Microreview
Protein secretion systems in Mycobacteria
Patricia A.DiGiuseppe Champion*and Jeffery S.Cox
Department of Microbiology and Immunology,Program in Microbial Pathogenesis and Host Defense,University of California,San Francisco,60016th Street,Campus Box 2200,San Francisco,CA 94143-2200,USA.Summary
Mycobacteria have a unique cell-envelope structure which protects the bacteria from the extracellular environment by limiting access to noxious molecules from the outside.This extremely hydrophobic and thick barrier also poses a unique problem for the export of bacterial products.Here we review the mul-tiple protein secretion pathways in Mycobacteria,including the general secretion pathway and the Twin-Arginine Transporter,with an emphasis on the ESX-1alternate secretion system.This newly identi?ed protein secretion system is required for growth during infection and has provided insight into how M.tuberculosis manipulates the host immune response during infection.Introduction
Mycobacteria have a unique cell-envelope structure which insulates the bacteria from the extracellular environment.This envelope includes the inner plasma membrane and a unique cell wall composed of dual polymer layers (peptidoglycan and arabinogalactan)sur-rounded by a lipid-rich mycolate layer (Brennan and Nikaido,1995).In the case of mycobacterial pathogens the cell wall plays a critical role in protecting the bacteria from the physical assaults mediated by the mammalian immune system.However,while limiting access to noxious molecules from the outside,this extremely hydro-phobic and thick barrier also poses a unique problem for the export of bacterial products.Here we review the mul-tiple protein secretion pathways in Mycobacteria with an emphasis on systems important for virulence.We high-light a newly identi?ed protein secretion system required
for growth during infection that has provided insight into how M.tuberculosis manipulates the host immune response during infection.The general secretion pathway
Like all other bacteria,Mycobacteria have an essential general secretion pathway (GSP ,or the Sec secretion system),which functions to secrete unfolded proteins with N-terminal signal sequences across the cytosolic membrane.The Sec system consists of the SecY ,SecE,SecG,SecD and SecF membrane components and the SecA ATPase,which recognizes the signal sequence.As expected,all indications are that this highly conserved system functions in Mycobacteria analogously to that of other bacteria.Mycobacteria,like Gram-positive bacteria,appear to lack the SecB chaperone and thus may use other cytosolic chaperones to escort proteins in their unfolded state from the ribosome to SecA (see Scott and Barnett,2006).In Gram-negative bacteria,additional pathways are used to direct proteins across the outer membrane,including the Omp85/YaeT complex (see Gentle et al .,2005).However,in Mycobacteria,it is unknown how GSP-dependent substrates get across the mycolylarabinogalactan (mAGP)layer.It seems likely that comparable systems exist in Mycobacteria but this area requires further study.
As in other prokaryotes,the Mycobacterial Sec signal sequence contains three domains including a positively charged N-terminus,followed by a hydrophobic region,and an uncharged polar region.In Gram-negative bacte-ria,signal sequences are approximately 20amino acids long,while in Gram-positive bacteria they can be up to 60amino acids long.Mycobacterial signal sequences resemble those of Gram-positive bacteria,although the functional consequence of this is unclear (Wiker et al .,2000).Interestingly,most proteins secreted from the M.tuberculosis cell contain an aspartic acid-proline sequence at the N-terminus (the DP motif)that is revealed after signal sequence cleavage.Wiker et al .suggest that this motif may be recognized by a second system for translocation across the cell wall.This raises a tantalizing model in which bipartite signal sequences dictate a protein’s ?nal localization,analogous to mitochondrial import signals.However,this notion remains untested.
Received 6March,2007;revised 19March,2007;accepted 26March,2007.*For correspondence.E-mail Patricia.Champion@https://www.doczj.com/doc/2f17375769.html,;Tel.(+1)4154765293;Fax (+1)4155024315.Cellular Microbiology (2007)9(6),1376–1384
doi:10.1111/j.1462-5822.2007.00943.x First published online 25April 2007
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In stark contrast to most Gram-negative bacteria,all sequenced Mycobacterial genomes encode for a second, non-essential homologue of SecA,termed SecA2(Braun-stein et al.,2001;McDonough et al.,2005).An emerging number of Gram-positive bacteria have been found to contain both SecA1(the SecA homologue with the highest sequence similarity to that of E.coli)and SecA2,including the human pathogen Listeria monocytogenes(Braunstein et al.,2001;Lenz and Portnoy,2002).In M.tuberculosis, SecA2is only~50%similar and~38%identical to SecA1, suggesting that it is not simply a duplicate allele(Braun-stein et al.,2001).In Mycobacterium smegmatis,the secA2mutant has a growth defect on rich media,sug-gesting that its removal may have pleiotropic effects on cell?tness(Braunstein et al.,2001).The M.tuberculosis mutant grows normally in minimal liquid media but poorly during infections of macrophages and mice(Braunstein et al.,2003;Kurtz et al.,2006).It is tempting to speculate that the SecA2requirement during infection re?ects a speci?c interaction of SecA2-dependent substrates with host cells.However,the speci?c role of SecA2in patho-genesis is unclear.
What are the possible ways that the SecA2pathway could promote virulence?Clues from two other patho-gens with SecA2raise interesting possibilities.First,in L.monocytogenes,SecA2functions as an accessory SecA to secrete enzymes that remodel the peptidoglycan layer(Lenz et al.,2003).Breakdown products of digested peptidoglycan catalysed by these enzymes are recog-nized by host cells and modify the innate immune response to create an environment favouring bacterial colonization(Lenz et al.,2003).Interestingly,the secA2 mutant of M.tuberculosis elicits a more pronounced in?ammatory immune response in infected macrophages, consistent with this hypothesis(Kurtz et al.,2006). Second,the SecA2of Streptococcus gordonii functions to accomodate the secretion of a highly glycosylated adhe-sion molecule,GspB(Bensing and Sullam,2002;Bensing et al.,2004;2005).Thus,the evolution of SecA2may re?ect structural constraints of certain substrates,and perhaps is suited to preferentially translocate proteins that cannot be accommodated by SecA1,such as those with extensive post-translational modi?cations.Ultimately, understanding the speci?c effects of this secretion pathway in M.tuberculosis pathogenesis will require iden-ti?cation of the substrates secreted by SecA2.
The twin-arginine transporter
Mycobacteria also use a twin-arginine transporter(Tat) pathway,a Sec-independent secretion machine that has the unique ability to translocate folded protein substrates across the plasma membrane.Substrates are targeted to the Tat machine by a signal sequence with the same overall structure of the Sec signal sequence,but include a double arginine motif followed by two uncharged residues near the N-terminus(Berks,1996).Tat is best understood in E.coli,but is also present and functional in Gram-positive bacteria,and is required for the virulence of many pathogens including Pseudomonas aeruginosa,Agro-bacterium tumefaciens,enteropathogenic E.coli and Legionella pneumophila(Voulhoux et al.,2001;Ding and Christie,2003;Pradel et al.,2003;Rossier and Cianci-otto,2005)(for a recent review see Lee et al.,2006).The Mycobacterial Tat system is homologous to Tat systems in other bacteria,and has been shown to be functional in both M.tuberculosis and M.smegmatis(McDonough et al.,2005;Posey et al.,2006).In M.smegmatis,dele-tion of any of the three main tat genes yields colonies that grow slowly on solid media.However,in liquid media,the mutant cells have no apparent growth defect(McDonough et al.,2005).In M.tuberculosis,the orthologous genes appear to be essential for growth in culture,precluding phenotypic analysis of null alleles in the pathway(Saint-Joanis et al.,2006).
Predictions using various algorithms have estimated from11to31potential Tat substrates encoded by the M.tuberculosis genome(Cole,2002;Dilks et al.,2003). Two classes of substrates have received attention and suggest that this pathway may be used to interact directly with the host.First,four phospholipase C enzymes are secreted by Tat and,together,are required for full viru-lence in the mouse model of M.tuberculosis(Raynaud et al.,2002;McDonough et al.,2005).Although their role in pathogenesis is not entirely clear,two compelling hypotheses are that they act on host membranes either to alter host signalling pathways or to release lipids making them available for the bacterium to catabolize(Munoz-Elias and McKinney,2006).Second,the Rv2525c protein was recently shown to be secreted by Tat and a Rv2525c mutant displayed a hyper-virulent phenotype during infec-tion of immuno-de?cient mice(Saint-Joanis et al.,2006). The mechanism of Rv2525c is not understood and homol-ogy searches of the primary amino acid sequence have not revealed any putative functional domains.However, using the Phyre secondary structure matching program (Kelley et al.,2000),we?nd that this protein likely has structural homology to the transglycosidase protein superfamily,enzymes involved in peptidoglycan syn-thesis.This is seemingly consistent with the observation that the mutant is slightly more sensitive to certain beta-lactam antibiotics and suggests yet another possible link between peptidoglycan metabolism and virulence.
The ESAT-6secretion system
Over the past decade of bacterial pathogenesis research, the alternative secretion systems of Gram-negative
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bacteria have generated a remarkable amount of attention.In addition to Sec and Tat,Gram-negative pathogens use a variety of secretion systems(Types I-V), which function to deliver bacterial proteins into the host cell and manipulate the host response to infection(for recent reviews on alternate secretion see Backert and Meyer,2006;Galan and Wolf-Watz,2006).Substrates of the individual systems differ between pathogens but the secretory systems themselves are mostly conserved. Although homologues of these systems are absent from the M.tuberculosis genome,the likely functional equiva-lent of these systems,termed ESX-1,has recently been identi?ed and has been a burgeoning focus in the Myco-bacterial?eld.
Initial clues that a specialized secretion system exists in M.tuberculosis came from studies that identi?ed secreted proteins that lack obvious Sec signal sequences (Sorensen et al.,1995).Most notably,ESAT-6(Early secreted antigen target6kDaA)and CFP-10(Culture ?ltrate protein,10kDa)are two secreted proteins of unknown function originally identi?ed as immunodominant antigens of M.tuberculosis.Since their identi?cation, several studies suggested that these proteins are impor-tant for virulence.Deletion of the genes encoding ESAT-6 and CFP-10from the virulent Mycobacterium bovis strain results in a diminution of virulence(Wards et al.,2000). Furthermore,all strains of the attenuated vaccine strains of bacillus Calmette–Guerin(BCG)have deletions encompassing the ESX-1locus,also known as the RD1region(Mahairas et al.,1996).Importantly,deletion of RD1from M.tuberculosis attenuates the organism and,conversely,incorporation of the RD1region from M.tuberculosis into BCG restores ESAT-6and CFP-10 expression and increases virulence and immunogenicity (Pym et al.,2002;Lewis et al.,2003;Pym et al.,2003).
Components of the ESX-1secretion system
In silico analysis led to the suggestion that the genes surrounding esxBA,the operon which encodes for CFP-10and ESAT-6,may be important for secretion of these proteins(Gey van Pittius et al.,2001;2002;Pallen, 2002).Importantly,this notion was proven to be true when individual genes at the RD1locus were identi?ed in genetic screens to de?ne virulence genes of M.tuberculosis(Hsu et al.,2003;Sassetti et al.,2003; Stanley et al.,2003;Guinn et al.,2004).Disruption of individual genes(Rv3870,Rv3871and Rv3877)within this locus prevented secretion of ESAT-6and CFP-10, providing the?rst genetic evidence that this region encodes for a secretion system(Stanley et al.,2003; Guinn et al.,2004).Rv3870,Rv3871or Rv3877mutant strains are phenotypically indistinguishable from RD1 deletion strains;they are attenuated for growth in mac-rophages and elicit an altered immune response during macrophage infection(Stanley et al.,2003).
The ESX-1locus is conserved in several pathogenic and non-pathogenic Mycobacterial species,allowing for more rapid progress to be made than in the slow-growing and highly infectious https://www.doczj.com/doc/2f17375769.html,ing Mycobacte-rium marinum,Rv3868,Rv3878and Rv3879were shown to be required for ESAT-6/CFP-10secretion(Gao et al., 2004).In M.smegmatis,the homologues of Rv3866, Rv3869,Rv3882c and MycP1are also required for ESAT-6/CFP-10export(Converse and Cox,2005)(see Fig.1).
Finally,using M.bovis BCG and Mycobacterium microti strains complemented with an‘extended RD1’region from M.tuberculosis,each gene was systemically disrupted and monitored for changes in ESAT-6/CFP-10secretion (Brodin et al.,2006).Deleting Rv3872(PE35)resulted in no expression of ESAT-6and CFP-10.As found previ-ously,Rv3868through Rv3871,and Rv3877were required for secretion of ESAT-6/CFP-10,although not for their expression.Strains missing Rv3864,Rv3867, Rv3873(ppe68)and Rv3876had no virulence or secre-tion defects whereas disruptions in Rv3865and Rv3866 resulted in attenuation but did not affect ESAT-6/CFP-10 secretion.In contrast to M.smegmatis and M.marinum, Rv3866,Rv3878and Rv3879were not required for ESX-1secretion in these studies.Therefore,although there is some disagreement between these studies,it is clear that most of the genes at the RD1locus are required for ESX-1secretion.
In addition to RD1,another genetic other locus has recently been identi?ed that is required for ESAT-6/ CFP-10secretion in M.tuberculosis(MacGurn et al., 2005;Sassetti and Rubin,2003).Strains bearing disrup-tions in Rv3616c-Rv1614c are attenuated for growth in the mouse lung and spleen.Rv3616c(EspA)and Rv3615c were found to be secreted into the culture?ltrate in an ESX-1-dependent manner(Fortune et al.,2005;J.A. MacGurn and J.S.Cox,unpubl.obs.).Rv3614c is also required for secretion of ESAT-6and CFP-10(MacGurn et al.,2005).This operon likely arose from a gene dupli-cation event originating from the RD1locus as Rv3616c-Rv1614c are homologous to Rv3864,Rv3865and Rv3866.
Clearly,there are many genes,identi?ed in a number of distinct mycobacterial species,which are required for the export of ESAT-6,CFP-10and other small proteins from Mycobacteria.Some of these genes encode for proteins with readily predictable function.These include the ATPases Rv3868,Rv3870and Rv3871,which likely provide the energy required for translocation.MycP1is a putative transmembrane serine protease whose active site is predicted to be extracytoplasmic.Finally,there are a number of transmembrane proteins without obvious
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functional domains.Although the role of these proteins is unknown,these ?ndings suggest that the ESX-1system is very complex and requires multiple protein complexes for its proper functioning.However,a complete catalogue of the required ESX-1components is likely still not complete.Molecular mechanisms of ESX-1secretion
How do the large number of components required for ESX-1secretion function together to target and translo-cate substrates across the cytosolic membrane and the cell wall?Studies on how the components and substrates interact have provided insight into the molecular mecha-nisms of the ESX-1system.First,ESAT-6and CFP-10interact to form a tight dimer (Renshaw et al .,2002;Stanley et al .,2003),and in the mycobacterial cell,these two proteins are interdependent on each other for stability.Second,yeast two-hybrid experiments revealed that Rv3870interacts with Rv3871,a cytosolic protein,and together these two proteins are thought to function as an AAA ATPase of the SpoIIIE/FtsK family.Rv3871also interacts with CFP-10,and was hypothesized to escort CFP-10and ESAT-6to Rv3870and Rv3877,a multitrans-membrane protein,which may make up the pore that spans the cytosolic membrane (Stanley et al .,2003;Champion et al .,2006).Therefore,it is likely that Rv3871functions to recognize the CFP-10/ESAT-6substrate pair,and deliver it in an ATP-dependent manner to Rv3870which is at the membrane (Fig.1).
The interaction of Rv3871with CFP-10was exploited to identify a C-terminal signal sequence on CFP-10,which is necessary for targeting both CFP-10and ESAT-6for secretion (Champion et al .,2006).In fact,single amino acid changes at the extreme C-terminus of CFP-10blocks the secretion of both proteins.This seven amino acid signal sequence is also sufficient for targeting an unre-lated protein,ubiquitin,for secretion (Champion et al .,2006).Interestingly,the solution structure of the ESAT-6/CFP-10pair revealed that the C-terminal 15amino acids of CFP-10is unstructured and does not participate in interactions with ESAT-6(Renshaw et al .,2005).Thus,the C-terminal signal sequence is unstructured,
and
Fig.1.A model for ESX-1mediated secretion in Mycobacteria.All known components of the ESX-1system from a number of Mycobacteria are shown here.Those discovered in M.tuberculosis are annotated with the Rv number.Those from M.marinum are designated ‘Mh’for
Marinum https://www.doczj.com/doc/2f17375769.html,ponents identi?ed in M.smegmatis are noted with ‘Sm’.Proteins of unknown function are in light blue.Putative AAA ATPases are in orange.The three known substrates are in Red.SmMycP1is a putative mycosin-like protease,Rv3866has potential DNA binding domains,and Rv3872is a PE related protein.Although no components outside of the cytoplasm are known to date,we have indicated potential components with a question mark.
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resembles a handle by which Rv3871can grab onto the ESAT-6/CFP-10dimer and target it for secretion.
A curious observation is that all the known ESX-1sub-strates,Rv3616c,Rv3615c,ESAT-6and CFP-10are mutually dependent on each other for secretion.Although the basis for this phenomenon is unknown,the answer to this puzzle will likely shed light on the mechanism of substrate recognition and secretion.For example,it may indicate that these four substrates interact prior to secre-tion,perhaps via Rv3871.Alternatively,these proteins may be components of the secretion machine itself and the true substrates have yet to be identi?ed(Ize and Palmer,2006).
Role of the ESX-1secretion system
What role does the ESX-1system play in pathogenesis? Clearly,this system is a major determinant of Mycobac-terial pathogenesis but the way in which this system affects the biology of the host cell is unknown.Many groups have suggested that this system functions to modulate early events during M.tuberculosis infection. ESX-1mutants are attenuated for growth during the?rst few days of infection of mice and cultured macrophages, after which the bacteria eventually begin to grow.Despite the late growth,however,these mutants are severely attenuated(Brodin et al.,2006;Guinn et al.,2004; Stanley et al.,2003).
We believe that the ESX-1secretion system mediates early contact with the host cell,and functions to modulate the host cell immune response.Indeed,ESX-1mediated secretion is important for controlling the macrophage cytokine response during infection by M.tuberculosis. Whether this is a functional intention of the bacterium or an inadvertent readout of other perturbations on the host cell remains to be elucidated.Additionally,ESX-1is responsible for the elicitation of the cytokine interferon-beta and the resulting induction of a set of interferon responsive genes by wild-type M.tuberculosis(Stanley et al.,2007).We hypothesize that these differences in macrophage signalling re?ects active perturbations of the host cell by molecules secreted by ESX-1.In contrast, Hsu et al.suggest that ESAT-6functions as a toxin to directly lyse cellular membranes(Hsu et al.,2003).This is consistent with studies demonstrating that the RD1region is required for tissue necrosis in lungs of infected mice (Junqueira-Kipnis et al.,2006).Further work is required to reconcile these two seemingly very different models of ESX-1function.
Mycobacterium marinum causes a systemic tuberculosis-like infection in ectotherms and is closely related to M.tuberculosis phylogenetically.Real time studies of M.marinum infections in a zebra?sh model revealed that wild-type M.marinum recruits macroph-ages to form granulomas in an RD1-dependent manner (Volkman et al.,2004).This is consistent with ESX-1 functioning to manipulate macrophage responses, perhaps by activating cytokines or chemokines early during infection that lead to macrophage aggregation later during infection.However,M.marinum differs from M.tuberculosis in that following replication in the host phagosome,M.marinum escapes and propels itself through the host cytosol via actin-based motility,much like L.monocytogenes(Stamm et al.,2003).It is thought that the ESX-1system plays a role in phagosome escape and cell to cell spread in M.marinum,which may be distinct from how this system functions in M.tuber-culosis(Gao et al.,2004).These studies also provided no evidence that ESAT-6functions directly as a pore-forming cytotoxin.
It has also been suggested by many groups that,like alternate secretion systems in Gram-negative bacteria, the ESX-1system functions to secrete effector proteins, including ESAT-6/CFP-10,directly into the host cell pha-gosome or cytosol(Lewinsohn et al.,2006;Stanley et al., 2007).The only evidence for this,however,comes from the ESX-5-dependent secretion of PPE41into the host macrophage by M.marinum(Abdallah et al.,2006). Proving that ESX-1substrates are secreted directly into the host cell has proven difficult as the known substrates are likely secreted into the macrophage at very low levels and are extremely difficult to epitope tag without disrupt-ing secretion.
The role of the ESX-1secretion machines in Gram-positive pathogenic bacteria is less clear.ESX-1secretion is absolutely required for Staphylococcus aureus patho-genesis(Burts et al.,2005)but,in contrast,is not required for L.monocytogenes virulence in mice(Way and Wilson, 2005).This disparity may be due to differences in the pathogenic strategies employed by these two different pathogens or due to limitations in the virulence models (Way and Wilson,2005).
Any model of the role of ESX-1in pathogenesis must take into account the fact that this pathway is also present in non-pathogenic Gram-positive and mycobacterial species.One possibility is that the pathway plays a fun-damental role in both pathogenic and non-pathogenic organisms,for example,in cell-to-cell communication.In support of this,Flint et al.reported that M.smegmatis ESX-1mutants display increased conjugation efficiency compared with wild-type cells,and they present indirect evidence that ESAT-6/CFP-10secretion in trans sup-presses this hyperconjugation phenotype(Flint et al., 2004).Alternatively,the ESX-1pathway may be modular, allowing substrates to evolve for the particular needs of each organism(Converse and Cox,2005).It is therefore necessary to study the ESX-1system in both pathogenic and non-pathogenic bacteria to further elucidate the roles
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this system may play in addition to promoting bacterial virulence.
Parallels between ESX-1and Gram-negative secretion systems
Although it appears that ESX-1evolved independently of the Gram-negative systems,we believe there are signi?-cant parallels between the mechanisms of the ESX-1 system and Type IV secretion at the molecular level.First, like CFP-10,Type IV substrates,including those of the Dot/Icm system in L.pneumophila and the VirB/D4 system in A.tumefaciens,are targeted for secretion using unstructured C-terminal transport signals(Amor et al., 2005;Christie et al.,2005;Nagai et al.,2005;Vergunst et al.,2005).
Second,Type IV translocators use the coupling protein (CP)to facilitate the interaction between the substrate and the secretion machine,and to link cytosolic and mem-brane components.CPs are integral membrane proteins of the SpoIIIE/FtsK family of ATPases.Rv3870and Rv3871are members of the same family of ATPases,and it has been previously suggested that Rv3870and Rv3871function together as a single membrane bound AAA ATPase(Stanley et al.,2003;Guinn et al.,2004). Finally,ESAT-6and CFP-10are targeted for secretion through the ESX-1system as a substrate pair.Type IV secretion systems have examples of chaperone-substrate pairs that are targeted and secreted from the bacterial cell (Dumenil and Isberg,2001;Sundberg and Ream,1999). Speci?cally,like ESAT-6and CFP-10,the chaperones of these systems are small proteins(10–15kDa)with an acidic pI,are encoded by a gene adjacent to the gene that encodes the secreted substrate,and bind the substrate with high affinity.These chaperones typically function to keep the substrate in a secretion competent conformation and to prevent interaction with other proteins or aggregation.
It is therefore likely that the ESX-1machine represents a novel class of secretion machines in Mycobacteria and Gram-positive bacteria but which share some character-istics of Type IV machines of Gram-negative bacteria.
Additional ESX systems in Mycobacterial genomes
In addition to the ESX-1locus,there are10additional operons paralogous to the esxBA operon as a result of numerous duplications in the M.tuberculosis genome (Cole et al.,1998).Many of these CFP-10/ESAT-6paral-ogues(Esx proteins)have been identi?ed in the secreted proteome of M.tuberculosis but are not ESX-1substrates (Champion et al.,2006).Interestingly,?ve of the paralo-gous CFP-10/ESAT-6pairs are embedded within loci with synteny to the ESX-1locus(ESX-2-ESX-5)(Cole et al.,1998;Gey Van Pittius et al.,2001),raising the possibility that other ESX-1-like secretion systems function to secrete these Esx paralogues.
The ESX-5locus has recently been shown to operate in M.marinum and is the?rst report of a functional ESX locus besides ESX-1(Abdallah et al.,2006).They suggest that elements of this system are essential for viability,and that this system functions to secrete proteins of the PPE family during infection.However,because many ESAT-6/CFP-10paralogues are secreted from the Mycobacterial cell,it is likely that at least some of the other ESX loci encode for functional secretion systems,some of which may be essential for virulence or even viability.
Remaining questions
Despite many recent advances in the?eld of protein secretion in Mycobacteria,many questions remain.First, how are proteins that are destined for export out of the bacterial cell secreted across the cell wall and lipid layers?Clearly,Mycobacteria use many different secre-tion systems to export proteins from the cytosol.In Gram-positive bacteria,chaperones and foldases are localized to the Sec machinery and assist in folding and directing proteins to the?nal destination(e.g.Rosch and Caparon,2004;2005).In Gram-negative bacteria,fol-lowing translocation to the periplasm by the Sec system, proteins destined for the outer membrane interact with a series of chaperones that fold the proteins and escort them to sites in the outer membrane which functions to target and assemble the OMPs(e.g.Werner and Misra, 2005).There are no known components for any secre-tion machine in Mycobacterium outside of the cytoplas-mic membrane,and how these systems interact with proteins outside of the cytosol has yet to be discovered.
Type III and Type IV systems in Gram-negative bacteria have protein components that span the periplasm and the outer membrane to deliver bacterial effectors into the host cell.It is possible that the secreted proteins including ESAT-6,CFP-10,Rv3616c and Rv3615c,are actually extracytoplasmic components of the ESX-1secretion system(Ize and Palmer,2006).This could explain the mutually dependent secretion of these substrates.It could also be that the extracytoplasmic components of the secretion system are as of yet unknown,and further work is required to identify these.Finally,it is possible that ESX-1substrates,all of which are small,can passively work their way through the cell wall.
Second,how does the ESX-1secretion machine assemble,and how is it regulated?The Type III and IV machines require the ordered assembly of multiprotein complexes and are activated upon host cell contact. Similarly,it is likely that the many ESX-1components
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assemble and are regulated by environmental signals during infection.
Third,in addition to ESAT-6,CFP-10and EspA,what are the other substrates secreted by the ESX-1system? Proteomic analysis of the ESX-1system has generally failed to identify substrates of the ESX-1system outside of ESAT-6,CFP-10and Rv3616c.This may be because the other substrates,if they exist,are present at very low levels.It is also possible that these substrates are not present under the in vitro growth conditions used in the laboratory,and are only secreted during host-cell infection.This would suggest that substrates of this system are regulated by some as yet undiscovered mechanism.
Certainly,many more questions will arise as the exciting ?eld of Mycobacterial protein secretion unfolds.There is clearly much fertile ground for discovering both basic mechanisms of protein secretion and new insights into how these systems mediate interactions between host and pathogen.
Acknowledgements
P.A.C.is supported by NIH under Ruth L.Kirschstein National Research Service Award A105155.J.S.C gratefully acknowl-edges the support of the Sandler Family Supporting Foundation, the W.M.Keck Foundation,and NIH Grants AI63302and AI51667.
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蛋白质组学研究方法选择及比较 目前研究蛋白组学的主要方法有蛋白质芯片及质谱法，本文将从多方面对两种研究方法进行了解与比较； 蛋白质芯片（Protein Array） 将大量不同的蛋白质有序地排列、固定于固相载体表面，形成微阵列。利用蛋白质分子间特异性结合的原理，实现对生物蛋白质分子精准、快速、高通量的检测。 主要类型： ●夹心法芯片(Sandwich-based Array) ●标记法芯片(Label-based Array) ●定量芯片(Quantitative Array) ●半定量芯片(Semi-Quantitative Array) 质谱（Mass Spectrometry） 用电场和磁场将运动的离子按它们的质荷比分离后进行检测，测出离子准确质量并确定离子的化合物组成，即通过对样品离子质荷比的分析而实现对样品进行定性和定量的一种方法。 主要类型：
●二维电泳+质谱(2D/Mass Spectrometry, MS) ●表面增强激光解吸电离飞行时间质谱(Surface-enhanced laser desorption/ionization- time of flight, SELDI) ●同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantitation, iTRAQ) Protein Array or Mass Spectrometry？ 如何选择合适的研究方法？以下将从六个方面进行比较与推荐： 1.筛查蛋白组学表达差异 建议选择：RayBiotech（1000个因子的芯片）+质谱 a)不同的方法学有不同的特点：对于质谱，可以筛查到未知的蛋白，但是对于分子量大、 低丰度的蛋白质，质谱的灵敏度和准确性有一定的限制。 b)不同的方法能筛查到的目标不同：根据Proteome Analysis of Human Aqueous Humor 一文中报道，质谱筛查到的差异蛋白集中在小分子与代谢物。而用RayBiotech芯片筛查到的结果，多是集中在细胞因子、趋化、血管、生长等等。 c)质谱筛查到355个蛋白，而RayBiotech抗体芯片也筛查到328个蛋白，且用定量芯片 验证25个蛋白有差异，这些蛋白是质谱找不到的。目前RayBiotech夹心法抗体芯片已经可以检测到1000个蛋白，采用双抗夹心法，尤其是对于低丰度蛋白，有很好的灵敏度和特异性，很多的低丰度蛋白是抗体芯片可以检测出来，而质谱检测不到的，且样品不经过变性和前处理，保持天然状态的样品直接检测，对于蛋白的检测准确度高。 d)质谱的重复性一直是质谱工作者纠结的问题，不同操作者的结果，不同样品处理条件， 峰值的偏移等影响因素都会产生大的影响；RayBiotech的夹心法芯片重复性高。
《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/2f17375769.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/2f17375769.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/2f17375769.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/2f17375769.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/2f17375769.html, 我国的北京大学生物信息中心(www.cbi.
第35卷 第1期2011年1月 南京林业大学学报(自然科学版) J o u r n a l o f N a n j i n g F o r e s t r y U n i v e r s i t y (N a t u r a l S c i e n c e E d i t i o n ) V o l .35,N o .1 J a n .,2011 h t t p ://w w w .n l d x b .c o m [d o i :10.3969/j .i s s n .1000-2006.2011.01.024] 收稿日期:2009-12-31 修回日期:2010-10-26 基金项目:国家自然科学基金项目(31000287);江苏省高校自然科学基础研究项目(10K J B 220002) 作者简介:甄艳(1976—),副教授,博士。＊施季森(通信作者),教授。E -m a i l :j s h i @n j f u .e d u .c n 。 引文格式:甄艳,施季森.质谱技术在蛋白质组学研究中的应用[J ].南京林业大学学报:自然科学版,2011,35(1):103-108. 质谱技术在蛋白质组学研究中的应用 甄 艳,施季森 ＊ (南京林业大学,林木遗传与生物技术省部共建教育部重点实验室,江苏 南京 210037) 摘要:随着蛋白质组学研究的迅速发展,质谱技术已成为应用于蛋白质组学研究中的强有力工具和核心技术。质谱技术的先进性在于为蛋白质组学研究提供的通量和分子信息。笔者重点概述了基于质谱路线的蛋白质组学研究,介绍了基于质谱的定量蛋白质组学﹑翻译后修饰蛋白质组学、定向蛋白质组学、功能蛋白质组学以及基于串联质谱技术的蛋白质组学数据解析的研究 进展。 关键词:质谱;蛋白质组学;定量蛋白质组学;翻译后修饰;定向蛋白质组学;功能蛋白质组学中图分类号:Q 81 文献标志码:A 文章编号:1000-2006(2011)01-0103-06 A p p l i c a t i o n o f m a s s s p e c t r o m e t r y i n p r o t e o m i c s s t u d i e s Z H E NY a n ,S H I J i s e n ＊ (K e y L a b o r a t o r y o f F o r e s t G e n e t i c s a n d B i o t e c h n o l o g y M i n i s t r y o f E d u c a t i o n , N a n j i n g F o r e s t r y U n i v e r s i t y ,N a n j i n g 210037,C h i n a ) A b s t r a c t :W i t ht h e r a p i d d e v e l o p m e n t o f p r o t e o m i c s ,m a s s s p e c t r o m e t r y i s m a t u r i n g t o b e a p o w e r f u l t o o l a n dc o r e t e c h -n o l o g y f o r p r o t e o m i c s s t u d i e s d u r i n g t h e r e c e n t y e a r s .T h e s u p e r i o r i t y o f m a s s s p e c t r o m e t r y l i e s i n p r o v i d i n g t h e t h r o u g h -p u t a n d t h e m o l e c u l a r i n f o r m a t i o n ,w h i c hn o o t h e r t e c h n o l o g y c a n b e m a t c h e di np r o t e o m i c s .I nt h i s r e v i e w ,w e m a d e a g l a n c e o n t h e o u t l i n e o f m a s s s p e c t r o m e t r y -b a s e d p r o t e o m i c s .A n dt h e nw e a d d r e s s e d o n t h e a d v a n c e s o f d a t a a n a l y s i s o f m a s s s p e c t r o m e t r y -b a s e dp r o t e o m i c s ,q u a n t i t a t i v em a s ss p e c t r o m e t r y -b a s e dp r o t e o m i c s ,p o s t -t r a n s l a t i o n a l m o d i f i c a t i o n s b a s e d m a s s s p e c t r o m e t r y ,t a r g e t e d p r o t e o m i c s a n df u n c t i o n a l p r o t e o m i c s b a s e d -m a s s s p e c t r o m e t r y . K e yw o r d s :m a s ss p e c t r o m e t r y ;p r o t e o m i c s ;q u a n t i t a t i v ep r o t e o m i c s ;p o s t -t r a n s l a t i o n m o d i f i c a t i o n ;t a r g e t e d p r o -t e o m i c s ;f u n c t i o n a l p r o t e o m i c s 蛋白质组学(P r o t e o m i c s )是从整体水平上研究细胞内蛋白质的组成、活动规律及蛋白质与蛋白质的相互作用,是功能基因组学时代一门新的学科。目前蛋白质组学的研究主要有两条路线:一是基于双向电泳的蛋白质组学;二是基于质谱的蛋白质组学,其中基于双向电泳的蛋白质组学研究路线最终也离不开质谱技术的应用。自20世纪80年代末,两种质谱软电离方式即电喷雾电离(e l e c t r o s p r a y i o n i z a t i o n ,E S I )和基质辅助激光解析离子化(m a -t r i x a s s i s t e d l a s e r d e s o r p t i o n i o n i z a t i o n ,M A L D I )的发明和发展解决了极性大、热不稳定蛋白质和多肽分 析的离子化和分子质量大的测定问题[1] ,蛋白质组学研究中常用的质谱分析仪包括离子阱(i o n t r a p ,I T ),飞行时间(t i m e o f f l i g h t ,T O F ),串联飞行时间(T O F -T O F ),四级杆/飞行时间(q u a d r u p o l e /T O F h y b r i d s ),离子阱/轨道阱(I T /o r b i t r a ph y b r i d ) 和离子阱/傅里叶变换串联质谱分析仪(I T /F o u r i e r t r a n s f o r m i o n c y c l o t r o nr e s o n a n c em a s s s p e c t r o m e t e r s h y b r i d s ,I T /F T M S ),这些质谱仪具有不同的灵敏度、分辨率、质量精确度和产生不同质量的M S /M S 谱[2] 。质谱作为蛋白质组学研究的一项强有力的工具日趋成熟,并作为样品制备及数据分析的信息学工具被广泛地应用。因此,有学者指出质谱技术 已在蛋白质组学研究中处于核心地位[3] 。目前在通量及所包含的分子信息内容上,基于质谱的蛋白质组学技术在细胞生物学研究中可以鉴定和量化
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。
蛋白质组学及其研究方法与进展 蛋白质是生命活动的体现者，基因的表达最后是通过蛋白质来体现的，在这个过程中，蛋白质起了连接基因与表现的功能。蛋白质是有氨基酸组成的，组成蛋白质的氨基酸的种类及排列顺序构成了蛋白质的一级结构，而在一级机构基础上的多肽链本身的折叠和盘绕方式构成了蛋白质的二级结构，考虑到多肽链上原子在空间的分布，由二级结构进一步形成了蛋白质的三级结构，对于有多个亚基的蛋白质还存在四级结构。 蛋白质的一级结构决定了高级结构，而高级结构则决定着蛋白质的生物学功能。如今对于蛋白质研究已经单独形成了一个活跃的生物学分支学科―――蛋白质组学，在蛋白质的研究中发挥着很重要的作用，下面将介绍蛋白质组学的一些基本内容及研究进展。 一.产生背景[1] 在20世纪中后期随着DNA双螺旋结构的提出和蛋白质空间结构的解析，生命科学研究进入了分子生物学时代，对遗传信息载体DNA和生命功能的体现者蛋白质的研究，成为了其主要内容。90年代初期启动的庞大的人类基因组计划．在经过各国科学家多年的努力下，已经取得了巨大的成就。10多种低等模式生物的基因组序列测定L三完成；第一个多细胞生物一线虫基因组的DNA全序列测定也在1998年年底完成；人类所有基因的部分序列测定(EST)已经完成；人类基因组的全序列测定有可能提前到2003年完成。生命科学已跨入了后基因组时代。在后基因组时代，研究重心将从揭示生命的所有遗传信息转移到在整体水平上对功能的研究。这种转向的第一个标志是产生了功能基因组学这一新学科，即从基因组整体水平上对基因的活动规律进行阐述。如在mRNA 水平上，通过DNA 芯片(DNA chips)和微阵列(Microarray)法等技术检测大量基因的表达模式，并取得了很好的进展。但是，mRNA的表达水平(包括mRNA的种类和含量)由于mRNA储存和翻译调控以及翻译后加工等的存在．并不能直接反映蛋白质的表达水平}蛋白质自身特有的活动规律，如蛋白质的修饰加工、转运定位结构形成、代谢、蛋白质与蛋白质及其他生物大分子的相互作用等．均无法从在基因组水平上的研究获知。因此，对生物功能的主要体现者或执行者一蛋白质的表达模式和功能模式的研究就成为生命科学发展的必然。在此背景下．80年代中期，国际上葫发了一门研究细胞内垒部蛋白质的组成及其活动规律的新兴学科- 蛋白质组学(Proteomic)。 蛋白质组(proteome)一词是马克.威尔金斯(Marc Wilkins)最先提出来的, 最早见诸于1995年7月的“Electrophoresis”杂志上它是指一个有机体的全部蛋白质组成及其活动方式。蛋白质组研究虽然尚处于初始阶段, 但已经取得了一些重要进展。当前蛋白质组学的主要内容是, 在建立和发展蛋白质组研究的技术方法的同时, 进行蛋白质组分析。对蛋白质组的分析工作大致有两个方面。一方面,通过二维凝胶电泳得到正常生理条件下的机体、组织或细胞的全部蛋白质的图谱, 相关数据将作为待检测机体、组织或细胞的二维参考图谱和数据库。一系列这样的二维参考图谱和数据库已经建立并且可通过联网检索。二维参考图谱
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