).Indeed,ROS levels increased up to a2-fold higher level after48hr in response to IFN-g in HeLa and T1cells(Figure S7A),suggesting that i-pro-teasomes may possess an important role in IFN-induced oxida-tive stress response.Endogenous ROS formation may cause oxidant-damage to proteins.Therefore,by detection of carbonyl groups,we tested whether IFN stimulation results in increased levels of oxidized proteins in HeLa(Figure6A)and T1and T2 (Figure S7C)cells.The amount of oxidized proteins coincided with increased amounts of poly-ub conjugates and was signi?-cantly higher in samples of cells lacking i-proteasomes,such as T2cells,whereas the levels were decreased in samples that exhibited high i-proteasome activity(Figure6A and Figures S7B and S7C).To study whether the oxidant-damaged proteins are indeed polyubiquitylated,we immunoprecipitated ub-modi-?ed proteins and stained the precipitates for oxidized proteins. Oxidized proteins could be precipitated from lysates of IFN-stim-ulated cells with an anti-ubiquitin antibody(Figure6B).Further-more,a known speci?c substrate of proteasomes,i k B a,was also oxidant damaged(Figure S7D).Thus,our results suggest that IFNs trigger oxidation of proteins,which would then be polyubiquitylated and degraded by i-proteasomes.The correla-tion between oxidant damage and ub modi?cation was supported through the use of the antioxidant sulforaphane, which impaired the IFN-induced accumulation of both poly-ub conjugates and oxidant-damaged proteins(Figure6C).
To investigate the impact of i-proteasomes on degradation of oxidant-damaged protein,WT and LMP7-de?cient cells were compared with respect to levels of oxidized proteins upon IFN-g exposure.Unchallenged WT skin?broblasts displayed low levels of oxidized proteins,which were transiently increased between8and24hr of IFN-g exposure(Figure6D).Notably, such a transient induction of oxidant-damaged proteins was also detectable in liver tissue in response to LPS-induced in?am-mation(Figure6E).In contrast,LMP7-de?cient?broblasts ex-hibited signi?cant amounts of oxidized proteins throughout the time course,with the highest accumulation at48hr upon IFN-g exposure(Figures6D).This was also observed in LMP7-de?cient MEF cells(Figures S7E and S7F)and T2cells(Figure S7C).Simi-larly,in?ammation resulted in a strong induction of oxidized proteins in vivo as revealed by the analysis of liver and brain tissue from in?ammation-challenged mice(Figure6F).Thus, proteins that are oxidant damaged by IFN-induced ROSs are polyubiquitylated and become a substrate of i-proteasomes. Because IFN-induced oxidative stress may result in enhanced cell death,we tested whether LMP7-de?cient cells are more susceptible to apoptosis in response to IFNs than WT cells.
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Indeed,caspase 3/7activity was enhanced 2-fold in response to IFN-g in LMP7-de?cient MEFs,whereas caspase activity remained almost unchanged in WT MEFs.Moreover,LMP7-de?-cient MEFs were highly susceptible to the potent apoptosis inducer etoposide (Figure 6G).
To examine the in vivo effects of LMP7de?ciency in EAE-induced in?ammation,we determined the clinical scores in WT,LMP7à/à,and LMP7+/àmice.Neurological de?cits in EAE were scored on a scale from 0to 5,with animals showing no abnormality at a score of 0and being increasingly affected by motor de?cits at higher disease scores (Zamvil and Steinman,1990).Accordingly,LMP7à/àmice suffered from EAE-induced in?ammation with approximately 3-fold higher clinical scores and earlier onset of clinical symptoms than WT mice.Heterozy-
gous LMP7+/àmice displayed an intermediate increase in the clinical scores.T cell-associated primary immune responses against the encephalitogenic MOG peptide,however,remained unaffected (Figure 6H and Figures S7G and S7H).
IFN-g Increases the Pool of Polyubiquitylated Nascent Proteins
Polyubiquitylated nascent defective proteins (DRiPs)represent the major but not the sole source for MHC class I peptide ligands (Reits et al.,2000;Schubert et al.,2000).However,what the ‘‘defect’’is in DRiPs has been unclear.In addition,there existed no explanation as to how,upon IFN-g stimulation,the amount of DRiPs could be increased to meet the substrate demands of a strongly upregulated antigen presentation machinery.Because
poly-Ub FK1proteasome merge
poly-Ub FK1proteasome
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Figure 5.i-Proteasomes Are Required for Ef?cient Removal of ALISs
(A and B)Poly-ub conjugates and formation of ALISs were visualized by immuno?uorescence with the poly-ub antibody FK1(green).Poly-ub conjugates were colocalized with proteasomes (red).In LMP7à/àMEFs (B),accumulation of poly-ub conjugates was detectable after up to 48hr of IFN-g exposure,whereas ALISs were removed in WT MEFs (A)at this time point.
(C)Statistical evaluation of ALISs per cell out of four independent series ±SD;p values from t test.
(D)Immunohistochemistry of in?ammation-challenged liver or brain tissue of C57BL/6WT or LMP7à/àmice revealed pronounced staining of in?amed tissue of LMP7à/àmice.Cryosections of liver tissue and EAE brain (cortex,cerebellum)were stained with FK1for poly-ub (green)and Hoechst (blue).Magni?cation illustrates increased levels of accumulated poly-ub-positive structures corresponding to ALISs in in?amed brain areas in LMP7-de?cient mice (white arrow-heads).The scale bar represents 25m m.Statistical evaluation of poly-ub-positive cells/total cells in 10mm 2of three animals ±standard error of the mean [SEM];p values from Mann-Whitney U test.See also Figures S5and S6.
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IFN-g stimulation enhanced the generation of ROSs and the formation of oxidant-damaged newly synthesized polyubiquity-lated proteins,we determined the pool of DRiPs in response to IFN-g after the experimental setup as initially reported (Schubert et al.,2000).Thus,for labeling of nascent proteins,murine ?broblasts were exposed to a short 30s radioactive pulse with
5mCi /ml -1[35S]methionine,which was followed by a nonradio-active chase period for the time points indicated.Newly trans-lated ubiquitylated proteins forming a high-molecular weight smear were visualized by autoradiography.
IFN-g stimulation resulted in an increased formation of nascent polyubiquitylated proteins (Figure 7A).Moreover,this
A B
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e a s e )
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Figure 6.The Levels of Protein-Oxidation and Apoptosis Are Induced in LMP7-De?cient Cells
(A)Levels of oxidized proteins were determined by Oxyblot in HeLa cells in response to IFN-g (GAPDH,loading control).
(B)Oxidative modi?cations of ub proteins could be demonstrated in immunoprecipitates of poly-ub proteins after IFN-g stimulation in HeLa cells (IgG hc,IgG heavy chain).
(C)The antioxidant sulforaphane abolished the IFN-g -induced accumulation of ub conjugates (left)and oxidant-damaged proteins (right)(densitometric evaluation of two series ±SD).
(D)Levels of oxidized proteins were determined by Oxyblot in C57/BL6WT or LMP7à/àskin ?broblasts.LMP7-de?cient skin ?broblasts display increased levels of oxidant-damaged proteins in comparison to WT ?broblasts.
(E)LPS-in?amed livers of C57BL/6WT mice treated with LPS or left naive were sacri?ced after 24or 48hr and stained for oxidant-damaged proteins by immunoblotting (GAPDH,loading control).Representative results out of three independent series are shown.
(F)LPS-in?amed livers or EAE-in?amed brains of C57/BL6WT or LMP7à/àmice stained for oxidant-damaged proteins in immunoblots.Formation of oxidant-damaged proteins is higher in LMP7à/àcells.Representative results out of four independent series are shown (statistic evaluation of oxy-blot ±SD;p values from t test).
(G)LMP7-de?cient MEFs showed increased apoptosis as indicated by enhanced caspase activity in response to IFNs and are more susceptible to etoposide than WT MEFs.Error bars represent the SD of three independent experiments.
(H)LMP7à/àmice suffer from EAE-induced in?ammation with signi?cantly higher clinical scores,whereas heterozygous LMP7+/àlittermates reveal intermediate clinical scores (six animals ±SEM).See also Figure S7.
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pool was rapidly degraded by proteasomes because degrada-tion was almost completely abrogated by proteasome inhibition in cells stimulated with IFN-g for 24hr (Figure 7B).
To study whether these nascent polypeptides are indeed defective,we used epigallocatechin-3-gallat (EGCG),a green tea compound that binds randomly to the backbone groups of unfolded proteins (Ehrnhoefer et al.,2008).Indeed,we observed an increase in EGCG-stained polypeptides in response to IFNs,ultimately demonstrating that IFN-induced oxidation of proteins contributes to the unfolding of nascent proteins (Figure 7C).DISCUSSION
As part of the MHC class I antigen-processing machinery,the function of i-proteasomes has been associated with improved antigen generation.However,the relevance of i-proteasomes in speci?c immune responses has been increasingly questioned,but no alternative functions have been proposed (Nussbaum et al.,2005;Yewdell,2005).We now show that a major function of i-proteasomes is the maintenance of protein homeostasis during in?ammatory conditions by rapidly degrading nascent oxidant-damaged proteins.
These ?ndings lead us to propose the following model (Figure 7D):IFNs induce oxidative stress and newly synthe-sized proteins are particularly sensitive to oxidation (Medicherla and Goldberg,2008).This causes their partial unfolding and consequent recognition by the UPS.Together with enhanced translation activated by the mTOR pathway (Kaur et al.,2008),this results in a strong increase of nascent,oxidant-damaged proteins or DRiPs.In this context,virus infection,radiation,the mTOR-pathway,and other signals also have been shown to induce the birth of DRiPs (Lelouard et al.,2007;Reits et al.,2006;Yewdell,2005).Thus,under cyto-kine-inducing conditions,DRiPs are indeed defective proteins.The resulting need for augmented K48-linked ubiquitylation and degradation of polyubiquitylated proteins is met by cytokine-induced upregulation of the ubiquitylation machinery.For example,UBE2L6,an important player in cross-presentation by human dendritic cells (Ebstein et al.,2009)and a target of the mTOR signaling cascades (Kaur et al.,2008),is strongly upregulated in response to IFNs.In addition,i-proteasomes exhibiting an accelerated degradation rate of polyubiquitylated proteins are formed.Ef?cient elimination of oxidant damaged and unfolded nascent proteins may prevent their potentially toxic effects.In contrast,in the absence of fully functional i-proteasomes as in MEFs of i-proteasome subunit LMP7-de?-cient mice,the degradation of oxidant damaged nascent proteins is severely impaired.This impairment leads to the accumulation of DRiPs,the formation of ALISs,and conse-quently to increased sensitivity toward apoptosis.Thus,forma-tion of i-proteasomes with their superior poly-ub conjugate degrading capacity is essential in the protection of cells against persistent formation of ALISs.
Previous studies have revealed that incorporation of i-subunits results in enhanced proteasomal peptide-hydrolyzing activity (Gaczynska et al.,1993;Strehl et al.,2008;Voigt et al.,2010).In addition,integration of i-subunits into the 20S core complex also induces slight structural changes,suggesting an improved accessibility of the active sites for protein substrates (Sijts et al.,2000).In consequence,i26S protea-somes may be better equipped for ef?cient poly-ub substrate turnover.In fact,our in vitro experiments with saturating amounts of poly-ub substrate,and excluding indirect substrate af?nity effects,demonstrate that the composition and
activity
nascent protein pool
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2
3IFN-γ(h)
00106001060++--Chase (min)
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----+++Epoxomicin -+-+24h IFN-γA
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C
Figure 7.IFN-g Stimulation Increases the Pool of DRiPs
(A)The nascent protein pool (DRiPs)is increased rapidly after IFN-g stimulation in murine ?broblasts.Cells were pulsed for 30s with 5mCi /ml à1[35S]methionine followed by a nonra-dioactive chase.
(B)The nascent protein pool (DRiPs)increased after IFN-g stimulation is rapidly degraded by i-proteasomes.Protein degradation could be prevented by addition of proteasome inhibitor epoxomicin.
(C)Defective proteins were increased after IFN-g treatment as detected by EGCG staining.
(D)IFN-g induces oxidative stress and newly synthesized proteins are sensitive to oxidation.In combination with enhanced translation,this induc-tion will lead to an increase in oxidant-damaged,partially unfolded proteins or DRiPs.The resulting increased requirement for ubiquitylation and protein degradation is met by cytokine-induced upregulation of the ubiquitylation machinery and the formation of i26S proteasomes that degrade polyubiquitylated proteins with higher ef?ciency.
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of the20S core modulates the degradation ef?ciency of26S proteasomes.
The molecular events underlying the dissociation of s26S proteasomes during the early phase of IFN induction(Figure3) are not clear at present.However,changes in26S proteasome levels in response to IFN-g have been observed previously and are thought to be due to different phosphorylation patterns of a subunits(Bose et al.,2001).Rapid replacement of the s26S proteasomes in response to IFN-g will require their disassembly and the reassembly of19S regulator complexes with de novo-formed i20S core complexes.Thus,cells tolerate a short adaptation phase of reduced proteolytic activity that is termi-nated by the rapidly increasing amounts of i-proteasomes to adjust the proteasome pool to the increased proteolytic require-ments of IFN-challenged cells.
Visual evidence for the importance of i-proteasome activity is obtained in IFN-g-treated LMP7-de?cient cells or in?amed tissues of LMP7à/àmice,which reveal a cellular accumulation of ub conjugates with persistent formation of ALISs.Our exper-iments show that as a result of IFN-induced ROS formation, oxidant-damaged proteins have the tendency to aggregate and consequently accumulate in such ub-rich inclusions.The formation of ALISs represents a severe physiological stress (Szeto et al.,2006)that requires i-proteasome function to protect cells against accumulation of protein aggregates.Most cell types and tissues constitutively express low levels of LMP7, whereas immune cells which actively produce ROSs and perma-nently have to deal with cytokine-induced oxidative stress express i-proteasomes even at constitutively high levels (Yewdell,2005).
Underlining the importance of i-proteasomes for the preserva-tion of cell viability under cytokine-induced oxidative stress conditions,b5i/LMP7à/àcells were considerably more suscep-tible to apoptosis.This protective role is also evidenced by studies showing that i-proteasome-de?cient mice severely suffer from impaired survival and clearance rates upon infection (Chou et al.,2008;Ishii et al.,2006;Strehl et al.,2006)or develop severe myocarditis upon Coxsackie virus B3infection(A.V.,E.K., U.K.,and P.-M.K.,unpublished data).As shown here,i-protea-somes also protect mice against the pathological consequences of EAE.Because neuronal damage is the major determinant of disease severity in EAE,i-proteasome de?ciency signi?cantly contributes to the susceptibility of the central nervous system to in?ammation-mediated oxidative damage.
i-subunits,apart from their induction by cytokines or infection, have also been reported to be induced by insults like heat stress (Callahan et al.,2006),arsenic trioxide(Zheng et al.,2005)and nitric oxide(Kotamraju et al.,2006)or in the course of neurode-generative diseases(D?′az-Herna′ndez et al.,2003;Ferrer et al., 2004;Puttaparthi and Elliott,2005).Thus,the expression of i-proteasomes may be initiated in heterogeneous damage scenarios,supporting the view of a more general physiological function of i-proteasomes apart from antigen processing. These insights point at alternative physiological functions of i-proteasomes,which are not primarily connected with a speci?c role in MHC class I antigen processing but rather with the protec-tion of cells against cytokine induced oxidative damage to preserve cell viability.EXPERIMENTAL PROCEDURES
Cell Culture and Mouse Experiments
Human and murine cell lines were cultivated under standard conditions and treated with100U/ml human or mouse IFN-g(recombinant,Roche),respec-tively.Proteasome activity was blocked by epoxomicin(1m M)4hr before harvesting of cells.siRNA knockdown of UBE2L6in comparison to control siRNA was performed24hr before stimulation by IFN-g according to the manufacturer’s instructions(Dharmacon).
C57BL/6LMP7à/àmice or C57BL/6LMP7+/+wild-type littermates were kept at local facilities.MEFs were obtained from day13.5embryos.For LPS-induced in?ammatory response,8-week-old mice were injected intraper-itoneally with LPS from E.coli(5mg/kg body weight).Survival until day3was 100%in both groups.For active EAE,mice were immunized subcutaneously with200m g MOG35–55and were sacri?ced after disease was established (day45).Animal care was in accordance with institutional guidelines.
Cells and tissue sections for immunocytochemistry and immunohistochem-istry were prepared according to standard protocols and stained with FK1 (mAb,Biomol),ubH2A(E6C5,Millipore),or anti-proteasome polyclonal anti-body(laboratory stock)followed by Alexa488-or Alexa568-conjugated secondary antibodies(Invitrogen).
Quanti?cation of experiments is shown as mean±SD or±SEM with p values from Student’s t test or Mann-Whitney U test,respectively.
In Vitro Ub Conjugation
Fifty microgram lysate of B8cells stimulated with or without IFN-g were incu-bated with1m M His-tagged ub(WT ub,K48only,K48to R,K63only,K63to R; Boston Biochemicals)for1hr in reaction buffer containing20mM Tris-HCl (pH7.6),20mM KCl,5mM MgCl2,10%glycerol,1mM DTT,2mM AMPPNP (Sigma),50m M MG-132(Calbiochem),and5m M ub-aldehyde(Boston Biochemicals).Ub conjugates were immunodetected by a penta-His antibody (QIAGEN).
Immunoprecipitation of Ub Conjugates and Detection of DRiPs
B8cells were stimulated with100U/ml IFN-g for12hr,pulsed with TRAN35S-Label(ICN)for45min,and chased with25m g/ml cycloheximide for up to6hr.Ub conjugates were precipitated with FK2antibody(Biomol) and processed as described(Heink et al.,2005).Detection of DRiPs in B8cells was accomplished as previously described(Schubert et al.,2000).Detection of defective proteins by EGCG(Sigma)staining was performed as previously described(Ehrnhoefer et al.,2008).
Determination of Oxidative Stress
Oxidized proteins were visualized with the OxyBlot protein oxidation detection kit(Chemicon International)via immunodetection of carbonyl groups.So that ROS formation could be prevented,cells were pretreated for24hr with the antioxidant sulforaphane(10m M;Sigma).
ACCESSION NUMBERS
The accession number for the microarray data reported in this paper is GSE21760.All primary data are available at the Gene Expression Omnibus database(GEO database).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, seven?gures,and one table and can be found with this article online at doi:10.1016/j.cell.2010.07.036.
ACKNOWLEDGMENTS
D.Ludwig and
E.Bu¨rger are acknowledged for their excellent technical assis-tance.This work was supported by the Deutsche Forschungsgemeinschaft (SFB421,SFB740,Transregio19,Transregio43,KR1915/4-1,SPP1365).
622Cell142,613–624,August20,2010a2010Elsevier Inc.
Received:October31,2008
Revised:April1,2010
Accepted:June22,2010
Published:August19,2010
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《基因组学与蛋白质组学》课程教学大纲 学时： 40 学分：2.5 理论学时： 40 实验学时：0 面向专业：生物科学、生物技 术课程代码：B7700005先开课程：生物化学、分子生物 学课程性质：必修/选修执笔人：朱新 产审定人： 第一部分：理论教学部分 一、课程的性质、目的和任务 《基因组学与蛋白质组学》是随着生物化学、分子生物学、结构生物学、晶体学和计算机技术等的迅猛发展而诞生的，是融合了生物信息学、计算机辅助设计等多学科而发展起来的新兴研究领域。是当今生命科学研究的热点与前沿领域。由于基因组学与蛋白质组学学科的边缘性，所以本课程在介绍基因组学与蛋白质组学基本基本技术和原理的同时，兼顾学科发展动向，讲授基因组与蛋白组学中的热点和最新进展，旨在使学生了解现代基因组学与蛋白质组学理论的新进展并为相关学科提供知识和技术。 二、课程的目的与教学要求 通过本课程的学习，使学生掌握基因组学与蛋白质组学的基本理论、基础知识、主要研究方法和技术以及生物信息学和现代生物技术在基因组学与蛋白质组学上的应用及典型研究实例，熟悉从事基因组学与蛋白质组学的重要方法和途
径。努力培养学生具有科学思维方式、启发学生科学思维能力和勇于探索，善于思考、分析问题的能力，激发学生的学习热情，并通过学习提高自学能力、独立思考能力以及科研实践能力，为将来从事蛋白质的研究奠定坚实的理论和实践基础。 三、教学内容与课时分配 第一篇基因组学
第一章绪论（1学时） 第一节基因组学的研究对象与任务； 第二节基因组学发展的历程； 第三节基因组学的分子基础； 第四节基因组学的应用前景。 本章重点： 1. 基因组学的概念及主要任务； 2. 基因组学的研究对象。 本章难点： 1.基因组学的应用及发展趋势； 2.基因组学与生物的遗传改良、人类健康及生物进化。建议教学方法：课堂讲授和讨论 思考题： 查阅有关资料，了解基因组学的应用发展。 第二章人类基因组计划（1学时） 第一节人类基因组计划的诞生； 第二节人类基因组研究的竞赛； 第三节人类基因组测序存在的缺口； 第四节人类基因组中的非编码成分； 第五节人类基因组的概观； 第六节人类基因组多样性计划。 本章重点： 1. 人类基因组的研究； 2. 人类基因组多样性。 本章难点： 人类基因组序列的诠释。 建议教学方法：课堂讲授和讨论 思考题：
通过校园网进入数据库例如维普期刊数据库、CNKI、超星电子图书等。完成 A、任选一题，检索相关资料，截取检索过程图片，做成一个ppt文件（50分）。 B、写综述形式的学术论文(学术论文格式，字数不限，正文字体小四)，做成word文件（50分）。要求：按照自己的思路组织成文件，严禁抄袭。 写明班级学号，打印纸质版交给老师。 1、对检索课题“磷酸对草莓生长和开花的影响”检索中文信息。提示：磷酸的化学物质名称是“Phosphonic acid ”普通商业名称是“ethephon”， 2、基因组学和蛋白质组学对新药研发的影响 3、红霉素衍生物的设计、合成与抗菌活性研究 4、HPLC法测定复方谷氨酰胺肠溶胶囊中L-谷氨酰胺的释放度 姓名：朱艳红 班级： 11生科师范 学号： 11223074 学科教师：张来军
基因组学和蛋白质组学对新药研发的影响琼州学院生物科学与技术学院 11生科师范2班朱艳红 11223074 摘要 20世纪末伴随着人类基因组计划的实施，相继产生了基因组学和蛋白质组学，基因组学和蛋白质组学的迅速发展，对药学科学产生着深远的影响。文章在简介蛋白质组学基本概念、核心技术的基础上，综述了基因组学和蛋白质组学对新药研发带来的影响。 关键词：基因组学；蛋白质组学；药物研发 The impact of genomics and proteomics on the research and development of innovative drug abstract With the implementation of the 20th century,Genomics and proteomics had emerged one after the other. Driven by Soaring development of the omits，pharmaceutical industry presents a new vision，all human life faces a promising future. On the basis of proteomics Introduction to basic concepts, core technology, reviewed the genomics and proteomics research on the impact of new drugs. Keywords:Genomics; proteomics; drug development
蛋白质预测在线分析常用软件推荐 蛋白质预测分析网址集锦 物理性质预测： Compute PI/MW http://expaxy.hcuge.ch/ch2d/pi-tool.html Peptidemasshttp://expaxy.hcuge.ch/sprot/peptide-mass.html TGREASE ftp://https://www.doczj.com/doc/7311389676.html,/pub/fasta/ SAPS http://ulrec3.unil.ch/software/SAPS_form.html 基于组成的蛋白质识别预测 AACompIdent http://expaxy.hcuge.ch ... htmlAACompSim http://expaxy.hcuge.ch/ch2d/aacsim.html PROPSEARCH http://www.e mbl-heidelberg.de/prs.html 二级结构和折叠类预测 nnpredict https://www.doczj.com/doc/7311389676.html,/~nomi/nnpredict Predictprotein http://www.embl-heidel ... protein/SOPMA http://www.ibcp.fr/predict.html SSPRED http://www.embl-heidel ... prd_info.html 特殊结构或结构预测 COILS http://ulrec3.unil.ch/ ... ILS_form.html MacStripe https://www.doczj.com/doc/7311389676.html,/ ... acstripe.html 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。 由NCBI检索蛋白质序列 可联网到：“http://www.ncbi.nlm.ni ... gi?db=protein”进行检索。 利用SRS系统从EMBL检索蛋白质序列 联网到：https://www.doczj.com/doc/7311389676.html,/”，可利用EMBL的SRS系统进行蛋白质序列的检索。 通过EMAIL进行序列检索 当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。 蛋白质基本性质分析 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的
问:基因组学、转录组学、蛋白质组学、结构基因组学、功能基因组学、比较基因组学研究有哪些特点? 答:人类基因组计划完成后生物科学进入了人类后基因组时代,即大规模开展基因组生物学功能研究和应用研究的时代。在这个时代,生命科学的主要研究对象是功能基因组学,包括结构基因组研究和蛋白质组研究等。以功能基因组学为代表的后基因组时代主要为利用基因组学提供的信息。 基因组研究应该包括两方面的内容:以全基因组测序为目标的结构基因组学(struc tural genomics和以基因功能鉴定为目标的功能基因组学(functional genomics。结构基因组学代表基因组分析的早期阶段,以建立生物体高分辨率遗传、物理和转录图谱为主。功能基因组学代表基因分析的新阶段,是利用结构基因组学提供的信息系统地研究基因功能,它以高通量、大规模实验方法以及统计与计算机分析为特征。 功能基因组学(functional genomics又往往被称为后基因组学(postgenomics,它利用结构基因组所提供的信息和产物,发展和应用新的实验手段,通过在基因组或系统水平上全面分析基因的功能,使得生物学研究从对单一基因或蛋白质的研究转向多个基因或蛋白质同时进行系统的研究。这是在基因组静态的碱基序列弄清楚之后转入基因组动态的生物学功能学研究。研究内容包括基因功能发现、基因表达分析及突变检测。 基因的功能包括:生物学功能,如作为蛋白质激酶对特异蛋白质进行磷酸化修饰;细胞学功能,如参与细胞间和细胞内信号传递途径;发育上功能,如参与形态建成等采用的手段包括经典的减法杂交,差示筛选,cDNA代表差异分析以及mRNA差异显示等,但这些技术不能对基因进行全面系统的分析。新的技术应运而生,包括基因表达的系统分析,cDNA微阵列,DNA芯片等。鉴定基因功能最有效的方法是观察基因表达被阻断或增加后在细胞和整体水平所产生的表型变异,因此需要建立模式生物体。 功能基因组学
蛋白质结构预测和序列分析软件蛋白质数据库及蛋白质序列分析 第一节、蛋白质数据库介绍 一、蛋白质一级数据库 1、 SWISS-PROT 数据库 SWISS-PROT和PIR是国际上二个主要的蛋白质序列数据 库，目前这二个数据库在EMBL和GenBank数据库上均建 立了镜像 (mirror) 站点。 SWISS-PROT数据库包括了从EMBL翻译而来的蛋白质序 列，这些序列经过检验和注释。该数据库主要由日内瓦大 学医学生物化学系和欧洲生物信息学研究所(EBI)合作维 护。SWISS-PROT的序列数量呈直线增长。 2、TrEMBL数据库： SWISS-PROT的数据存在一个滞后问题，即 进行注释需要时间。一大批含有开放阅读 了解决这一问题，TrEMBL(Translated E 白质数据库，它包括了所有EMBL库中的 质序列数据源，但这势必导致其注释质量 3、PIR数据库： PIR数据库的数据最初是由美国国家生物医学研究基金 会（National Biomedical Research Foundation, NBRF） 收集的蛋白质序列，主要翻译自GenBank的DNA序列。 1988年，美国的NBRF、日本的JIPID（the Japanese International Protein Sequence Database日本国家蛋 白质信息数据库）、德国的MIPS（Munich Information Centre for Protein Sequences摹尼黑蛋白质序列信息 中心）合作，共同收集和维护PIR数据库。PIR根据注释 程度(质量)分为4个等级。 4、 ExPASy数据库： 目前，瑞士生物信息学研究所（Swiss I 质分析专家系统（Expert protein anal 据库。 网址：https://www.doczj.com/doc/7311389676.html, 我国的北京大学生物信息中心(www.cbi.
蛋白质预测分析网址集锦? 物理性质预测：? Compute PI/MW?? ?? SAPS?? 基于组成的蛋白质识别预测? AACompIdent???PROPSEARCH?? 二级结构和折叠类预测? nnpredict?? Predictprotein??? SSPRED?? 特殊结构或结构预测? COILS?? MacStripe?? 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。? 由NCBI检索蛋白质序列? 可联网到：“”进行检索。? 利用SRS系统从EMBL检索蛋白质序列? 联网到：”，可利用EMBL的SRS系统进行蛋白质序列的检索。? 通过EMAIL进行序列检索?
当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。? 蛋白质基本性质分析? 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的例子是在羧基端含有KDEL序列特征的蛋白质将被引向内质网。WEB中有很多此类资源用于帮助预测蛋白质的功能。? 疏水性分析? 位于ExPASy的ProtScale程序（?）可被用来计算蛋白质的疏水性图谱。该网站充许用户计算蛋白质的50余种不同属性，并为每一种氨基酸输出相应的分值。输入的数据可为蛋白质序列或SWISSPROT数据库的序列接受号。需要调整的只是计算窗口的大小（n）该参数用于估计每种氨基酸残基的平均显示尺度。? 进行蛋白质的亲/疏水性分析时，也可用一些windows下的软件如，bioedit,dnamana等。? 跨膜区分析? 有多种预测跨膜螺旋的方法，最简单的是直接，观察以20个氨基酸为单位的疏水性氨基酸残基的分布区域，但同时还有多种更加复杂的、精确的算法能够预测跨膜螺旋的具体位置和它们的膜向性。这些技术主要是基于对已知
蛋白质结构预测方法综述 卜东波陈翔王志勇 《计算机不能做什么？》是一本好书，其中文版序言也堪称佳构。在这篇十余页的短文中，马希文教授总结了使用计算机解决实际问题的三步曲，即首先进行形式化，将领域相关的实际问题抽象转化成一个数学问题；然后分析问题的可计算性；最后进行算法设计，分析算法的时间和空间复杂度，寻找最优算法。 蛋白质空间结构预测是很有生物学意义的问题，迄今亦有很多的工作。有意思的是，其中一些典型工作恰恰是上述三步曲的绝好示例，本文即沿着这一路线作一总结，介绍于后。 1 背景知识 生物细胞种有许多蛋白质（由20余种氨基酸所形成的长链），这些大分子对于完成生物功能是至关重要的。蛋白质的空间结构往往决定了其功能，因此，如何揭示蛋白质的结构是非常重要的工作。 生物学界常常将蛋白质的结构分为4个层次：一级结构，也就是组成蛋白质的氨基酸序列；二级结构，即骨架原子间的相互作用形成的局部结构，比如alpha螺旋,beta片层和loop区等；三级结构，即二级结构在更大范围内的堆积形成的空间结构；四级结构主要描述不同亚基之间的相互作用。 经过多年努力，结构测定的实验方法得到了很好的发展，比较常用的有核磁共振和X光晶体衍射两种。然而由于实验测定比较耗时和昂贵，对于某些不易结晶的蛋白质来说不适用。相比之下，测定蛋白质氨基酸序列则比较容易。因此如果能够从一级序列推断出空间结构则是非常有意义的工作。这也就是下面的蛋白质折叠问题： 1蛋白质折叠问题（Protein Folding Problem） 输入: 蛋白质的氨基酸序列
输出: 蛋白质的空间结构 蛋白质结构预测的可行性是有坚实依据的。因为一般而言，蛋白质的空间结构是由其一级结构确定的。生化实验表明：如果在体外无任何其他物质存在的条件下，使得蛋白质去折叠，然后复性，蛋白质将立刻重新折叠回原来的空间结构，整个过程在不到1秒种内即可完成。因此有理由认为对于大部分蛋白质而言，其空间结构信息已经完全蕴涵于氨基酸序列中。从物理学的角度讲，系统的稳定状态通常是能量最小的状态，这也是蛋白质预测工作的理论基础。 2 蛋白质结构预测方法 蛋白质结构预测的方法可以分为三种： 同源性（Homology ）方法：这类方法的理论依据是如果两个蛋白质的序列比较相似，则其结构也有很大可能比较相似。有工作表明，如果序列相似性高于75％，则可以使用这种方法进行粗略的预测。这类方法的优点是准确度高，缺点是只能处理和模板库中蛋白质序列相似性较高的情况。 从头计算（Ab initio ） 方法：这类方法的依据是热力学理论，即求蛋白质能量最小的状态。生物学家和物理学家等认为从原理上讲这是影响蛋白质结构的本质因素。然而由于巨大的计算量，这种方法并不实用，目前只能计算几个氨基酸形成的结构。IBM 开发的Blue Gene 超级计算机，就是要解决这个问题。 穿线法(Threading )方法：由于Ab Initio 方法目前只有理论上的意义，Homology 方法受限于待求蛋白质必需和已知模板库中某个蛋白质有较高的序列相似性，对于其他大部分蛋白质来说，有必要寻求新的方法。Threading 就此应运而生。 以上三种方法中，Ab Initio 方法不依赖于已知结构，其余两种则需要已知结构的协助。通常将蛋白质序列和其真实三级结构组织成模板库，待预测三级结构的蛋白质序列，则称之为查询序列(query sequence)。 3 蛋白质结构预测的Threading 方法 Threading 方法有三个代表性的工作：Eisenburg 基于环境串的工作、Xu Ying 的Prospetor 和Xu Jinbo 、Li Ming 的RAPTOR 。 Threading 的方法：首先取出一条模版和查询序列作序列比对(Alignment)，并将模版蛋白质与查询序列匹配上的残基的空间坐标赋给查询序列上相应的残基。比对的过程是在我们设计的一个能量函数指导下进行的。根据比对结果和得到的查询序列的空间坐标，通过我们设计的能量函数，得到一个能量值。将这个操作应用到所有的模版上，取能量值最低的那条模版产生的查询序列的空间坐标为我们的预测结果。 需要指出的是，此处的能量函数却不再是热力学意义上的能量函数。它实质上是概率的负对数，即 ，我们用统计意义上的能量来代替真实的分子能量，这两者有大致相同的形式。 p E log ?=如果沿着马希文教授的观点看上述工作 ，则更有意思：Eisenburg 指出如果仅仅停留在简单地使用每个原子的空间坐标(x,y,z)来形式化表示蛋白质空间结构，则难以进一步深入研究。Eisenburg 创造性地使用环境串表示结构，从而将结构预测问题转化成序列串和环境串之间的比对问题；其后，Xu Ying 作了进一步发展，将蛋白质序列表示成一系列核（core ）组成的序列，Core 和Core 之间存在相互作用。因此结构就表示成Core 的空间坐标，以及Core 之间的相互作用。在这种表示方法的基础上，Xu Ying 开发了一种求最优匹配的动态规划算法，得到了很好的结果。但是由于其较高的复杂度，在Prospetor2上不得不作了一些简化；Xu Jinbo 和Li Ming 很漂亮地解决了这个问题，将求最优匹配的过程表示成一个整数规划问题，并且证明了一些常用
【摘要】基因组相对较稳定，而且各种细胞或生物体的基因组结构有许多基本相似的特征；蛋白质组是动态的，随内外界刺激而变化。对蛋白质组的研究可以使我们更容易接近对生命过程的认识。蛋白质组学是在细胞的整体蛋白质水平上进行研究、从蛋白质整体活动的角度来认识生命活动规律的一门新学科，简要介绍功能基因组学和蛋白质组学的科学背景、概念及其应用。 【关键词】基因组；功能基因组学；蛋白质组学； 一、基因组及基因组学的概念 基因组(genome)一词系由德国汉堡大学H．威克勒教授于1920年首创，用以表示真核生物从其亲代所继承的单套染色体，或称染色体组。更准确地说，基因组是指生物的整套染色体所含有的全部DNA序列。由于在真核细胞的线粒体和植物的叶绿体中也发现存在遗传物质，因此又将线粒体或叶绿体所携带的遗传物质称为线粒体基因组或叶绿体基因组。原核生物基因组则包括细胞内的染色体和质粒DNA。此外非独立生命形态的病毒颗粒也携带遗传物质，称为病毒基因组。所有生命都具有指令其生长与发育，维持其结构与功能所必需的遗传信息，本书中将生物所具有的携带遗传信息的遗传物质总和称为基因组。[1] 基因组学(genomic)一词系由T．罗德里克(T．Roderick)于1986年首创，用于概括涉及基因组作图、测序和整个基因组功能分析的遗传学学科分支，并已用来命名一个学术刊物Genomics。基因组学是伴随人类基因组计划的实施而形成的一个全新的生命科学领域。[1] 基因组学与传统遗传学其他学科的差别在于，基因组学是在全基因组范围研究基因的结构、组成、功能及其进化，因而涉及大范围高通量收集和分析有关基因组DNA的序列组成，染色体分子水平的结构特征，全基因组的基因数目、功能和分类，基因组水平的基因表达与调控以及不同物种之间基因组的进化关系。基因组学的研究方法、技术和路线有许多不同于传统遗传学的特点，各相关领域的研究仍处于迅速发展和不断完善的过程中。 基因组学的主要工具和方法包括：生物信息学，遗传分析，基因表达测量和基因功能鉴定。 二、功能基因组学的概念及应用
蛋白质结构预测网址 物理性质预测： Compute PI/MW Peptidemass TGREASE SAPS 基于组成的蛋白质识别预测 AACompIdent PROPSEARCH 二级结构和折叠类预测 nnpredict Predictprotein SSPRED 特殊结构或结构预测 COILS MacStripe 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。 由NCBI检索蛋白质序列 可联网到：“”进行检索。 利用SRS系统从EMBL检索蛋白质序列 联网到：”，可利用EMBL的SRS系统进行蛋白质序列的检索。 通过EMAIL进行序列检索 当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。 蛋白质基本性质分析 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的例子是在羧基端含有KDEL序列特征的蛋白质将被引向内质网。WEB中有很多此类资源用于帮助预测蛋白质的功能。 疏水性分析 位于ExPASy的ProtScale程序（）可被用来计算蛋白质的疏水性图谱。该网站充许用户计算蛋白质的50余种不同属性，并为每一种氨基酸输出相应的分值。输入的数据可为蛋白质序列或SWISSPROT数据库的序列接受号。需要调整的只是计算窗口的大小（n）该参数用于估计每种氨基酸残基的平均显示尺度。 进行蛋白质的亲/疏水性分析时，也可用一些windows下的软件如， bioedit,dnamana等。 跨膜区分析 有多种预测跨膜螺旋的方法，最简单的是直接，观察以20个氨基酸为单位的疏水性氨基酸残基的分布区域，但同时还有多种更加复杂的、精确的算法能够预测跨膜螺旋的具体位置和它们的膜向性。这些技术主要是基于对已知跨膜螺旋的研究而得到的。自然存在的跨膜螺旋Tmbase 数据库，可通过匿名FTP获得()，参见表一
实习 5 ：蛋白质结构预测 学号20090***** 姓名****** 专业年级生命生技**** 实验时间2012.6.21 提交报告时间2012.6.21 实验目的： 1.学会使用GOR和HNN方法预测蛋白质二级结构 2.学会使用SWISS-MODEL进行蛋白质高级结构预测 实验内容： 1.分别用GOR和HNN方法预测蛋白质序列的二级结构，并对比异同性。 2.利用SWISS-MODEL进行蛋白质的三级结构预测，并对预测结果进行解释。 作业： 1. 搜索一条你感兴趣的蛋白质序列，分别用GOR和HNN进行二级结构预测，解释预测结果，分析两个方法结果有何异同。 答：所选用蛋白质序列为>>gi|390408302|gb|AFL70986.1| gag protein, partial [Human immunodeficiency virus] （1）GOR预测结果： 图1 图1是每个氨基酸在序列中所处的状态，可以看出序列的二级结构预测结果为： 1到9位个氨基酸为无规卷曲，10到33位氨基酸为α螺旋,34到37位为β折叠，38到45位为无规卷曲，46到49位为α螺旋，50到53位为无规卷曲，54到65为α螺旋，66到72位为无规卷曲，73到95位为α螺旋，96到101位为无规卷曲，102到108为β折叠，109到115位为无规卷曲，117位为β折叠。 图2 图2为各种结构在序列中所占的比例，其中Alpha helix占53.85%，Extended strand占11.11%，Random coil占35.04%，无他二级结构。
图3 图3为各个氨基酸在序列中的状态以及二级结构在全序列中二级结构分布情况。 （2）HNN预测： 图4 图4是每个氨基酸在序列中所处的状态，可以看出序列的二级结构预测结果为： 1到6位个氨基酸为无规卷曲，7到34位氨基酸为α螺旋,35到37位为β折叠，38位为α螺旋，39到44位为无规卷曲，45到49位为α螺旋，50到55位为无规卷曲，56到65为α螺旋，66到71位为无规卷曲，72到83位为α螺旋，84到86位为无规卷曲，87到95位为α螺旋，96到102为无规卷曲，103到108位为β折叠，108到117位为无规卷曲。 图5 图5为各种结构在序列中所占的比例，其中Alpha helix占55.56%，Extended strand占7.69%，Random coil占36.75%，无他二级结构。
第26卷第3期2006年3月生 态 学 报ACTA EC OLOGI CA SI NICA Vol .26,No .3Mar .,2006生态毒理基因组学和生态毒理蛋白质组学研究进展 戴家银,王建设 (中国科学院动物研究所,北京 100080) 基金项目:中国科学院知识创新工程重要方向性资助项目(KSCX2-SW -128) 收稿日期:2005-08-30;修订日期:2005-12-05 作者简介:戴家银(1965～),男,安徽怀宁人,博士,研究员,主要从事生态毒理学和生物化学研究.E -mail :daijy @ioz .ac .cn Foundation item :The project was supported by the Innovation Project of Chines e Academy of Sciences (No .KSCX2-SW -128) Received date :2005-08-30;Accepted date :2005-12-05 Biography :DAI Jia -Yin ,Ph .D .,Professor ,mainly engaged in ecotoxicology and biochemis try .E -mail :daijy @ioz .ac .cn 摘要:将基因组学和蛋白质组学知识整合到生态毒理学中形成了生态毒理基因组学和生态毒理蛋白质组学。通过生态毒理基因组学和生态毒理蛋白质组学的研究能够在基因组和蛋白质组水平更深入理解毒物的作用机制,寻找更敏感、有效的生物标记物,形成潜在的强有力的生态风险评价工具。介绍了生态毒理基因组学和生态毒理蛋白质组学的研究进展,以及DNA 芯片技术和2D -凝胶电泳技术在持久性有毒污染物的生态毒理学研究中的应用。 关键词:生态毒理基因组学;生态毒理蛋白质组学;DNA 芯片技术;2D -凝胶电泳;持久性有机污染物 文章编号:1000-0933(2006)03-0930-05 中图分类号:X171 文献标识码:A Progress in ecotoxicogenomics and ecotoxicoproteomics DAI Jia -Yin ,WANG Jian -She (Institut e of Zoology ,C hines e Acade my of Sci ence s ,Beijing 100080,C hina )..Acta Ecologica Sinica ,2006,26(3): 930～934.Abstract :Ec otoxicogeno mics and ecotoxic oproteo mics are integration of genomics and proteomics into ec otoxicology .Ecotoxic ogenomics is defined as the study of gene and pr otein expr ession in non -target organisms that is impor tant in responses to environmental toxicant exposures .Ecotoxic ogenomic toolsmay provide us with a better mechanistic understanding of ec otoxicology ,and they are likely to provide a vital r ole in ecological risk assessment .Pr ogress in ec otoxicogenomics and ecotoxicoprote omics are discussed in this paper .DNA gene c hip and 2D -gel usually used in ecotoxicogeno mics and ecotoxicoproteomics ar e also e xpounded by exa mples . Key words :ec otoxicogeno mics ;ecotoxic oproteo mics ;D NA micr oarra y ;2D -gel ;persistent organic pollutants 随着生态学和环境科学的深入发展,生态毒理学已成为生态学和环境科学前沿研究领域,正从基因、蛋白质、器官和整体水平深入开展研究工作。 在人类基因组计划实施的短短几年间,以“组学(-omics )”构成的学科及其相关研究如雨后春笋般在生命科学界迅速蔓延、蓬勃发展。在环境科学领域中也出现了环境基因组学(environmental genomics )、毒理基因组学(toxicogenomics )等学科。Snape 等人[1～3]将基因组学知识整合到生态毒理学中,于2004年提出了“生态毒理基因组学(ecotoxicogenomics )”的概念,通过生态毒理基因组学研究确认一系列毒物效应基因,从而在基因组水平更深入理解毒物的作用机制,并在基因和蛋白质水平寻找更敏感、有效的生物标记物(biomarkers ),形成潜在的强有力的生态风险评价工具。 持久性有机污染物(Persistent Organic Pollutants ,POPs )是指能持久存在于环境中、通过食物链蓄积、逐级传递,经直接或间接途径进入人体的化学物质。POPs 具有致癌、致畸、致突变性、内分泌干扰等毒作用。POPs 对人体健康和生态环境带来的危害受到全社会的普遍关注,引起世界各国的决策者和科学家的高度重视,也成为环境科学和生态毒理学研究的热点课题之一[4,5]。我国已于2001年5月签署了控制12种P OPs 对人类健康
蛋白质组学 蛋白质是生物体的重要组成部分，参与几乎所有生理和细胞代谢过程。此外，与基因组学和转录组学比较，对一个细胞或组织中表达的所有蛋白质，及其修饰和相互作用的大规模研究称为蛋白质组学。 蛋白质组学通常被认为是在基因组学和转录组学之后，生物系统研究的下一步。然而，蛋白质组的研究远比基因组学复杂，这是由于蛋白质内在的复杂特点，如蛋白质各种各样的翻译后修饰所决定的。并且，研究基因组学的技术要比研究蛋白质组学的技术强得多，虽然在蛋白质组学研究中，质谱技术的研究已取得了一些进展。 尽管存在方法上的挑战，蛋白质组学正在迅速发展，并且对癌症的临床诊断和疾病治疗做出了重要贡献。几项研究鉴定出了一些蛋白质在乳腺癌、卵巢癌、前列腺癌和食道癌中表达变化。例如，通过蛋白质组学技术，人们可以在患者血液中明确鉴定出肿瘤标志物。表1列出了更多的蛋白质组学技术用于研究癌症的例子。 另外，高尔基体功能复杂。最新研究表明，它除了参与蛋白加工外，还能参与细胞分化及细胞间信号传导的过程，并在凋亡中扮演重要角色，其功能障碍也许和肿瘤的发生、发展有某种联系。根据人类基因组研究，约1000多种人类高尔基体蛋白质中仅有500~600种得到了鉴定，建立一条关于高尔基体蛋白质组成的技术路线将有助于其功能的深入研究。 蛋白质组学是一种有效的研究方法，特别是随着亚细胞器蛋白质组学技术的迅猛发展，使高尔基体的全面研究变为可能。因此研究人员希望能以胃癌细胞中的高尔基体为研究对象，通过亚细胞器蛋白质组学方法，建立胃癌细胞中高尔基体的蛋白质组方法学。 研究人员采用蔗糖密度梯度的超速离心方法分离纯化高尔基体，双向凝胶电泳（2-DE）分离高尔基体蛋白质，用ImageMaster 2D软件分析所得图谱，基质辅助激光解吸离子化飞行时间质谱（MALDI-TOF MS）鉴定蛋白质点等一系列亚细胞器蛋白质组学方法建立了胃癌细胞内高尔基体的蛋白图谱。 最后，人们根据分离出的纯度较高的高尔基体建立了分辨率和重复性均较好的双向电泳图谱，运用质谱技术鉴定出12个蛋白质，包括蛋白合成相关蛋白、膜融合蛋白、调节蛋白、凋亡相关蛋白、运输蛋白和细胞增殖分化相关蛋白。通过亚细胞器分离纯化、双向电泳的蛋白分离及MALDI-TOF MS蛋白鉴定分析，研究人员首次成功建立了胃癌细胞SGC7901中高尔基体的蛋白质组学技术路线。 3.1 蛋白质功能预测工具 也许生物信息学方法在癌症研究中最常用的就是基因功能预测方法，但是这些数据库只存储了基因组的大约一半基因的功能。为了在微阵列资料基础上完成功能性的富集分析，基因簇的功能注解是非常重要的。近几年生物学家研发了一些基因功能预测的方法，这些方法旨在超越传统的BLAST搜索来预测基因的功能。基因功能预测可以以氨基酸序列、三级结构、与之相互作用的配体、相互作用过程或基因的表达方式为基础。其中最重要的是基于氨基酸序列的分析，因为这种方法适合于微阵列分析的全部基因。 在表3中，前三项列举了三种同源搜索方法。FASTA方法虽然应用还不太广泛，但它要优于BLAST，或者至少相当。FASTA程序是第一个使用的数据库相似性搜索程序。为了达到较高的敏感程度，程序引用取代矩阵实行局部比对以获得最佳搜索。美国弗吉尼亚大学可以提供这项程序的地方版本，当然数据库搜索结果依赖于要搜索的数据库序列。如果最近的序列数据库版本在弗吉尼亚大学不能获得，那么就最好试一下京都大学（Kyoto University）的KEGG站点。PSI-BLAST（位点特异性反复BLAST）是BLAST的转化版本，PSI-BLAST的特色是每次用profile 搜索数据库后再利用搜索的结果重新构建profile，然后用新的profile再次搜索数据库，如此反复直至没有新的结果产生为止。PSI-BLAST先用带空位的BLAST搜索数据库，将获得的序列通过多序列比对来构建第一个profile。PSI-BLAST自然地拓展了BLAST方法，能寻找蛋白质序列中的隐含模式，有研究表明这种方法可以有效地找到很多序列差异较大而结构功能相似的相关蛋白，所以它比BLAST和FASTA有更好的敏感性。PSI-BLAST服务可以
实验名称：蛋白质结构与功能的生物信息学研究 实验目的：1.掌握运用BLAST工具对指定蛋白质的氨基酸序列同源性搜索的方法。 2.掌握用不同的工具分析蛋白质的氨基酸序列的基本性质 3掌握蛋白质的氨基酸序列进行三维结构的分析 4.熟悉对蛋白质的氨基酸序列所代表蛋白的修饰情况、所参与的 代谢途径、相互作用的蛋白，以及与疾病的相关性的分析。实验方法和流程： 一、同源性搜索 同源性从分子水平讲则是指两个核酸分子的核苷酸序列或两个蛋白质分子的氨基酸序列间的相似程度。BLAST工具能对生物不同蛋白质的氨基酸序列或不同的基因的DNA序列极性比对，并从相应数据库中找到相同或相似序列。对指定的蛋白质的氨基酸序列进行同源性搜索步骤如下： ↓ 登录网址https://www.doczj.com/doc/7311389676.html,/blast/ ↓ 输入序列后,运行blast工具 ↓ 序列比对的图形结果显示
序列比对的图形结果：用相似性区段（Hit）覆盖输入序列的范围判断两个序列 的相似性。如果图形中包含低得分的颜色（主要是红色） 区段，表明两序列的并非完全匹配。 ↓ 匹配序列列表及得分
各序列得分 可选择不同的比对工具 备注: Clustal是一款用来对()的软件。可以用来发现特征序列，进行蛋白分类，证明序列间的同源性，帮助预测新序列二级结构与三级结构，确定PCR引物，以及 在分子进化分析方面均有很大帮助。Clustal包括Clustalx和Clustalw(前者是 图形化界面版本后者是命令界面)，是生物信息学常用的多序列比对工具。 该序列的比对结果有100条，按得分降序排列，其中最大得分2373，最小得分 分为1195. ↓ 详细的比对序列的排列情况 第一个匹配 序列 第一个序列的匹配率为100% Score表示打分矩阵计算出来的值，由搜索算法决定的，值越大说明匹配程度
蛋白质结构与功能的关系 专业：植物学 摘要：蛋白质特定的功能都是由其特定的构象所决定的，各种蛋白质特定的构象又与其一级结构密切相关。天然蛋白质的构象一旦发生变化，必然会影响到它的生物活性。由于蛋白质的构象的变化引起蛋白质功能变化，可能导致蛋白质构象紊乱症，当然也能引起生物体对环境的适应性增强。而分子模拟技术为蛋白质的研究提供了一种崭新的手段。在理论上解决了结构预测和功能分析以及蛋白质工程实施方面所面临的难题。它在蛋白质的结构预测和模建工作中占有举足轻重的地位，实现了生物技术与计算机技术的完美结合。 关键词：蛋白质的结构、功能；折叠/功能关系；蛋白质构象紊乱症；分子模拟技术；同源建模 RNase是由124个氨基酸残基组成的单肽链，分子中 8 个Cys的-SH构成4对二硫键，形成具有一定空间构象的蛋白质分子。在蛋白质变性剂和一些还原剂存在下，酶分子中的二硫键全部被还原，酶的空间结构破坏，肽链完全伸展，酶的催化活性完全丧失。当用透析的方法除去变性剂和巯基乙醇后，发现酶大部分活性恢复，所有的二硫键准确无误地恢复原来状态。若用其他的方法改变分子中二硫键的配对方式，酶完全丧失活性。这个实验表明，蛋白质的一级结构决定它的空间结构，而特定的空间结构是蛋白质具有生物活性的保证。前体与活性蛋白质一级结构的关系，由108个氨基酸残基构成的前胰岛素原，在合成的时候完全没有活性，当切去N-端的24个氨基酸信号肽，形成84个氨基酸的胰岛素原，胰岛素原也没活性，在包装分泌时，A、B链之间的33个氨基酸残基被切除，才形成具有活性的胰岛素。 功能不同的蛋白质总是有着不同的序列；种属来源不同而功能相同的蛋白质的一级结构，可能有某些差异，但与功能相关的结构也总是相同。若一级结构变化，蛋白质的功能可能发生很大的变化。蛋白质特定的功能都是由其特定的构象所决定的，各种蛋白质特定的构象又与其一级结构密切相关。天然蛋白质的构象一旦发生变化，必然会影响到它的生物活性。由于蛋白质的构象的变化引起蛋白质功能变化，可能导致蛋白质构象紊乱症，当然也能引起生物体对环境的适应性增强。 虽然蛋白质结构与生物功能的关系比序列与功能的关系更加紧密，但结构与功能的这种关联亦若隐若现，并不能排除折叠差别悬殊的蛋白质执行相似的功能，折叠相似的蛋白质执行差别悬殊功能的现象的存在。无奈，该领域仍不得不将100多年前Fisher提出的“锁一钥
基因组学与蛋白质组学之间的关系 1 基因组学概述 基因组学，研究生物基因组和如何利用基因的一门学问。用于概括涉及基因作图、测序和整个基因组功能分析的遗传学分支。该学科提供基因组信息以及相关数据系统利用，试图解决生物，医学，和工业领域的重大问题。 基因组研究应该包括两方面的内容：以全基因组测序为目标的结构基因组学）和以基因功能鉴定为目标的功能基因组学，又被称为后基因组研究，成为系统生物学的重要方法。 基因组学能为一些疾病提供新的诊断，治疗方法。例如，对刚诊断为乳腺癌的女性，一个名为“Oncotype DX”的基因组测试，能用来评估病人乳腺癌复发的个体危险率以及化疗效果，这有助于医生获得更多的治疗信息并进行个性化医疗。基因组学还被用于食品与农业部门。 基因组学的主要工具和方法包括：生物信息学，遗传分析，基因表达测量和基因功能鉴定。 2 蛋白质组学概述 蛋白质组学（Proteomics）一词，源于蛋白质（protein）与基因组学（genomics）两个词的组合，意指“一种基因组所表达的全套蛋白质”，即包括一种细胞乃至一种生物所表达的全部蛋白质。蛋白质组本质上指的是在大规模水平上研究蛋白质的特征，包括蛋白质的表达水平，翻译后的修饰，蛋白与蛋白相互作用等，由此获得蛋白质水平上的关于疾病发生，细胞代谢等过程的整体而全面的认识，这个概念最早是由Marc Wilkins 在1995年提出的。 3 两者之间的关系 90年代初期开始实施的人类基因组计划，在经过各国科学家近10年的努力下，已经取得了巨大的成就。不仅完成了十余种模式生物（从大肠杆菌、酿酒酵母到线虫）基因组全序列的测定工作，还有望在2003年提前完成人类所有基因的全序列测定。那么，知道了人类的全部遗传密码即基因组序列，就可以任意控制人的生老病死吗？其实并不是这么简单。基因组学虽然在基因活性和疾病的相关性方面为人类提供了有力根据,但实际上大部分疾病并不是因为基因改变所造成。并且，基因的表达方式错综复杂，同样的一个基因在不同条件、不同时期可能会起到完全不同的作用。关于这些方面的问题，基因组学是无法回答的。所以，随着人类基因组计划的逐步完成，科学家们又进一步提出了后基因组计划，蛋白质组研究是其中一个很重要的内容。 目前，在蛋白质功能方面的研究是极其缺乏的。大部分通过基因组测序而新发现的基因编码的蛋白质的功能都是未知的，而对那些已知功能的蛋白而言，它们的功能也大多是通过同源基因功能类推等方法推测出来的。有人预测，人类基因组编码的蛋
蛋白质结构与功能的关系 （The relationship between protein structure and function） 摘要蛋白质特定的功能都是由其特定的构象所决定的，各种蛋白质特定的构象又与其一级结构密切相关。天然蛋白质的构象一旦发生变化，必然会影响到它的生物活性。由于蛋白质的构象的变化引起蛋白质功能变化，可能导致蛋白质构象紊乱症，当然也能引起生物体对环境的适应性增强！现而今关于蛋白质功能研究还有待发展，一门新兴学科正在发展，血清蛋白组学，生物信息学等！本文仅就蛋白质结构与其功能关系进行粗略阐述。 关键词：蛋白质结构；折叠/功能关系；蛋白质构象紊乱症；分子伴侣 Keywords：protein structure；fold／function relationship；protein conformational disorder；molecular chaperons 虽然蛋白质结构与生物功能的关系比序列与功能的关系更加紧密，但结构与功能的这种关联亦若隐若现，并不能排除折叠差别悬殊的蛋白质执行相似的功能，折叠相似的蛋白质执行差别悬殊功能的现象的存在。无奈，该领域仍不得不将100多年前Fisher提出的“锁一钥匙”模型(“lock—key”model)和50多年前Koshand提出的诱导契合模型(induce fitmodel)作为蛋白质实现功能的理论基础。这2个略显粗糙的模型只是认为蛋白质执行功能的部位局限在结构中的一个或几个小区域内，此类区域通常是蛋白质表面上的凹洞或裂隙。这种凹洞或裂隙被称为“活性部位(active site)”或“别构部位（fallosteric site）”，凹陷部位与配体分子在空间形状和静电上互补。此外，在酶的活性部位中还存在着几个作为催化基团(catalyticgroup)的氨基酸残基。对蛋白质未来的研究应从实验基本数据的归纳和统计入手，从原始的水平上发现蛋白质的潜藏机制【1】。 蛋白质结构与功能关系的研究主要是以力求刻画蛋白质的3D结构的几何学为基础的。蛋白质结构既非规则的几何形，又非完全的无规线团(randomcoil)，而是有序(α一螺旋和β一折叠)与无序(线团或环域loop)的混合体。理解蛋白质3D结构的技巧是将结构简化，只保留某种几何特征或拓扑模式，并将其数字化。探求数字中所蕴含的规律，且根据这一规律将蛋白质进行分类，再将分类的结构与蛋白质的功能进行比较，以检验蛋白质抽象结构的合理性。如果一种对蛋白质结构的简化、比较和分类能与蛋自质的功能有较好地对应关系，那么这就是一种对蛋白质结构的有价值的理解。蛋白质结构中，多种弱力(氢键、范德华力、静电相互作用、疏水相互作用、堆积力等)和可逆的二硫键使多肽链折叠成特定的构象。从某种意义上说，共价键维系了蛋白质的一级结构；主链上的氢键维系了蛋白质的二级结构；而氨基酸侧链的相互作用和二硫桥维系着蛋白质的三级结构。亚基(subunit)内部的侧链相互作用是构象稳定的基础，蛋白质链之间的侧链的相互作用是亚基组装(四级结构)的基础，而蛋白质中侧链与配体基团问的相互作用是蛋白质行使功能的基础。 牛胰核糖核酸酶(RNase)变性和复性的实验是蛋白质结构与功能关系的很好例证。蛋白质空间结构遭到破坏；，可导致蛋白质的理比性质和生物学性质的变化，这就是蛋白质变性。变性的蛋白质，只要其一级结构仍然完好，可在一定条件下恢复其空间结构，随之理化性质和生物学性质也可重现，这被称为复性。RNase是由124个氨基酸残基组成的一条肽链，分子中8个半胱氨酸的巯基构成4对二硫键，进而形成具有一定空间构象的活性蛋白质。天然RNase遇尿素和β巯基乙醇时发生变性，其分子中的氢键和4个二硫键解开，严密的空间结构遭破坏，丧失了生物学活性，但一级结构完整无损。若去除尿素和β巯基乙醇，RNase又可恢复其原有构象和生物学活性。RNase分子中的8个巯基若随机排列成二硫键可有105种方式。有活性的RNase只是其中的一种，复性时之所以选择了自