Recognition and Signaling by T oll-Like Receptors
A.Phillip West,?Anna Alicia Koblansky,?and Sankar Ghosh
Section of Immunobiology and Department of Molecular Biophysics and
Biochemistry,Yale University School of Medicine,New Haven,Connecticut 06520;email:phillip.west@https://www.doczj.com/doc/2d9149375.html,,alicia.koblansky@https://www.doczj.com/doc/2d9149375.html,,sankar.ghosh@https://www.doczj.com/doc/2d9149375.html,
Annu.Rev.Cell Dev.Biol.2006.22:409–37First published online as a Review in Advance on July 5,2006
The Annual Review of
Cell and Developmental Biology is online at https://www.doczj.com/doc/2d9149375.html,
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These authors contributed equally to this work.
Key Words
innate immunity,leucine-rich repeat,TIR domain,signal transduction,MyD88,TRIF
Abstract
T oll-like receptors (TLRs)are transmembrane proteins that de-tect invading pathogens by binding conserved,microbially derived molecules and that induce signaling cascades for proin?ammatory gene expression.A critical component of the innate immune sys-tem,TLRs utilize leucine-rich-repeat motifs for ligand binding and a shared cytoplasmic domain to recruit the adaptors MyD88,TRIF ,TIRAP ,and/or TRAM for downstream signaling.Despite signi?-cant domain conservation,TLRs induce gene programs that lead not only to the robust production of general proin?ammatory me-diators but also to the production of unique effectors,which provide pathogen-tailored immune responses.Here we review the mecha-nisms by which TLRs recognize pathogens and induce distinct sig-naling cascades.
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Contents
INTRODUCTION.................410TLR LIGAND SPECIFICITIES....411TLR1,TLR2,and TLR6.........411TLR3...........................412TLR4...........................413TLR5...........................413TLR7and TLR8.................414TLR9...........................414TLR11..........................414DIVERSITY OF TLR LIGAND RECOGNITION................414Leucine-Rich-Repeat Diversity ...415Cooperative Interactions Between TLRs.........................416Coreceptors and TLR Binding Speci?city.....................417TLR SIGNALING PATHWAYS....418MyD88-Dependent Signaling.....419MyD88-Independent/TRIF-Dependent Signaling..........421Additional TIR Adaptor Proteins..422TLR-Mediated Induction
of IRFs .......................424Chromatin Remodeling and TLR Signaling Speci?city...........425NEGATIVE REGULATION OF TLR SIGNALING ..............425FINAL THOUGHTS ..............
427
INTRODUCTION
Metchnikoff ?rst described the innate im-mune system more than a century ago,yet research into this critical arm of immunity was largely overshadowed by the discov-ery of antibodies,B and T cells,and other Innate immunity:the initial,rapidly induced immune response of multicellular
organisms;utilizes nonclonal,
germline-encoded receptors for pathogen
recognition and the activation/recruitment of phagocytic cells
components of the adaptive immune sys-tem (Silverstein 2003).Recent work,how-ever,has yielded substantial insight into the composition and function of innate immu-nity,and it is now apparent that the innate immune system utilizes an intricate network of effector mechanisms to clear or moder-ate pathogen replication until the adaptive
immune system can mount a more speci?c and robust response.In 1989,Janeway (1989)proposed that innate effector mechanisms are initiated via the speci?c detection of mi-crobes by germline-encoded,nonclonal re-ceptors,which are essential for the imme-diate detection and control of infection in mammals.These molecules,termed pattern-recognition receptors (PRRs),primarily func-tion in recognizing microbial structures re-ferred to as pathogen-associated molecular patterns (PAMPs).
Building on this foundational hypothe-sis,researchers have shown that the innate immune system is phylogenetically con-served and is present in all multicellular or-ganisms (Hoffmann 1999,Medzhitov 2001,Medzhitov &Janeway 1997).As proposed by Janeway (1989),innate immunity is in fact predicated upon PRR detection of abun-dantly expressed PAMPs,which are con-served among broad classes of microor-ganisms.These molecules are critical for pathogen replication and/or survival and are unique to microorganisms.Thus,they are ab-sent from host cells and thereby endow the host with an ef?cient,nonself-reactive means to detect invading pathogens.Examples of such microbial products include lipopolysac-charide (LPS),lipoproteins,and viral or bac-terial nucleic acids.Signaling through PRRs induces a cascade of events including pro-duction of proin?ammatory chemokines and cytokines,activation of complement,recruit-ment of phagocytic cells,and mobilization of professional antigen-presenting cells.
In the late 1990s,three key discoveries signi?cantly advanced the understanding and de?nition of PRR-mediated innate immunity.First,in 1996,the Drosophila melanogaster pro-tein T oll,identi?ed previously for its role in dorso-ventral embryo patterning,was shown to be critical for effective immune responses in adult ?ies against the fungus Aspergillus fu-migatus (Lemaitre et al.1996).The following year,a mammalian homolog of the Drosophila T oll receptor,initially termed human T oll and now known as T oll-like receptor 4(TLR4),
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was identi?ed.A constitutively active form of this receptor could activate the transcrip-tion factor nuclear factor-kappa B (NF-κB),leading to the expression of proin?ammatory genes encoding interleukin (IL)-1,IL-6,and IL-8and the upregulation of costimulatory molecules (Medzhitov et al.1997).Finally,identi?cation of a point mutation in the Tlr4gene that rendered mice unresponsive to LPS challenge,and more susceptible to Gram-negative bacterial sepsis,de?nitively linked TLRs to innate immune responses (Poltorak et al.1998).
TLRs are type I transmembrane proteins of the Interleukin-1receptor (IL-1R)fam-ily that possess an N-terminal leucine-rich-repeat (LRR)domain for ligand binding,a sin-gle transmembrane domain,and a C-terminal intracellular signaling domain.The TLR C terminus is homologous to the intracellular domain of the IL-1R and is thus referred to as the T oll/IL-1receptor (TIR)domain.TLRs are expressed at the cell membrane and in subcellular compartments such as the endo-some.TLRs are widely expressed in many cell types,including nonhematopoietic ep-ithelial and endothelial cells,although most cell types express only a select subset of these receptors.Hematopoietically derived sentinel cells,such as macrophages,neutrophils,and dendritic cells (DCs),however,express most of the TLRs,with some variation in differ-ent subsets,e.g.,between conventional DCs and plasmacytoid DCs.Thus far,13mam-malian TLRs,10in humans and 13in mice,have been identi?ed (Beutler 2004).TLRs 1–9are conserved among humans and mice,yet TLR10is present only in humans and TLR11is functional only in mice.Although much is known about the ligands and signaling path-ways of TLRs 1–9and 11,the biological roles of TLRs 10,12,and 13remain unclear,as their expression patterns,ligands,and modes of signaling have yet to be de?ned.
Over the past few years,several excellent reviews detailing the many aspects of TLR bi-ology have been published.T o avoid duplica-tion,we focus our review on the mechanisms
PRR:pattern
recognition receptor Pathogen-associated
molecular pattern (PAMP):a
molecular pattern common to classes of microorganisms but not found in host cells
LPS:
lipopolysaccharide Cytokine:proteins such as TNF αand IL-6that affect the function and behavior of
neighboring cells via receptor-mediated signaling.Immune cytokines are often referred to as interleukins (IL)TLR:T oll-like receptor
Nuclear
factor-kappa B (NF-κB):an evolutionarily conserved
transcription factor critical for the production of effector cytokines,growth factors,and enzymes required for the initiation and resolution of
immune responses LRR:leucine-rich repeat
TIR:T oll/IL-1receptor homology domain
by which TLRs recognize ligands and in-duce speci?c signaling cascades for unique an-timicrobial gene programs and general proin-?ammatory responses.We provide a brief overview of known TLR ligands,a discussion of the mechanisms by which TLRs achieve diversity in ligand recognition,and a dis-cussion of our current knowledge about the molecular pathways that determine signaling speci?city.
TLR LIGAND SPECIFICITIES TLR1,TLR2,and TLR6
LPS was originally considered to be the ligand for TLR2,but subsequent studies revealed that contaminating bacterial lipoprotein in LPS preparations is the actual ligand (Wetzler 2003).In agreement with these later ?ndings,TLR2-de?cient macrophages were found to be hyporesponsive to several Gram-positive bacterial cell wall components as well as Staphylococcus aureus peptidoglycan (T akeuchi et al.2000).Additional work has shown that TLR2is involved in the recognition of a wide range of microbial products and gen-erally functions as a heterodimer with either TLR1or TLR6(Ozinsky et al.2000,Wyllie et al.2000).The TLR2/TLR1heterodimer recognizes a variety of lipoproteins,includ-ing those from mycobacteria and meningo-cocci (T akeuchi et al.2002,Wetzler 2003),whereas the TLR2/TLR6complex recog-nizes mycoplasma lipoproteins and peptido-glycan (T akeuchi et al.2001).Recent reports have demonstrated that triacylated lipopro-teins from bacteria are preferentially recog-nized by the TLR1/TLR2complex,whereas diacylated lipoproteins are recognized by the TLR2/TLR6complex (T akeuchi et al.2002).However,additional TLR2ligands do not ap-pear to require TLR1or TLR6for signal-ing,implying that TLR2may recognize some ligands as homodimers or heterodimers with other non-TLR molecules.Such TLR2lig-ands include the Gram-positive cell wall com-ponent lipoteichoic acid;the mycobacterial
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Figure 1
TLR ligand speci?cities.TLRs recognize a diverse array of PAMPs from bacteria,viruses,protozoa,and fungi.For detection of bacteria,heterodimeric TLR2/1binds triacyl lipopeptides,whereas TLR2/6
dimers bind diacyl lipopeptides and lipoteichoic acid.Homodimeric TLR2binds peptidoglycan,atypical LPS,phenol-soluble modulin from Staphylococcus epidermidis ,and porin proteins from Neisseria .In
addition,TLR4binds LPS,TLR5binds ?agellin,and TLR9binds bacterial CpG DNA.TLR11detects an unidenti?ed protein(s)from uropathogenic Escherichia coli .Viral dsRNA,RSV F protein,ssRNA,and unmethylated CpG DNA are sensed by TLRs 3,4,7/8,and 9,respectively.Finally,heterodimeric TLR2/6binds fungally derived zymosan and Trypanosoma cruzi GIPLs,whereas TLR11also senses a pro?lin-like protein from T oxoplasma gondii .Abbreviations:dsRNA,double-stranded RNA;LPS,lipopolysaccharide;GIPLs,glycolipids;PAMP ,pathogen-associated molecular pattern;RSV F ,respiratory syncytial virus fusion;ssRNA,single-stranded RNA;TLR,T oll-like receptor.
cell wall component lipoarabinomannan;atypical LPS produced by Legionella ,Lep-tospira interrogans,Porphyromonas gingivitis,and Bordetella ;and porins present in the outer membrane of Neisseria (Figure 1)(Massari et al.2002,Wetzler 2003).
In addition to bacterial PAMPs,TLR2het-ero/homodimers recognize fungal and pro-tozoan molecules.Zymosan (a crude mix-ture of glucans,mannan,proteins,chitin,and glycolipids extracted from the cell mem-brane of fungi)induces signaling through TLR2/6(Kataoka et al.2002,Underhill
et al.1999a).In addition,glycosylphos-phatidylinositol (GPI)anchors and glycoinos-itolphospholipids from the parasitic protozoa Trypanosoma cruzi induce signaling through TLR2(Campos et al.2001).
TLR3
Double-stranded RNA (dsRNA)was long hypothesized to be a viral PAMP ,as it is pro-duced during the course of many viral in-fections.Expression of human TLR3con-fers responsiveness to puri?ed dsRNA and
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polyinosinic-polycytidylic acid [poly(I:C)]in HEK293cells,and TLR3-de?cient mice dis-play impaired responses to these ligands.TLR3signaling results in the activation of NF-κB and interferon regulatory factor 3(IRF3),ultimately leading to the production of antiviral molecules,such as type I interfer-ons (IFN-α/β)(Alexopoulou et al.2001).Recent research,however,has demon-strated TLR3-independent recognition of viral dsRNA via the helicases RIG-I (retinoic acid–inducible gene I)and MDA5(melanoma-differentiation-associated gene 5),which are cytoplasmic PRRs expressed abundantly in multiple cell types (Andrejeva et al.2004,Y oneyama et al.2004).RIG-I and MDA5differentially recognize different groups of RNA viruses and are thus critical for a robust antiviral response (Kato et al.2006).These receptors contain a helicase domain for RNA binding and two caspase recruitment domains (CARDs)for signal transduction.Upon ligand binding,RIG-I and MDA5bind and activate the adaptor IPS-1(interferon-βpromoter stimulator 1;also termed CARDIF ,MAVS,and VISA)for NF-κB and IRF3activation and subsequent production of IFN-β(Kawai et al.2005,Meylan et al.2005,Seth et al.2005,Sun et al.2006,Xu et al.2005).The importance of RIG-I-and MDA5-mediated viral recog-nition is further supported by gene-targeting experiments demonstrating that TLR3and its adaptor TRIF are not required for type I IFN production in some virally infected cells,such as ?broblasts and conventional DCs (Honda et al.2003).However,plasmacytoid dendritic cells exclusively utilize TLR3/TRIF signaling for type I IFN production in re-sponse to RNA viruses and poly(I:C)(Kato et al.2005).
TLR4
LPS is the most thoroughly studied TLR lig-and.LPS,a glycolipid component of the outer membrane of Gram-negative bacteria,ex-hibits the most potent immunostimulatory ac-
Polyinosinic:poly-cytidylic acid [poly(I:C)]:a synthetic,
double-stranded RNA mimic that binds and signals through TLR3IRF:interferon regulatory factor Interferon (IFN):cytokines that block viral replication and infection of
surrounding cells
tivity among all known TLR ligands (Miyake 2004).T race amounts of LPS activate the in-nate immune system via TLR4,leading to the production of numerous proin?ammatory mediators,such as TNF α,IL-1,and IL-6.As discussed below,TLR4alone is not suf?-cient for a robust response to LPS;additional components of the LPS recognition com-plex,CD14and MD-2,are required.Other TLR4ligands include Lipid A analogs (Lien et al.2000),taxol (Perera et al.2001),my-cobacterial components (Means et al.1999),Aspergillus hyphae (Mambula et al.2002),Cryptococcus neoformans capsular polysaccha-ride (Shoham et al.2001),and respiratory syn-cytial virus (RSV)F protein (Kurt-Jones et al.2000).
TLR5
Flagellin,a protein component of Gram-negative bacterial ?agella,is the cognate lig-and for TLR5(Hayashi et al.2001).TLR5recognizes a highly conserved,central core structure of ?agellin that is essential for proto?lament assembly and bacterial motil-ity (Smith et al.2003).Interestingly,the TLR5recognition site is masked in the ?l-amentous ?agellar structure,thus indicat-ing that TLR5recognizes only monomeric ?agellin (Smith et al.2003).Furthermore,?agellin appears to bind directly to TLR5at residues 386–407of the extracellular do-main (ED),as TLR5-ED mutants lacking this domain are unable to interact with ?agellin in biochemical assays (Mizel et al.2003b).Recent articles have demonstrated TLR5-independent recognition of cytosolic Salmonella typhimurium ?agellin via Ipaf,a member of the nucleotide-binding oligomer-ization domain (NOD)-LRR (Franchi et al.2006,Miao et al.2006).Ipaf-mediated recog-nition of cytosolic ?agellin is critical for caspase-1activation and subsequent IL-1βse-cretion by macrophages,further highlighting the signi?cance of TLR-independent pattern recognition in the overall complexity of the innate immune response.
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CpG DNA:short DNA sequences consisting of
cytosine-guanosine sequences ?anked by additional
nucleotides that bind and signal via TLR9
TLR7and TLR8
Mouse TLR7recognizes a class of synthetic antiviral compounds,such as imidazoquino-lines and loxoribine (Hemmi et al.2002).Furthermore,TLR7and human TLR8de-tect the antiviral azoquinoline compound R-848(Jurk et al.2002).These synthetic compounds are structurally related to nu-cleic acids;TLR7and human TLR8rec-ognize guanosine-or uridine-rich single-stranded RNA (ssRNA)derived from RNA viruses (Diebold et al.2004,Heil et al.2003,Lund et al.2004).Interestingly,mammalian RNA,which contains many modi?ed nu-cleosides,is signi?cantly less stimulatory via TLRs 7and 8than bacterial RNA,suggest-ing that the mammalian host utilizes nucle-oside modi?cation as a means to distinguish between endogenous and pathogen-derived RNA (Kariko et al.2005).Similar to the func-tion of TLR3,engagement of these receptors leads to the production of type I IFNs,which are obligate components of antiviral innate immunity.
TLR9
TLR9recognizes bacterial DNA contain-ing unmethylated CpG motifs,and TLR9-de?cient mice are not responsive to CpG DNA challenge (Hemmi et al.2000).TLR9expressed in plasmacytoid DCs recognizes vi-rally derived CpG sequences for the induction of IFN-α(Krug et al.2004,Lund et al.2003,T akeshita et al.2001).The low frequency and high rate of methylation of CpG mo-tifs prevent recognition of mammalian DNA by TLR9under physiological circumstances.In certain autoimmune disorders,however,IgG2a/chromatin complexes containing CpG DNA can engage the B cell receptor and TLR9concomitantly,leading to the pro-duction of rheumatoid factor and other au-toreactive molecules (Leadbetter et al.2002,Viglianti et al.2003).A recent report indi-cated that the intracellular,endosomal restric-tion of TLR9is critical for discriminating be-tween self and nonself DNA,as host DNA,unlike microbial DNA,does not usually en-ter the endosomal compartment (Barton et al.2006).Finally,Coban et al.(2005)reported that TLR9recognizes a novel non-DNA lig-and called hemozoin,which is a hydrophobic heme polymer produced by malaria parasites as they digest host hemoglobin.
In addition to TLR9-mediated detection of CpG DNA in the endosomal compartment,the mammalian innate immune system also responds to foreign DNA in the cytosol.This response is important for type I IFN produc-tion in response to viruses and intracellular pathogens,such as Listeria monocytogenes and Shigella ?exneri ,that replicate in the cytoplasm (Ishii et al.2006,Stetson &Medzhitov 2006).As membrane restriction prevents TLRs from sampling the cytosol for PAMPs,cytosolic PRRs have evolved to provide comprehensive innate immune recognition that offsets TLR restriction.
TLR11
Gene-targeting studies revealed that TLR11-de?cient mice are susceptible to uropathogenic Escherichia coli infection (Zhang et al.2004).Although the bacterial ligand for TLR11remains undiscovered,the stimulatory activity of uropathogenic bacteria can be destroyed by proteinase K treatment,suggesting that TLR11recognizes a protein ligand.TLR11also recognizes a class of pro?lin-like molecules expressed by apicomplexan protozoans,including T oxoplasma gondii (Yarovinsky et al.2005).
DIVERSITY OF TLR LIGAND RECOGNITION
As described above,TLRs sense an extremely diverse repertoire of molecular structures from pathogens.Because TLRs recognize conserved molecules shared among members of a particular class of microbes (e.g.,LPS from Gram-negative pathogens and ssRNA from RNA viruses),the entire pathogenic
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universe can be readily sampled by a small group of receptors (Medzhitov &Janeway 1997).However,the mechanism by which this limited group of germline-encoded re-ceptors uniquely recognizes its ligands is still unclear,and it is remarkable that TLRs rec-ognize such varied molecules utilizing a con-served LRR motif.In addition,TLRs 2and 4are unique in their ability to recognize multiple,diverse PAMPs.In light of this di-versity of recognition,we discuss below the identi?ed and otherwise possible mechanisms by which TLRs bind their ligands,namely through diversi?cation of LRR motifs,coop-erative interactions between different TLRs,and the use of non-TLR coreceptors or accessory molecules.
Leucine-Rich-Repeat Diversity
The basic LRR motif comprises 24amino acids that form a β-strand and an α-helix joined by a loop,and it is present in several prokaryotic and eukaryotic receptors (Kobe &Kajava 2001).TLRs contain a unique consen-sus LRR,xLxxLxLxx [N/L ]x +xx +xxxxFxxLx ,where x represents any amino acid and +rep-resents any hydrophobic amino acid (Bell et al.2003).This consensus sequence appears re-peatedly throughout the extracellular domain of TLRs,although other variants of this mo-tif are present.In a recent article,Bell et al.(2003)thoroughly characterize each LRR in TLRs 1–10and propose that nonconsensus LRRs containing insertions after residues 10and 15confer ligand speci?city.Insertions in LRRs after the tenth amino acid are predicted to provide ligand binding sites at the concave surface of TLRs,whereas insertions at posi-tion 15are predicted to create binding sites at the convex surface.
There are several compelling examples in support of this model,including TLR9and TLR5LRR insertions that potentially create binding sites for CpG DNA and ?agellin,re-spectively.With regard to TLR9,insertions at position 10of LRRs 2,5,8,and 11occur on the concave face of the receptor and perhaps
create potential CpG binding sites.Notably,the insertion in the eighth LRR contains two cysteine-rich CXXC motifs,which mediate direct binding of CpG DNA by a transcrip-tional activator termed CpG-binding protein (Lee et al.2001).Thus,the location and sequence of these nonconsensus LRRs sug-gest that they function in TLR9-dependent CpG DNA binding,although this hypothe-sis has not been tested experimentally (Bell et al.2003,Lee et al.2001).As mentioned above,Mizel et al.(2003b)recently mapped the TLR5extracellular domain required for ?agellin binding (amino acids 386–407),and they also showed that the domain encom-passes an LRR.Interestingly,the predicted ?agellin binding site resides within LRR 14of the extracellular domain,and it contains a six-residue insertion after position 15,which provides further experimental support for the insertion speci?city model (Bell et al.2003).Finally,mutational analysis of the TLR2extracellular domain illustrated that deletion of the ?rst seven N-terminal LRRs did not drastically affect its ability to transduce sig-nals for NF-κB and mitogen-activated pro-tein kinase (MAPK)activation in 293cells;therefore,its ability to interact with microbial polypeptides and peptidoglycan remains un-affected (Meng et al.2003,Mitsuzawa et al.2001).However,N-terminal mutant TLR2displayed downstream signaling de?ciencies in response to many other TLR2ligands,pro-viding evidence that speci?c LRR domains on TLR2interact with different PAMPs,thereby increasing recognition capacity.Therefore,LRRs on each TLR seem to function to create unique ligand speci?cities for the respective receptors,which aids in explaining the diverse array of PAMPs recognized by this family of PRRs.
Most data regarding LRRs and ligand binding are predicated on some form of muta-tional analysis in which large portions of var-ious TLR ectodomains are removed and the degree of inhibition between PAMP/receptor interaction is observed biochemically.Although these experiments are highly
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informative,they do not address the concern that mutating portions of any protein may result in an overall altered conformation.Thus,the altered binding of ligands to mutated TLRs may not be the direct re-sult of removing the binding site per se.Ultimately,cocrystallographic studies are needed to provide concrete proof of speci?c PAMP/TLR interaction domains,but given the dif?culty in purifying large amounts of TLR extracellular domains,this proof has been slow to arrive.
Recently,Bell et al.(2005)and Choe et al.(2005)independently solved the crystal struc-ture of human TLR3-ED,providing a ma-jor breakthrough in the structural analysis of TLRs.Both groups concluded that the struc-ture of human TLR3is a horseshoe-shaped solenoid that is heavily glycosylated,and both identi?ed possible dsRNA binding sites.Dis-agreeing with the current model of TLR lig-and binding,Choe et al.(2005)concluded that glycosylation and the overall negative charge of the concave surface would likely prevent dsRNA binding.They proposed that the binding site of dsRNA exists on the con-vex,glycosylation-free face of TLR3,which consequently has many basic residues to ac-commodate the net negative charge of nucleic acids.In contrast,Bell et al.(2005)argued that the binding site for dsRNA may reside in the concave region of the solenoid because sulfate ions that mimic the phosphate backbone of nucleic acids were found attached to the side chains of several amino acids on the concave face.These authors also proposed that loops created from insertions in LRRs 12and 20yield a pocket on the glycosylation-free,con-vex face of TLR3suitable for dsRNA bind-ing.Because these insertions are highly con-served across TLR3from different species,these sites likely participate in ligand binding (Bell et al.2005).Although no cocrystalliza-tion data were provided,the structural anal-yses present substantial insight into the po-tential means by which TLR3binds dsRNA.As the LRR sequence and crystal structure of TLR3indicate multiple ligand binding sites,
it is interesting to consider whether this re-ceptor binds ligands in addition to dsRNA.Additional evidence is needed to determine whether this idea is correct.However,other TLRs bind multiple ligands,and the large sur-face area and spatial distribution of putative binding sites on TLR3may explain the di-verse ligand speci?cities of other TLRs.
Cooperative Interactions Between TLRs
As described above,TLR2functions as a heterodimer with TLRs 1and 6for recog-nition of a diverse group of PAMPs.Al-though bacterial lipopeptides are important ligands for these heterodimeric pairs,data from many other groups suggest that TLR2/1or TLR2/6dimers recognize additional lig-ands,such as zymosan,Y ersinia V antigen (Sing et al.2002),and Escherichia coli en-terotoxins (Hajishengallis et al.2005).These ligands are structurally unrelated to bacte-rial lipopeptides,and the diversity of recog-nition is likely provided by the combination of LRR domains on each TLR.Recently,Omueti et al.(2005)showed that LRRs 9–12of human TLRs 1and 6mediate the abil-ity of these receptors to discriminate between acylated lipoproteins,as domain exchange be-tween the two receptors alters lipopeptide re-sponsiveness in transfected cells.Additionally,LRRs 7–10of TLR2were shown to be crit-ical for responses to tri-lauroylated lipopep-tides (Grabiec et al.2004,Meng et al.2003).T aken in combination,these data suggest that TLR2/1and TLR2/6heterodimers bind lipopeptide PAMPs cooperatively at regions predicted to lie on the convex,outer loops of both receptors (Omueti et al.2005).Although these studies clarify heterodimeric TLR re-sponses to lipopeptides,they do not readily explain the requirement of both TLR2and either TLR1or TLR6receptor pairs for the recognition of structurally unrelated PAMPs.Analysis of all three TLRs reveals that several nonconsensus LRRs containing insertions lie outside of the predicted lipopeptide binding
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domains (Bell et al.2003),thereby raising the possibility that these LRRs,acting singly or in concert with regions from their heterodimeric partners,function in the recognition of ad-ditional PAMPs.Thus,through cooperative interaction,TLRs may signi?cantly increase their PAMP speci?cities,further ensuring detection of numerous pathogens.
TLR heterodimerization may also have functional consequences for signaling path-ways induced upon ligand binding.As de-scribed in detail below,TLRs utilize one of two adaptors for signal transduction,myeloid differentiation factor 88(MyD88)or TIR domain–containing adaptor inducing interferon-β(TRIF),although other adaptors contribute to signaling speci?city.MyD88-dependent and TRIF-dependent signaling pathways result in the induction of distinct innate immune effectors,and signaling from heterodimeric receptor pairs may engage both adaptors for additional or varied responses.For example,both TLR4and TLR5signal-ing are critical for the ?agellin-induced pro-duction of nitric oxide (NO),but not TNF α,in macrophage-like lines (Mizel et al.2003a).TLR4can signal via both MyD88-dependent and -independent pathways,and engagement of TRIF-induced signaling through the het-erodimeric TLR4/TLR5pair may lead to the activation of molecules,such as IFN-β,for increased NO production.Because NO has important antimicrobial properties,its induc-tion is likely critical for the control of ?ag-ellated bacteria,and combinatorial signaling through TLRs 4and 5may augment the an-tibacterial NO response.Although examples of TLR heterodimerization inducing unique proin?ammatory pathways are limited,thor-ough analysis of TLR and/or TLR-adaptor knockout mice may con?rm and yield further insight into this interesting hypothesis.
Coreceptors and TLR Binding Speci?city
Although several TLRs have been suggested to bind their respective PAMPs directly (Bell
et al.2005,Iwaki et al.2002,Lien et al.2000,Mizel et al.2003b,Poltorak et al.2000),researchers have also identi?ed addi-tional molecules that enhance recognition,and thus signaling,by some TLRs.Notably,TLR4forms a complex at the cell mem-brane with several membrane-linked and sol-uble molecules,including CD14and MD-2.CD14,an LRR-containing,GPI-linked molecule,binds LPS binding protein/LPS complexes and is thought to transfer LPS to the TLR4complex (Ulevitch 1993,Wright et al.1990).Genetic ablation of the Cd14gene in mice leads to reduced clearance of Gram-negative bacteria and decreased susceptibility to LPS challenge,supporting the notion that CD14participates in TLR4-mediated innate immune responses (Haziot et al.1996).More-over,several groups have demonstrated that CD14expression enhances TLR2-mediated responses to many PAMPs,including pep-tidoglycan and lipopeptides,and they have postulated that CD14also acts to transfer these ligands to TLR2for signaling (Sellati et al.1998,Vasselon et al.2004,Wetzler 2003,Wooten et al.1998).CD14was also shown to play a critical role in TLR3-mediated re-sponses to poly(I:C)by binding extracellular poly(I:C)and inducing its uptake and delivery into the endosomal compartment for TLR3signaling (Lee et al.2006).Another molecule,MD-2,interacts with TLR4intracellularly and at the cell surface,and its presence is re-quired for LPS responsiveness (Nagai et al.2002,Shimazu et al.1999).Although MD-2contains LRRs,it appears to function in the targeting of TLR4to the cell membrane,and it is not known whether this molecule enhances binding of TLR4ligands.
More recently,dectin-1,a C-type lectin receptor for β-glucans,was shown to bind and induce internalization of zymosan in cells of the monocyte lineage (Brown &Gordon 2001,Brown et al.2003).Phagocytosis of zy-mosan leads to the recruitment of TLR2/6heterodimers to intracellular phagosomes for signaling and the subsequent induction of proin?ammatory cytokines.With regard to
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TLR2/6-mediated recognition of diacylated lipopeptides and lipotechoic acid,CD36,a type B scavenger receptor,is necessary for ro-bust NF-κB activation and proin?ammatory cytokine production as well as Gram-positive bacterial clearance (Hoebe et al.2005).Al-though the mechanism by which CD36en-hances responses to speci?c TLR2ligands is unknown,CD36may function analogously to CD14by bringing ligands into proxim-ity with the receptor for signaling.Therefore,TLRs 2,3,and 4utilize additional recep-tors and/or cofactors for the recognition of some PAMPs,as these molecules increase the ef?ciency and speci?city of PAMP/TLR in-teractions.Additional TLRs may also utilize accessory molecules to increase their ligand binding potential.
TLR SIGNALING PATHWAYS
A plethora of PAMPs stimulate various TLRs to induce the transcription of distinct tar-get genes required for effective immune re-sponses,and the molecular pathways by which TLRs initiate speci?c gene programs have been studied extensively in recent years.Be-low we succinctly review the ever-growing body of knowledge encompassing TLR sig-naling by characterizing key components and pathways leading to transcription factor ac-
tivation and subsequent induction of proin-?ammatory gene products.
PAMP binding by TLRs is thought to fa-cilitate dimerization of the receptors to induce conformational changes required for adaptor recruitment,although PAMP/TLR interac-tion may also induce structural changes in the cytoplasmic domains of preformed membrane dimers (Akira &T akeda 2004).Nevertheless,ligand binding initiates signal transduction cascades that originate from the cytoplasmic TIR domains of TLRs.The critical impor-tance of the TIR domain was ?rst shown in C3H/HeJ mice,which have a point mutation in cytoplasmic proline residue 712and are hy-poresponsive to LPS owing to the inability of the TLR4TIR domain to recruit downstream effectors (Poltorak et al.1998).Further stud-ies showed that this residue is conserved among most TLRs,except for TLR3,and transiently transfected mutant TLRs harbor-ing the proline-to-histidine mutation do not signal (Hoshino et al.1999,Underhill et al.1999b).This is due to the inability of TIR mu-tants to recruit downstream molecules such as MyD88,a critical adaptor in TLR signaling pathways.Research over the past few years has demonstrated MyD88-dependent and -independent TLR signaling pathways;a de-tailed discussion of both is provided below (Figure 2).
?????????????????????????????????????????????????????????????????????→
Figure 2
TLR signaling pathways.TLRs 1/2/6,3,4,and 5are localized at the plasma membrane of most cells,whereas TLRs 7–9are localized exclusively in the endosomal compartments of conventional and/or plasmacytoid dendritic cells.PAMP binding initiates con?rmational changes to the TLRs,such that TIR-containing adaptors are recruited to the TLR TIR domain.Adaptors TIRAP and TRAM link MyD88and TRIF to speci?c TLRs.Signaling through TLRs 1/2/6,5,and 7–9is exclusively mediated by MyD88.Receptor-associated MyD88recruits IRAK-4and IRAK-1,which then associate with TRAF6.TRAF6then activates TAK1,which subsequently activates IKK,JNK,and p38,ultimately leading to early-phase NF-κB and AP-1activation and the transcription of effectors of the innate immune response.In addition,the MyD88/IRAK/TRAF6complex mediates the activation of
transcription factors IRF5and IRF7for cytokine and IFN-αproduction,respectively.TLR3utilizes TRIF for signaling exclusively,whereas TLR4utilizes both MyD88and TRIF .TRIF promotes the
activation of both TRAF6and RIP1,which activate TAK1,resulting in NF-κB and AP-1activity and the production of innate immune effector molecules.Additionally,TRIF-dependent signaling activates TBK1for IRF3phosphorylation,which results in IFN-βproduction.
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MyD88-Dependent Signaling
MyD88was originally shown to be crit-ical for signaling through the IL-1R,as overexpression of dominant-negative MyD88blocked signaling,and thus NF-κB activation,
in response to IL-1(Muzio et al.1997,Wesche et al.1997).MyD88possesses a C-terminal TIR domain that interacts with TLR or IL-1R TIR domains and an N-terminal death domain that binds other
death
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domain–containing molecules,such as mem-bers of the Interleukin-1receptor–associated kinase (IRAK)family.The importance of MyD88in LPS-induced TLR4signaling was demonstrated through the generation and analysis of MyD88-de?cient mice (Adachi et al.1998,Kawai et al.1999).LPS-and IL-1-induced proin?ammatory cytokine pro-duction by macrophages and ?broblasts from these mice is drastically decreased,if not ab-lated.Furthermore,MyD88-de?cient cells do not respond to peptidoglycans,?agellin,CpG DNA,ssRNA,or T oxoplasma pro?lin–like protein,indicating that TLR2,TLR5,TLR7/8,TLR9,and TLR11signaling pro-ceeds exclusively through MyD88(Adachi et al.1998,Beutler et al.2005,T akeda 2003,Yarovinsky et al.2005).
MyD88-dependent signaling is initiated by a PAMP-induced conformational change in the TLR cytoplasmic domain,which leads to the association of MyD88with the TLR via a homotypic interaction between their TIR domains.Subsequent downstream sig-naling is mediated through the interaction of MyD88with the serine/threonine IL-1receptor–associated kinase-4(IRAK-4)via their respective death domains (Burns et al.2003,Medzhitov et al.1998,Muzio et al.1997,Suzuki et al.2002).Once IRAK-4binds MyD88,it recruits and phosphorylates IRAK-1,which activates the kinase function of IRAK-1.IRAK-1then autophosphory-lates itself,recruiting tumor necrosis fac-tor receptor–associated factor-6(TRAF6)to the MyD88/IRAK-4/IRAK-1complex.Next,IRAK-1and TRAF6dissociate from the re-ceptor complex and interact with additional molecules,resulting in c-Jun N-terminal kinase (JNK)and inhibitor of κB kinase (IKK)activation.These proteins then induce AP-1(activator protein-1)and NF-κB acti-vation,ultimately leading to the transcrip-tion of genes encoding proin?ammatory cy-tokines and chemokines such as TNF α,IL-6,IL-8,and IL-1β(Chen &Goeddel 2002,Hayden &Ghosh 2004,T akeda &Akira 2005).
The mechanism by which TRAF6acti-vates IKK is still somewhat unclear.However,it has been proposed that ubiquitination of dimerized TRAF6leads to the recruit-ment and subsequent activation of a complex made up of transforming growth factor-β–activated kinase-1(TAK1)and two adaptor proteins,TGF-βbinding protein-1(TAB1)and TAB2(Deng et al.2000,Shibuya et al.1996,Sun et al.2004,T akaesu et al.2000).Upon dimerization,TRAF6interacts with ubiquitin-conjugating enzyme 13(UBC13)and a UBC-like protein UEV1A,both of which facilitate lysine 63ubiquitination of TRAF6(Chen 2005).Ub-TRAF6then binds TAB2and phosphorylates TAK1,which re-sults in the TAK1-dependent phosphoryla-tion and activation of IKK,p38,and JNK (Wang et al.2001).Owing to the embry-onic lethality of TAK1-,TAB1-,and TAB2-de?cient animals,genetic evidence for the role of TAK1in TLR/IL-1R-induced NF-κB and AP-1activation was dif?cult to ob-tain.However,recent analysis of embryonic ?broblasts isolated from knockout animals il-lustrated that TAK1,but not TAB1or TAB2,is necessary for the activation of IL-1R-and TLR-mediated NF-κB and AP-1induction (Sato et al.2005,Shim et al.2005).Thus,it ap-pears that TAK1is localized upstream of IKK and JNK and is critical for their activation.The exact means by which this occurs is un-clear,and additional experiments are needed to elucidate the biochemical mechanisms by which TAK1functions.
Another molecule,ECSIT (evolutionary conserved signaling intermediate in T oll path-ways),was identi?ed as a TRAF6-interacting protein by yeast two hybrid and is involved in IL-1R-and TLR-induced NF-κB activation (Kopp et al.1999,Xiao et al.2003).ECSIT interacts with both TAK1and the mitogen-activated protein kinase MEKK3(A.P .West &S.Ghosh,unpublished observations),which are critical mediators of TLR and IL-1R sig-naling (Huang et al.2004).Thus,ECSIT may regulate the activity of MEKK3and/or TAK1,and studies are currently under way to
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elucidate the exact mechanism by which EC-SIT mediates TLR signaling.
MyD88-Independent/TRIF-Dependent Signaling
As indicated above,targeted deletion of MyD88results in aberrant signaling and dras-tically decreased cytokine production from most TLRs.However,LPS stimulation of MyD88-de?cient cells does result in NF-κB and MAPK activation,albeit with de-layed kinetics.In addition,DC maturation,as measured by costimulatory molecule up-regulation in response to LPS,is unaf-fected in MyD88knockout cells (Hoshino et al.2002,Kaisho et al.2001).Furthermore,TLR4and TLR3stimulation results in the MyD88-independent activation of IRF3,a key transcription factor necessary for IFN-βproduction and the delayed-phase NF-κB activation via TLR4(Covert et al.2005,Kawai et al.1999).Thus,these data sug-gest the existence of TLR signaling through pathways not dependent on MyD88,and they led to the characterization of the TIR domain–containing adaptor inducing IFN-β(TRIF)/TIR-containing adaptor molecule-1(TICAM-1),which is a critical transducer of MyD88-independent signaling (Akira et al.2001,Hoebe et al.2003,Yamamoto et al.2003a,Yamamoto et al.2002b).
Although it has been studied extensively,the mechanism by which TRIF activates NF-κB and IRF3is not completely understood.Initial characterization of TRIF suggested that the N-and C-terminal domains me-diate distinct functional roles.With respect to the N-terminal domain,two noncanon-ical I κB kinases,IKKi/IKK εand TRAF-family-member-associated NF-κB activator (TANK)-binding kinase (TBK-1),have been linked to IRF3activation downstream of TRIF (Fitzgerald et al.2003a).The N ter-minus of TRIF is believed to form a com-plex with TBK-1,IRF3,and possibly IKKi for the speci?c phosphorylation and activa-tion of IRF3(Fitzgerald et al.2003a).Corre-
TLR-INDUCED NF-κB ACTIVATION:A DETAILED LOOK
Signaling from the IL-1R and TLRs induces the activation of NF-κB,an inducible transcription factor critical for proper immune function.Upon PAMP binding and subsequent re-cruitment of adaptors MyD88or TRIF and TRAF6to TLRs,the TAK1kinase is activated,thus initiating the classical NF-κB pathway (Hayden &Ghosh 2004).TAK1phosphorylates the βsubunit of the I κB kinase (IKK)complex,which leads to IKK-mediated phosphorylation of the inhibitor of κB (I κB)molecules,which sequester NF-κB subunits p65(REL-A)and p50in the cytoplasm.Upon phosphorylation,I κB is recog-nized by ubiquitin ligase machinery,polyubiquitinated,and subsequently degraded by the proteasome (Karin &Ben-Neriah 2000).This results in the release of p65/p50het-erodimers for nuclear translocation;once at the nucleus,they bind speci?c sequences in the promoter or enhancer regions of immune genes (Hayden &Ghosh 2004).
spondingly,TBK-1-de?cient ?broblasts dis-play marked de?ciencies in IRF3activa-tion and IFN-βproduction in response to TLR3and TLR4ligands (Hemmi et al.2004,McWhirter et al.2004,Perry et al.2004).IKKi ?/?and TBK-1?/?cells display no dis-cernable defects in NF-κB transcriptional ac-tivity,suggesting that these kinases are not involved in TRIF-mediated NF-κB activa-tion (Hemmi et al.2004,McWhirter et al.2004).
With regard to TLR4-and TRIF-dependent NF-κB activation,some studies have suggested that TRAF6associates with the N-terminal domain of TRIF for NF-κB induction (Sato et al.2003).However,cells doubly de?cient in MyD88and TRAF6par-tially activate NF-κB in response to LPS,indi-cating the existence of TRAF6-independent,TRIF-mediated signaling (Kawai et al.2001).Moreover,another study demonstrated that TRAF6is dispensable for late-phase NF-κB activation in response to LPS,suggesting that TRAF6is not involved in the TRIF-mediated TLR4signaling pathway (Gohda et al.2004).
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Concerning the C-terminal domain of TRIF ,recent studies demonstrated that a kinase critical for TNFR-mediated IKK acti-vation,receptor-interacting protein 1(RIP1),can associate with TRIF via its respective RIP interaction domain (Meylan et al.2004).Anal-ysis of the TRIF C-terminal sequence iden-ti?ed a small region of homology to RIP1,referred to as the RIP homotypic interac-tion motif (RHIM),and mutational studies showed that the RHIM is required for the as-sociation of TRIF and RIP1(Meylan et al.2004).In the absence of RIP1,poly(I:C)-induced NF-κB activity is signi?cantly de-creased,although JNK and IRF3activity are unchanged.This indicates that RIP1is criti-cal for TLR3-mediated signaling to NF-κB.RIP1may also play a critical role in the TRIF-dependent activation of NF-κB from TLR4because RIP1/MyD88double-knockout cells do not activate NF-κB in response to LPS (Cusson-Hermance et al.2005).Further-more,upon poly(I:C)stimulation,RIP1is polyubiquitinated and recruited along with TRAF6and TAK1to TLR3,suggesting that RIP1links TRIF to TRAF6and thus to TAK1.
As mentioned above,TRIF-dependent TLR4signaling results in late-phase NF-κB activation,although the molecular mecha-nisms underlying this phenomenon have re-mained unclear.However,recent publica-tions from independent groups address this issue,suggesting that initial TRIF-mediated IRF3activation is required for subsequent NF-κB activity (Covert et al.2005,Werner et al.2005).Late-phase NF-κB activation via TLR4/TRIF ,in particular,requires de novo protein synthesis,speci?cally the production of TNF α(Covert et al.2005,Werner et al.2005).siRNA depletion of IRF3drastically decreases MyD88-independent activation of NF-κB;therefore,TRIF-mediated signaling in response to LPS appears to trigger the ac-tivation of IRF3,which binds to the TNF αpromoter and facilitates transcription (Covert et al.2005).TNF αthen signals via the TNF receptor in an autocrine feedback loop,result-ing in the activation of IKK for late-phase NF-κB activity.Future analysis of IRF3and/or TBK-1knockout mice may substantiate and further clarify these ?ndings.
Additional TIR Adaptor Proteins
TLR signaling is far more intricate than ini-tially anticipated:Activation of speci?c TLRs leads to varied patterns of gene expression re-sulting from the differential activation of tran-scription factors such as NF-κB,AP-1,and IRFs.As described above,signaling via TLR3or TLR4,but not TLR2or TLR5,leads to the activation of IRF3,which regulates the expression of IFN-βand is critical for mediat-ing anti-viral immune responses.In addition,speci?c signaling via TLRs 7–9leads to the induction of IFN-α,which also plays a vital role in the clearance of viral pathogens.These observations highlight the complexity of the mechanisms underlying differential signaling by TLRs.
The identi?cation of the MyD88-and TRIF-dependent pathways helps explain some aspects of signaling speci?city from TLRs;however,additional TIR-containing adapters,TIRAP (TIR domain–containing adaptor protein)and TRAM (TRIF-related adaptor molecule),have been characterized as critical linkers between speci?c TLRs and MyD88and/or TRIF .Genetic ablation of these adapters has yielded signi?cant insights into their role in TLR signaling.
TIRAP/Mal.Noting the LPS responsive-ness of MyD88-de?cient cells,investiga-tors searched for alternate TIR domain–containing adaptors,resulting in the discovery of TIRAP ,also called Mal (MyD88-adaptor-like)(Horng et al.2001,Yamamoto et al.2002b).Researchers initially hypothesized that TIRAP was the adaptor responsible for MyD88-independent signaling;however,they later determined that this molecule acts as an adaptor in the MyD88-dependent path-way from TLRs 2and 4(Horng et al.2002,Oshiumi et al.2003a).
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In TIRAP-de?cient mice,signaling via TLR2/1or TLR2/6heterodimers and TLR4—but not TLRs 3,5,7,or 9—results in abrogated early-phase NF-κB activation and,consequently,severely atten-uated TNF α,IL-6,and IL-12p40produc-tion (Horng et al.2002,Yamamoto et al.2002a).As a result,TIRAP-de?cient mice are resistant to cytokine-mediated endotoxin shock (Yamamoto et al.2002a).However,LPS-induced activation of late-phase NF-κB and MAPKs as well as IFN-βproduction are unaffected,indicating that TIRAP is not involved in TRIF-dependent TLR4signal-ing (Fitzgerald et al.2001,Yamamoto et al.2002a).
Recently,Mansell et al.(2004)showed that TIRAP has a putative TRAF6interaction domain and that TIRAP coimmunoprecipi-tates with TRAF6.A mutation of the TIRAP TRAF6-binding motif leads to inhibition of TLR2-and TLR4-mediated NF-κB activa-tion,indicating that TIRAP may assist in bridging TLRs 2and 4to TRAF6and,sub-sequently,to IKK.Therefore,MyD88and TIRAP may function redundantly in signal-ing to NF-κB;however,this hypothesis seems unlikely,as overexpression of MyD88,and not TIRAP ,in MyD88?/?/TIRAP ?/?cells leads to NF-κB activation (Akira &T akeda 2004).Why TIRAP speci?cally interacts with TLR2and TLR4and why it is required to bridge MyD88and the TIR domains of TLRs 2and 4remain unclear.Because TLR4in-teracts with both MyD88and TRIF ,TIRAP binding to TLR4may preferentially recruit MyD88for early-phase NF-κB activity lead-ing to proin?ammatory cytokines,which may prohibit TRIF-dependent late-phase NF-κB and IFN-βproduction.Future work may yield a more thorough understanding of the role of TIRAP in TLR signaling.
TRAM/TIRP/TICAM-2.In addition to TIRAP ,the adaptor TRAM,also called TIR domain–containing protein (TIRP)/TIR-containing adaptor molecule-2(TICAM-2),also participates in signal transduction from certain TLRs.Several concurrent studies con-cluded that TRAM functions in the TLR4-mediated activation of IRF3,effectively bridging TRIF and TLR4(Fitzgerald et al.2003b,Oshiumi et al.2003b,Yamamoto et al.2003b).Using yeast two-hybrid analysis,in-vestigators determined that TRAM could not bind to the TIR domains of TLR2or TLRs 5–9but could bind to TLR4and weakly to TLR3(Oshiumi et al.2003b).Another study showed that TRAM is required for TLR4,but not TLR3,TRIF-dependent signaling,as dominant-negative TRAM and siRNA knockdown inhibited NF-κB and IRF3acti-vation in response to LPS,but not poly(I:C)(Fitzgerald et al.2003b).Likewise,cells from TRAM-de?cient mice display decreased cytokine production in response to LPS,yet they produce normal levels of cytokines in response to IL-1and TLR 2,3,7,and 9ligands (Yamamoto et al.2003b).MyD88-dependent LPS signaling is unaffected in TRAM-de?cient mice,but TRIF-mediated NF-κB activation and IFN-βproduction are abolished,therefore supporting the notion that TRAM bridges TRIF to TLR4,but not to TLR3.Finally,dominant-negative TRIF ,IKKi,and TBK-1prevent TRAM-induced activation of IRF3and NF-κB,indicating that TRAM functions upstream of these molecules (Oshiumi et al.2003b).
It remains unclear why TRAM is specif-ically utilized in TLR4signaling,although a recent study addressed the mechanism by which TRAM is speci?cally targeted to the cell membrane for TLR4signaling.TRAM contains an N-terminal myristoy-lation site that is critical for colocalization with TLR4,and mutation of this residue abrogates TRAM-induced signaling to IRF3and NF-κB (Rowe et al.2006).Never-theless,differential recruitment of TIRAP or TRAM may determine whether TLR4signaling proceeds through MyD88or TRIF .Varying degrees of receptor conformational change induced by different TLR4ligands may speci?cally recruit TRAM or TIRAP to the TIR domain,resulting in skewing
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Adaptive immunity:the delayed immune response in higher vertebrates that results in the clonal expansion of
antigen-speci?c B and T cells and the development of immunological memory for
long-lived protection against pathogens
toward MyD88-dependent proin?ammatory cytokine production or TRIF-dependent IFN-βproduction.This skewing may translate into a more tailored innate effec-tor response necessary for the priming of adaptive immunity and clearance of speci?c pathogens.Thus,TIR-containing adaptors provide an initial level of signaling speci?city from TLRs.
TLR-Mediated Induction of IRFs
T ype I IFNs (IFN-α/β)are integral compo-nents of innate antiviral responses,and their expression is governed by IRF transcription factors.T wo members of this family,IRF3and IRF7,are absolutely required for tran-scription of IFN-α/βgenes.IRF3is critical for both non-TLR-(via RIG-I/MDA5)and TLR3/4-mediated production of IFN-βvia TRIF ,and it is activated by upstream kinases TBK1and IKKi in many cell types (Fitzger-ald et al.2003a,Sato et al.2000).Upon vi-ral infection,latent IRF3is phosphorylated at C-terminal serine residues,which leads to its dimerization and subsequent translo-cation into the nucleus.Upon entering the nucleus,IRF3synergizes with coactivator molecules and binds DNA elements at the IFN-βpromoter to induce gene transcrip-tion.In contrast,IRF7is basally present only in plasmacytoid DCs,which are speci?cally adapted to detect viruses and synthesize IFN-αupon infection,but is strongly induced in many cells after viral infection and subse-quent type I IFN autocrine/paracrine signal-ing (Izaguirre et al.2003,Kerkmann et al.2003).Plasmacytoid DCs express TLRs 7,8,and 9in the endosomal compartment,and upon viral PAMP uptake and TLR signal-ing,IRF7,along with the αsubunit of IKK,is recruited to the MyD88/IRAK-1/TRAF6complex (Kawai et al.2004,Uematsu et al.2005).This complex licenses the kinase activ-ity of IKK α,which then mediates IRF7phos-phorylation and activation (Hoshino et al.2006).Phosphorylated IRF7then dimerizes and translocates into the nucleus,where it
acts to induce IFN-αexpression.Genetic ab-lation of IRF7illustrates that the produc-tion of both IFN-αfrom pDCs and IFN-βfrom embryonic ?broblasts in response to vi-ral infection/PAMPs is severely impaired,in-dicating that IRF7is the ultimate regulator of type I IFN responses in both stromal and hematopoietic compartments (Honda et al.2005).Therefore,speci?c TLR signaling re-sults in the differential induction of IRF3and/or IRF7for tailored antiviral responses.T wo recent articles suggest that TRAF3,not TRAF6,is critical for TRIF-and MyD88-dependent activation of IRF3and 7via TLR3/4and TLRs 7–9,respectively (Hacker et al.2006,Oganesyan et al.2006).TRAF3bridges TRIF with downstream kinases IKKi and TBK1for TLR3/4-dependent IRF3activation and IFN-βproduction,whereas it associates with IRAK-1,and presumably IKK α,for the TLR7–9-dependent activation of IRF7and IFN-αproduction (Oganesyan et al.2006).Thus,bifurication in the TLR signaling pathway occurs at the level of TRAF proteins,as differential utilization of TRAF6or TRAF3results in NF-κB activa-tion for proin?ammatory cytokine production or IRF activation for type I IFN production,respectively.
In addition to IRF7,IRF5is recruited to the MyD88/TRAF6complex,which me-diates its activation (Schoenemeyer et al.2005,T akaoka et al.2005).Upon activa-tion and nuclear translocation,IRF5binds IFN-stimulated response element motifs in the promoter regions of proin?ammatory genes,thereby promoting transcription.Con-sequently,DCs and macrophages from IRF5knockout mice display markedly decreased IL-6,IL-12,and TNF-αproduction in re-sponse to various TLR agonists,whereas IFN-αinduction in CpG-stimulated pDCs is normal (T akaoka et al.2005).These data indicate that IRF5serves as a gen-eral transducer that synergizes with NF-κB and other transcription factors for MyD88-dependent,proin?ammatory cytokine gene induction.
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Chromatin Remodeling and TLR Signaling Speci?city
TLR ligands induce the production of general proin?ammatory cytokines,such as TNF αand IL-6,but their binding to TLRs also elicits the production of factors that induce speci?c immune responses.For example,IL-12is critically important for DC/macrophage priming of T-helper type 1cells of the adap-tive immune system,and its production is dependent on TLR signaling.Because TLR signaling results in the general activation of transcription factors such as AP-1and NF-κB,it is somewhat unclear how the expres-sion of speci?c genes,such as IL-12,is con-trolled.Weinmann et al.(2001)provided some insight into this dilemma in a recent study,which showed that LPS stimulation of macrophages results in nucleosome remod-eling around the IL-12p40promoter.This region of DNA is normally masked when complexed tightly with its respective histones,and LPS/TLR4signaling initiates its un-masking for increased accessibility to tran-scription factors,presumably leading to en-hanced transcription of the IL-12p40gene.Another study further indicated that stimu-lation of bone marrow–derived DCs and a macrophage-like cell line with CpG DNA,LPS,or lipoteichoic acid resulted in the dif-ferential induction of IL-12p40when nor-malized to TNF αproduction (Albrecht et al.2004).CpG induced drastically higher IL-12expression than did LPS or L TA,although NF-κB activation was equivalent after treat-ment with each agonist.In addition,CpG in-duced modestly higher nucleosome remod-eling around the IL-12p40promoter,which possibly accounts for the differential cytokine production observed after ligand treatment.Therefore,nucleosome/chromatin remodel-ing may provide an additional level of TLR signaling speci?city.
Although neither study identi?ed the ex-act mechanism by which nucleosome remod-eling occurred,it is interesting to speculate that differential signaling from TLRs leads to the cytoplasmic activation of molecules that
enter or signal through the nucleus to in-duce chromatin restructuring,thus augment-ing or decreasing gene expression of spe-ci?c immunomodulatory factors.Concurrent with this hypothesis is the recent observa-tion that TLR4signaling induces the expres-sion of ATF3,a member of the ATF/CREB family of transcription factors.ATF3specif-ically decreases transcription of proin?am-matory cytokine genes Il6and Il12b by recruiting histone deacetylases to possibly al-ter chomatin structure at their promoters (Gilchrist et al.2006).This restricts access of NF-κB and AP-1to promoter regions and thus serves to negatively regulate TLR4-mediated proin?ammatory cytokine produc-tion.Future studies may yield further insight into the precise processes and signaling cas-cades that lead to TLR-induced chromatin remodeling.
NEGATIVE REGULATION OF TLR SIGNALING
As discussed above,TLR ligand recognition and adaptor speci?city result in the upregu-lation of speci?c proin?ammatory cytokines and chemokines for pathogen control and clearance;however,excessive in?ammation is extremely harmful or even fatal to the host.Bacterial sepsis,autoimmune disorders,and chronic in?ammatory diseases are examples of hyperimmune conditions that result from excessive,dysregulated activity of the innate and adaptive immune systems (Liew et al.2005).TLRs have been extensively implicated in the immunopathology of many of these diseases.Therefore,it is not surprising that the host has evolved mechanisms to modu-late the TLR-mediated innate immune re-sponse.Brie?y summarized below are some of the most well-characterized negative regu-lators of TLR signaling (Figure 3).
IRAK-M,a member of the IRAK family of serine/threonine kinases,is expressed only in monoctyes and macrophages and is up-regulated upon stimulation with TLR ago-nists (Wesche et al.1999).Unlike the other
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Figure 3
Negative regulation of TLR signaling.Negative regulators function to dampen TLR signaling,thus preventing prolonged and potentially
harmful innate immune responses.TOLLIP ,IRAK-M,and SOCS-1are cytoplasmic molecules that block IRAK-1activation and/or
IRAK-1-mediated TRAF6activation.SOCS-1may also mediate TIRAP degradation and/or act indirectly by downmodulating type I IFN
signaling induced by TLRs.T ransmembrane receptors such as SIGRR and ST2L also downregulate TLR signaling by either blocking MyD88/IRAK-1/4activation or sequestering MyD88,respectively.
IRAK family members IRAK-1and IRAK-4,IRAK-M lacks kinase activity,as key residues within its putative kinase domains are absent (Muzio et al.1997).Although the inhibitory mechanism of IRAK-M is somewhat unclear,this molecule appears to block formation of the IRAK-1/TRAF6complex,but not the re-cruitment of IRAK-1to MyD88(Kobayashi et al.2002).Therefore,IRAK-M may inhibit the dissociation of IRAK-1and IRAK-4from TLRs by either blocking their phosphoryla-tion or stabilizing the TLR/MyD88/IRAK-4complex such that TRAF6is excluded and sig-naling to NF-κB and/or MAPKs cannot pro-ceed.As such,IRAK-M may act to regulate signaling generally from MyD88-dependent TLRs.
SOCS-1is a member of the SOCS family of proteins,which play an important role in the suppression of cytokine signaling through the JAK-STAT pathway (Alexander 2002).Macrophages from SOCS-1-de?cient mice produce increased levels of proin?ammatory cytokines in response to LPS and CpG DNA as a result of enhanced STAT1,I κB α,p38,and JNK phosphorylation in these cells (Kinjyo et al.2002,Nakagawa et al.2002).SOCS-1may function by blocking IRAK-1activation,but more recent data suggest that it negatively regulates signaling via TLRs 2and 4through degradation of TIRAP (Mansell et al.2006).Upon TLR2/4signaling,TIRAP is phos-phorylated by Bruton’s tyrosine kinase and then interacts with SOCS-1,which mediates its polyubiquitination and subsequent protea-somal degradation.Alternatively,two recent studies indicate that SOCS-1-dependent inhi-bition of TLR signaling is indirect and occurs by a block or decrease in type I IFN autocrine/paracrine signaling following TLR-mediated IFN-α/βsecretion (Baetz et al.2004,Gingras et al.2004).
The adaptor protein TOLLIP (T oll-interacting protein)was originally character-ized for its ability to interact with the IL-1R accessory protein (Burns et al.2000).Subse-quent research showed that TOLLIP inter-acts with several TLRs,including TLRs 2and 4,and blocks their activation of NF-κB (Bulut et al.2001,Zhang &Ghosh 2002).Overex-pression of TOLLIP results in the formation of a complex with IRAK-1,yielding decreased autophosphorylation of IRAK-1(Zhang &Ghosh 2002).Therefore,TOLLIP primarily functions at the level of IRAK-1,maintaining the quiescence of unstimulated immune cells and facilitating termination of IL-1R/TLR-induced cell signaling during in?ammation and infection.Analysis of TOLLIP-de?cient mice,however,suggests that the negative regulatory mechanism of TOLLIP is more
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complex than initially proposed,as NF-κB and MAPK activation in knockout cells is nor-mal (Didierlaurent et al.2006).
Finally,in addition to cytoplasmic molecules,membrane-bound proteins such as SIGIRR/TIR8and ST2/T1may also play a role in modulating TLR signaling.The single immunoglobulin IL-1R-related protein (SIGIRR)is an orphan receptor that does not induce NF-κB activation,as it possesses an intracellular TIR domain that lacks two amino acids essential for signaling by IL-1RI (Thomassen et al.1999,Wald et al.2003).SIGIRR is expressed in different cell types ranging from epithelial cells to immature DCs,but it is not expressed in macrophages,?broblasts,or endothelial cells.DCs from SIGIRR-de?cient mice display enhanced NF-κB activity in response to LPS and CpG,but not poly(I:C),and although the exact mechanism by which SIGIRR functions is unclear,it appears to be recruited to TLR4to block signaling by sequestering IRAK-1and TRAF6(Garlanda et al.2004,Wald et al.2003).Likewise,ST2/T1,a membrane-bound member of the IL-1receptor family,may inhibit IL-1R-and TLR4-mediated signaling,but not TLR3signaling,by seques-tering the TIR-containing adaptors MyD88and TIRAP (Brint et al.2004).
FINAL THOUGHTS
The innate immune system provides ini-tial protection from pathogens while appro-
priately licensing and shaping the adaptive immune response.The ?eld of innate im-munity research has undergone a veritable rebirth since the discovery of mammalian TLRs.TLRs detect PAMPs from a variety of microorganisms and viruses,and a con-served set of adaptor molecules is utilized for signal transduction,which leads to the upregulation of costimulatory molecules and the production of proin?ammatory cytokines,chemokines,antimicrobial peptides,and type I IFNs.The complex binding and signaling mechanisms of TLRs result in tailored effec-tor responses to combat most pathogenic in-sults,and additional speci?city is provided by the differential expression of various adaptors,transcription factors,and TLRs.
The past eight years have yielded sub-stantial insight into the ligand speci?cities,structure,and signaling cascades of TLRs,al-though many fundamental questions remain (see below).Ultimately,the continued char-acterization of TLR-and adaptor-de?cient mice,in combination with in-depth biochem-ical analysis of signaling pathways,will be necessary to round out our knowledge of TLR biology.A complete understanding of the molecular mechanisms governing TLR recognition and signaling will allow develop-ment of therapeutic approaches that target re-sponses from certain TLRs while leaving the greater innate immune response intact,thus allowing modulation of potentially harmful disorders such as sepsis and chronic in?am-mation.
SUMMARY POINTS
1.TLRs are critically important innate immune receptors.Thirteen mammalian TLRs that bind a variety of conserved bacterial,viral,fungal,and protozoan PAMPs have been identi?ed.
2.The N termini of TLRs comprise multiple LRRs,which provide ligand speci?city.Multiple-residue insertions in nonconsensus LRRs likely diversify the PAMP binding potential of TLRs.
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3.Heterodimeric TLR pairs,as well as non-TLR accessory molecules,may increase and diversify ligand recognition and/or lead to the recruitment of different adaptors for varied signaling.
4.Signaling through TLRs 1,2,5–9,and 11proceeds exclusively through the MyD88,resulting in the recruitment of IRAK/TRAF6complexes and the subsequent activation of NF-κB and MAPKs for proin?ammatory cytokine production.
5.TLR4differentially utilizes MyD88and TRIF ,and the TRIF-dependent pathway leads to the activation of IRF3for IFN-βproduction and delayed NF-κB activation.In addition,TLR3signaling is mediated exclusively by TRIF .
6.TIRAP and TRAM,two additional TIR-containing adaptors,bridge TLR4as well as MyD88and TRIF ,respectively.TIRAP also links TLR2to MyD88.
7.T ype I IFN production from both stromal and hematopoietic cells is regulated by the activation of the transcription factors IRF3and IRF7,whereas IRF5functions with NF-κB to induce the expression of proin?ammatory genes.
8.T o limit potent in?ammatory responses induced by TLRs,negative regulatory molecules exist to downregulate TLR signaling.
FUTURE ISSUES
1.Crystallographic analyses of complexes of PAMPs and their respective TLRs do not exist.Therefore,it remains unclear whether all TLRs bind their ligands directly,or whether other binding components are required.Structural studies of additional TLR ectodomains,possibly complexed with other TLRs or their ligands,may provide insight into the exact binding mechanisms of these receptors.
2.Still unclear is whether signaling molecules,such as MyD88and IRAK,are recruited to TLRs after PAMP binding,or whether they are always bound to their receptors.Knowledge of the exact nature of resting and active TLR complexes is critical for a complete understanding of signaling speci?city.
3.Adaptors TIRAP and TRAM are mainly required for signal transduction from TLR4,and why this receptor utilizes these molecules in addition to MyD88and TRIF is unclear.A thorough understanding of the functional signi?cance of these adaptors should yield additional information about the signaling speci?city of TLRs.
4.The extent to which TLR signaling induces chromatin/nucleosome remodeling,and thus promoter accessibility,at cytokine loci is unclear.Future work should characterize molecules that potentially signal to chromatin remodeling complexes.
ACKNOWLEDGMENTS
We thank members of our laboratory,notably Matthew Hayden,Elizabeth Ziegler,and Ian Strickland,for critically reviewing this manuscript.Research in the authors’laboratory is sup-ported by grants from the NIH (R37-AI33443and RO1-AI59440).
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第1章 信号与系统的基本概念 1.1 引言 系统是一个广泛使用的概念，指由多个元件组成的相互作用、相互依存的整体。我们学习过“电路分析原理”的课程，电路是典型的系统，由电阻、电容、电感和电源等元件组成。我们还熟悉汽车在路面运动的过程，汽车、路面、空气组成一个力学系统。更为复杂一些的系统如电力系统，它包括若干发电厂、变电站、输电网和电力用户等，大的电网可以跨越数千公里。 我们在观察、分析和描述一个系统时，总要借助于对系统中一些元件状态的观测和分析。例如，在分析一个电路时，会计算或测量电路中一些位置的电压和电流随时间的变化；在分析一个汽车的运动时，会计算或观测驱动力、阻力、位置、速度和加速度等状态变量随时间的变化。系统状态变量随时间变化的关系称为信号，包含了系统变化的信息。 很多实际系统的状态变量是非电的，我们经常使用各种各样的传感器，把非电的状态变量转换为电的变量，得到便于测量的电信号。 隐去不同信号所代表的具体物理意义，信号就可以抽象为函数，即变量随时间变化的关系。信号用函数表示，可以是数学表达式，或是波形，或是数据列表。在本课程中，信号和函数的表述经常不加区分。 信号和系统分析的最基本的任务是获得信号的特点和系统的特性。系统的分析和描述借助于建立系统输入信号和输出信号之间关系，因此信号分析和系统分析是密切相关的。 系统的特性千变万化，其中最重要的区别是线性和非线性、时不变和时变。这些区别导致分析方法的重要差别。本课程的内容限于线性时不变系统。 我们最熟悉的信号和系统分析方法是时域分析，即分析信号随时间变化的波形。例如，对于一个电压测量系统，要判断测量的准确度，可以直接分析比较被测的电压波形)(in t v （测量系统输入信号）和测量得到的波形)(out t v （测量系统输出信号），观察它们之间的相似程度。为了充分地和规范地描述测量系统的特性，经常给系统输入一个阶跃电压信号，得到系统的阶跃响应，图1-1是典型的波形，通过阶跃响应的电压上升时间（电压从10％上升至90％的时间）和过冲（百分比）等特征量，表述测量系统的特性，上升时间和过冲越小，系统特性越好。其中电压上升时间反映了系统的响应速度，小的上升时间对应快的响应速度。如果被测电压快速变化，而测量系统的响应特性相对较慢，则必然产生较大的测量误差。 信号与系统分析的另一种方法是频域分析。信号频域分析的基本原理是把信号分解为不
信号转导 061M5007H 学期：2015-2016学年秋| 课程属性：| 任课教师：谢旗等 教学目的、要求 本课程为细胞生物学专业研究生的专业基础课，同时也可作为相关专业研究生的选修课。细胞信号转导是细胞生物学学科进展最快的研究领域之一，信号转导的概念已经开始深入到生命科学的各个领域。本课程内容涵盖动植物受体、G蛋白、环核苷酸第二信使、质膜磷脂代谢产物胞内信使、酶活性受体、蛋白质可逆磷酸化、泛素蛋白化及其对基因表达的调控、信号转导途径的多样性、网络化和专一性等方面的研究现状和进展。 预修课程 生物化学、分子生物学 教材 生命科学学院 主要内容 第一章绪论（3学时，教师：谢旗）细胞信号转导的研究对象和研究意义，细胞信号的主要种类，细胞化学信号分子与信号传递途径的特征。真核生物的蛋白激酶，蛋白磷酸酶，蛋白质可逆磷酸化对信号转导的调节方式，蛋白质可逆磷酸化与基因表达调控，蛋白质可逆磷酸化在细胞信号中的意义。蛋白质稳定性与信号转导。第二章植物免疫的表观遗传调控（3学时，教师：郭惠珊）表观遗传调控包含RNA干扰、DNA修饰、组蛋白翻译后修饰和染色质重塑等各种过程互相交叠，共同调控基因组表观修饰的动态平衡；除了影响生长和发育，表观遗传调控的另一重要功能是抗病免疫作用。本讲将着重介绍植物表观遗传途径及其抗病免疫信号的调控作用。第三章MicroRNA介导的信号（3学时，教师：郭惠珊）microRNA 广泛存在于生物体内，是生物体保守机制RNA沉默过程产生并具有序列特异性调控功能的一类非编码小分子RNA。本课程主要讲授植物microRNA的产生、加工、特性及其调控作用的基本生物学过程；以及植物miRNAs和其他小分子RNA参与植物生长素信号途径和其他植物生理性状的调控作用。第四章钙离子通道及信号转导（3学时，教师：陈宇航）钙离子是生命活动的必需元素，基本分布和内稳，代谢平衡和疾病；钙离子发挥重要生物学功能，简述历史发现，作为第二信使的化学基础，功能调控的基本模式，以钙结合蛋白为例子展开介绍钙离子发挥功能调控的分子结构基础等；介绍钙离子信号转导系统的组成，
细胞信号转导途径研究方法 一、蛋白质表达水平和细胞内定位研究 1、信号蛋白分子表达水平及分子量检测: Western blot analysis. 蛋白质印迹法是将蛋白质混合样品经SDS-PAGE后,分离为不同条带，其中含有能与特异性抗体(或McAb)相应的待检测的蛋白质(抗原蛋白)，将PAGE胶上的蛋白条带转移到NC膜上此过程称为blotting，以利于随后的检测能够的进行，随后,将NC膜与抗血清一起孵育，使第一抗体与待检的抗原决定簇结合(特异大蛋白条带)，再与酶标的第二抗体反应,即检测样品的待测抗原并可对其定量。 基本流程： 检测示意图：
2、免疫荧光技术 Immunofluorescence （IF） 免疫荧光技术是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光素制成荧光标记物，再用这种荧光抗体（或抗原）作为分子探针检查细胞或组织内的相应抗原（或抗体）。在细胞或组织中形成的抗原抗体复合物上含有荧光素，利用荧光显微镜观察标本，荧光素受激发光的照射而发出明亮的荧光（黄绿色或桔红色），可以看见荧光所在的细胞或组织，从而确定抗原或抗体的性质、定位，以及利用定量技术测定含量。 采用流式细胞免疫荧光技术（FCM）可从单细胞水平检测不同细胞亚群中的蛋白质分子，用两种不同的荧光素分别标记抗不同蛋白质分子的抗体，可在同一细胞内同时检测两种不同的分子（Double IF），也可用多参数流式细胞术对胞内多种分子进行检测。 二、蛋白质与蛋白质相互作用的研究技术 1、免疫共沉淀（Co- Immunoprecipitation, Co-IP）
Co-IP是利用抗原蛋白质和抗体的特异性结合以及细菌蛋白质的“protein A”能特异性地结合到免疫球蛋白的FC片段的现象而开发出来的方法。目前多用精制的protein A预先结合固化在agarose的beads 上，使之与含有抗原的溶液及抗体反应后，beads上的prorein A就能吸附抗原抗体达到沉淀抗原的目的。 当细胞在非变性条件下被裂解时，完整细胞内存在的许多蛋白质－蛋白质间的相互作用被保留了下来。如果用蛋白质X的抗体免疫沉淀X，那么与X在体内结合的蛋白质Y也能沉淀下来。进一步进行Western Blot 和质谱分析。这种方法常用于测定两种目标蛋白质是否在体内结合，也可用于确定一种特定蛋白质的新的作用搭档。缺点：可能检测不到低亲和力和瞬间的蛋白质－蛋白质相互作用。 2、GST pull-down assay GST pull-down assay是将谷胱甘肽巯基转移酶（GST）融合蛋白（标记蛋白或者饵蛋白，GST, His6, Flag, biotin …）作为探针，与溶液中的特异性搭档蛋白(test protein或者prey被扑获蛋白)结合，然后根据谷胱甘肽琼脂糖球珠能够沉淀GST融合蛋白的能力来确定相互作用的蛋白。一般在发现抗体干扰蛋白质－蛋白质之间的相互作用时，可以启用GST沉降技术。该方法只是用于确定体外的相互作用。
信号与系统复习题 1、描述某系统的微分方程为 y ”(t) + 5y ’(t) + 6y(t) = f (t) y(0_)=2，y ’(0_)= -1 y(0_)= 1，y ’(0_)=0 求系统的零输入响应。 求系统的冲击相应 求系统的单位阶跃响应。 解： 2、系统方程 y (k)+ 4y (k – 1) + 4y (k – 2) = f (k) 已知初始条件y (0)=0，y (1)= – 1；激励k k f 2)(=，k ≥0。求方程的解。 解：特征方程为 λ2 + 4λ+ 4=0 可解得特征根λ1=λ2= – 2，其齐次解 y h(k )=(C 1k +C 2) (– 2)k 特解为 y p(k )=P (2)k , k ≥0 代入差分方程得 P (2)k +4P (2)k –1+4P (2)k –2= f (k ) = 2k ， 解得 P =1/4 所以得特解： y p(k )=2k –2 , k ≥0 故全解为 y (k )= y h+y p = (C 1k +C 2) (– 2)k + 2k –2 , k ≥0 代入初始条件解得 C 1=1 , C 2= – 1/4 3、系统方程为 y (k) + 3y (k –1) + 2y (k –2) = f (k) 已知激励k k f 2)(=, k ≥0，初始状态y (–1)=0, y (–2)=1/2, 求系统的零输入响应、零状态响应和全响应。 解：：（1）y zi(k )满足方程 y zi(k ) + 3y zi(k –1)+ 2y zi(k –2)= 0 y zi(–1)= y (–1)= 0, y zi(–2) = y (–2) = 1/2 首先递推求出初始值y zi(0), y zi(1), y zi(k )= – 3y zi(k –1) –2y zi(k –2) y zi(0)= –3y zi(–1) –2y zi(–2)= –1 y zi(1)= –3y zi(0) –2y zi(–1)=3 特征根为λ1= –1 ，λ2= – 2 解为 y zi(k )=C zi1(– 1)k + C zi2(–2)k 将初始值代入 并解得 C zi1=1 , C zi2= – 2 y zi(k )=(– 1)k – 2(– 2)k , k ≥0 （2）零状态响应y zs(k ) 满足：y zs(k ) + 3y zs(k –1) + 2y zs(k –2) = f (k ) y zs(–1)= y zs(–2) = 0 递推求初始值 y zs(0), y zs(1)， y zs(k ) = – 3y zs(k –1) – 2y zs(k –2) + 2k , k ≥0 y zs(0) = – 3y zs(–1) – 2y zs(–2) + 1 = 1 y zs(1) = – 3y zs(0) – 2y zs(–1) + 2 = – 1
第十五章细胞信号转导 复习测试 （一）名词解释 1. 受体 2. 激素 3. 信号分子 4. G蛋白 5. 细胞因子 6. 自分泌信号传递 7. 蛋白激酶 8. 钙调蛋白 9. G蛋白偶联型受体 10. 向上调节 11. 细胞信号转导途径 12. 第二信使 （二）选择题 A型题： 1. 关于激素描述错误的是： A. 由内分泌腺/细胞合成并分泌 B. 经血液循环转运 C. 与相应的受体共价结合 D. 作用的强弱与其浓度相关 E. 可在靶细胞膜表面或细胞内发挥作用 2. 下列哪种激素属于多肽及蛋白质类： A. 糖皮质激素 B. 胰岛素 C. 肾上腺素 D. 前列腺素 E. 甲状腺激素 3. 生长因子的特点不包括： A. 是一类信号分子 B. 由特殊分化的内分泌腺所分泌 C. 作用于特定的靶细胞 D. 主要以旁分泌和自分泌方式发挥作用 E. 其化学本质为蛋白质或多肽 4. 根据经典的定义，细胞因子与激素的主要区别是： A. 是一类信号分子 B. 作用于特定的靶细胞 C. 由普通细胞合成并分泌 D. 可调节靶细胞的生长、分化 E. 以内分泌、旁分泌和自分泌方式发挥作用 5. 神经递质、激素、生长因子和细胞因子可通过下列哪一条共同途径传递信号：
A. 形成动作电位 B. 使离子通道开放 C. 与受体结合 D. 通过胞饮进入细胞 E. 自由进出细胞 6. 受体的化学本质是： A. 多糖 B. 长链不饱和脂肪酸 C. 生物碱 D. 蛋白质 E. 类固醇 7. 受体的特异性取决于： A. 活性中心的构象 B. 配体结合域的构象 C. 细胞膜的流动性 D. 信号转导功能域的构象 E. G蛋白的构象 8. 关于受体的作用特点，下列哪项是错误的： A. 特异性较高 B. 是可逆的 C. 其解离常数越大，产生的生物效应越大 D. 是可饱和的 E. 结合后受体可发生变构 9. 下列哪项与受体的性质不符： A. 各类激素有其特异性的受体 B. 各类生长因子有其特异性的受体 C. 神经递质有其特异性的受体 D. 受体的本质是蛋白质 E. 受体只存在于细胞膜上 10. 下列哪种受体是催化型受体： A. 胰岛素受体 B. 甲状腺激素受体 C. 糖皮质激素受体 受体 D. 肾上腺素能受体 E. 活性维生素D 3 11. 酪氨酸蛋白激酶的作用是： A. 使蛋白质结合上酪氨酸 B. 使含有酪氨酸的蛋白质激活 C. 使蛋白质中的酪氨酸激活 D. 使效应蛋白中的酪氨酸残基磷酸化 E. 使蛋白质中的酪氨酸分解 12. 下列哪种激素的受体属于胞内转录因子型： A. 肾上腺素 B. 甲状腺激素 C. 胰岛素 D. 促甲状腺素 E. 胰高血糖素
细胞信号转导练习题 选择题：正确答案可能不止一个 1. NO直接作用于(B) A.腺苷酸环化酶 B.鸟苷酸环化酶 C.钙离子门控通道D．PKC 2.以下哪一类细胞可释放NO( B) A.心肌细胞 B.血管内皮细胞 C.血管平滑肌细胞 3.硝酸甘油作为治疗心绞痛的药物是因为它( C) A.具有镇痛作用 B.抗乙酰胆碱 C.能在体内转换为NO 4.胞内受体(A B) A.是一类基因调控蛋白 B.可结合到转录增强子上 C.是一类蛋白激酶 D.是一类第二信使 5.受体酪氨酸激酶RPTK( A B C D) A.为单次跨膜蛋白 B.接受配体后发生二聚化 C.能自磷酸化胞内段 D.可激活Ras 6. Sos属于(B) A.接头蛋白（adaptor protein） B.Ras的鸟苷酸交换因子（GEF） C.Ras的GTP酶活化蛋白（GAP）D：胞内受体 7.以下哪些不属于G蛋白(C)
A.Ras B.微管蛋白β亚基 C.视蛋白 D. Rho 8. PKC以非活性形式分布于细胞溶质中，当细胞之中的哪一种离子浓度升高时，PKC转位到质膜内表面(B) A.镁离子 B.钙离子 C.钾离子 D.钠离子 9.Ca2+载体——离子霉素（ionomycin）能够模拟哪一种第二信使的作用(A) A.IP3 B.IP2 C.DAG D.cAMP 10.在磷脂酰肌醇信号通路中，质膜上的磷脂酶C（PLC-β）水解4，5-二磷酸磷脂酰肌醇（PIP2），产生哪两个两个第二信使(A B) A.1，4，5-三磷酸肌醇（IP3） B.DAG C.4，5-二磷酸肌醇（IP2） 11.在磷脂酰肌醇信号通路中，G蛋白的直接效应酶是(B) A.腺苷酸环化酶 B.磷脂酶C-β C.蛋白激酶C D. 鸟苷酸环化酶 12.蛋白激酶A（Protein Kinase A，PKA）由两个催化亚基和两个调节亚基组成，cAMP能够与酶的哪一部分结合？(B) A.催化亚基 B.调节亚基 13.在cAMP信号途径中，环腺苷酸磷酸二酯酶（PDE）的作用是 (C) A.催化ATP生成cAMP B.催化ADP生成cAMP C.降解cAMP生成5’-AMP 14.在cAMP信号途径中，G蛋白的直接效应酶是(B)
G蛋白在信号转导中的作用 摘要：G蛋白是一种特殊的调节蛋白，它们都具有GTP结合位点，且活性受GTP的调节。G蛋白以其特定的方式偶联许多膜受体及其效应器，其中包括腺苷酸环化酶，cGMP磷酸二酯酶（PDE），离子通道以及磷脂肌醇特异的磷脂酶C（PLC）等，是跨膜信息传递机制中的一个关键因素。G蛋白也称GTP酶开关蛋白，属于GTP酶超大家族中的特殊亚型，可通过结合或水解GTP进行活性控制，是一类广泛分布在细胞中，并在许多生物学过程中执行重要功能的一类蛋白。G蛋白介导的信号转导系统是细胞中最常见的信号传递方式，G蛋白参与了G蛋白偶联受体所介导的信号转导途径和酶联受体信号传导途径，在信号转导中发挥的重要的作用。 关键词：G蛋白，信号转导，G蛋白偶联受体 G蛋白的种类和基本结构： G蛋白是一类能与鸟嘌呤核苷酸结合、具有GTP酶(GTPase)活性的蛋白。G蛋白位于质膜胞质侧，是一个超级家族，包括异源三聚体G蛋白(heterotrimeric G protein ) 或称大G蛋白和小G蛋白( Small G protein)。异源三聚G蛋白( heterotrmieric GTP binding protein )，由α，β，γ三个亚基组成。它变动于它的GDP形式(对环化酶无活性)及它的GTP 形式(有活性) 之间。根据不同的a亚基的功能特性可将大G蛋白分为四类：(1) Gs：其活性能被霍乱毒素抑制；(2) Gi：对腺苷酸环化酶有抑制效应；(3) Gq：百日咳毒素和霍乱毒素不能调节其活性；(4) G12：活化需通过血栓素和凝酶素的介导。目前已经确定了23种Gα，5种Gβ，10种Gγ，这样体内就有上千种G蛋白三聚体组合的可能性，这无疑增加了信号转导的可变性和灵活性。小分子G蛋白，它们的激活不是直接通过与激动型的G蛋白偶联受体相互作用而调节其活性，而是通过鸟嘌呤核苷交换因子（GEF）来控制这类小分子G蛋白的GTP交换，由GEF催化这类小分子单聚体G蛋白的无活性GDP结合状态向有活性的GTP结合状态转换。根据这类小分子G蛋白的蛋白质序列和功能的相似性，可分为Ras、Rho、Rab、Arf等亚家族。 Gα亚基为一多肽单链，含有一个GTP酶区( 结合和水解GTP ) 和一个α螺旋区( 该区将GTP埋藏在G蛋白的核心内) 。每种G蛋白的a亚基都有其独特的氨基酸序列和结构，但也都有一定的同源性，即5个关键功能区。它的N 端与βγ二聚体结合，C端参与和受体的相互作用，而与效应器结合的部位在他的功能区。Gβ亚基具有许多WD一40 (由β一片层结构组成的Trp - Asp结构域)和GH ( Gly - His )重复的保守序列形成的结构域，Gγ为伸展的单条链，与Gα和Gβ都紧密相连。在天然状态下，β和γ亚基以非共价键紧密结合在一起形成二聚体，只有在变性的条件下才能将其分离。 G蛋白作用过程中的分子机理，在受体未收到激素的作用之前，G蛋白与受体是各自分开的。作为基态，G蛋白以αβγ三聚体的形式存在，并有GDP结合在α亚基上。激素与受体的相互作用，导致激素·受体复合物与G蛋白结合，从而改变了G蛋白的构象，使α亚基上的鸟苷酸结合位点打开，GDP解离下来。在胞内GTP浓度较低时，由此可分离得到较为稳定的激素·受体·G蛋白高亲和态复合物，在胞内GTP浓度较高的情况下，GTP很容易结合到鸟苷酸结合位点上去。GTP结合导致G蛋白构象的进一步变化。
信号与系统重点概念公 式总结 文档编制序号：[KKIDT-LLE0828-LLETD298-POI08]
信号与系统重点概念及公式总结： 第一章：概论 1.信号：信号是消息的表现形式。（消息是信号的具体内容） 2.系统：由若干相互作用和相互依赖的事物组合而成的具有特定功能的整体。 第二章：信号的复数表示： 1.复数的两种表示方法：设C 为复数，a 、b 为实数。 常数形式的复数C=a+jb a 为实部，b 为虚部； 或C=|C|e j φ，其中，22||b a C +=为复数的模，tan φ=b/a ，φ为复 数的辐角。（复平面） 2.欧拉公式：wt j wt e jwt sin cos +=（前加-，后变减） 第三章：正交函数集及信号在其上的分解 1.正交函数集的定义：设函数集合)}(),(),({21t f t f t f F n = 如果满足：n i K dt t f j i dt t f t f i T T i T T j i 2,1)(0)()(2 1 21 2==≠=?? 则称集合F 为正交函数集 如果n i K i ,2,11==，则称F 为标准正交函数集。 如果F 中的函数为复数函数 条件变为：n i K dt t f t f j i dt t f t f i T T i i T T j i 2,1)()(0)()(21 21* * ==?≠=???
其中)(*t f i 为)(t f i 的复共轭。2.正交函数集的物理意义： 一个正交函数集可以类比成一个坐标系统； 正交函数集中的每个函数均类比成该坐标系统中的一个轴； 在该坐标系统中，一个函数可以类比成一个点； 点向这个坐标系统的投影（体现为该函数与构成坐标系的函数间的点积）就是该函数在这个坐标系统中的坐标。 3.正交函数集完备的概念和物理意义： 如果值空间中的任一元素均可以由某正交集中的元素准确的线性表出，我们就称该正交集是完备的，否则称该正交集是不完备的。 如果在正交函数集()()()()t g n ,t g ,t g ,t g 321之外，不存在函数x （t ） ()∞<
第六章?同化物的运输分配及信号传导单元自测 (一)填空 1．根据运输距离的长短，可将高等植物体内的运输可分为?????????????? 距离运输和?????????????? 距离运输。（短，长） 2．一般认为，胞间连丝有三种状态：(1)?????????????? 态，(2)?????????????? 态，(3)?????????????? 态。一般地说，细胞间的胞间连丝多、孔径大，存在的浓度梯度大，则?????????????? 于共质体的运输。（正常，开放，封闭，有利） 3．物质进出质膜的方式有三种：(1)顺浓度梯度的?????????????? 转运，(2)逆浓度梯度的??????????????转运，(3)依赖于膜运动的??????????????转运。(被动，主动，膜动) 4．以小囊泡方式进出质膜的膜动转运包括?????????????? ，?????????????? 和?????????????? 三种形式。(内吞，外排，出胞) 5．一个典型的维管束可由四部分组成：(1)以导管为中心，富有纤维组织的?????????????? ，(2)以筛管为中心，周围有薄壁组织伴联的?????????????? ，(3)穿插木质部和韧皮部间及四周的多种?????????????? ，(4)包围木质部和韧皮部。(木质部，韧皮部，细胞，维管束鞘) 6．目前测定韧皮部运输速度的常用的方法有两种。一种是利用?????????????? 作为示踪物，用显微注射技术将这种分子直接注入筛管分子内，追踪这种分子在筛管中的运输状况，根据单位时间中此分子的移动距离来计算运输速度。另一种是?????????????? 同位素示踪技术，常用的同位素是?????????????? 。将它的化合物饲喂叶片，然后追踪化合物在筛管中的运输状况、运输速度，用这种技术还可研究同化物的分配动态。(染料分子，放射性，14C） 8．筛管中糖的主要运输形式是?????????????? 糖和??????????????糖。(寡聚糖（棉子糖、水苏糖、毛蕊花糖等），蔗糖) 9．光合同化物在韧皮部的装载要经过三个区域：即（1）光合同化物?????????????? 区，指能进行光合作用的叶肉细胞；（2）同化物??????????????区，指小叶脉末端的韧皮部的薄壁细胞；（3）同化物?????????????? 区，指叶脉中的SE-CC。（生产，累积，输出，） 10．质外体装载是指?????????????? 细胞输出的蔗糖先进入质外体，然后通过位于SE-CC复合体质膜上的蔗糖载体??????????????蔗糖浓度梯度进入伴胞，最后进入筛管的过程。共质体装载途径是指??????????????细胞输出的蔗糖通过胞间连丝??????????????浓度梯度进入伴胞或中间细胞，最后进入筛管的过程。（光合，逆浓度，光合，顺蔗糖浓度） 11．韧皮部卸出的途径有两条：一条是?????????????? 途径，另一条是??????????????途径。（共质体，质外体） 12．光合碳代谢形成的磷酸丙糖可继续参与卡尔文循环的运转，或滞留在?????????????? 内，并在一系列酶作用下合成淀粉；或者通过位于叶绿体被膜上的?????????????? 进入细胞质，再在一系列酶作用下合成蔗糖。(叶绿体，磷酸丙糖转运器) 13．1930年E、Münch提出了解释韧皮部同化物运输的??????????????学说。该学说的基本论点是，同化物在筛管内是随液流流动的，而液流的流动是由?????????????? 两端的膨压差引起的。（压力流，输导系统） 14．转化酶是催化蔗糖?????????????? 反应的酶。根据催化反应所需的最适pH，可将转化酶分成两种，一种称为?????????????? 转化酶，该酶对底物蔗糖的亲和力较高，主要分布在液泡和细胞壁中；另一类称为?????????????? 转化酶，该酶主要分布在细胞质部分。(水解，酸性，碱性或中性) 15．光合细胞中蔗糖的合成是在?????????????? 内进行的。催化蔗糖降解代谢的酶有两类，一类是?????????????? ，另一类是?????????????? 。（细胞质，转化酶，蔗糖合成酶） 16．库细胞中淀粉合成的部位是?????????????? 。G1P在?????????????? 酶的作用下形成ADPG，
第十一章细胞的信号转导 一、名词解释 1、细胞通讯 2、受体 3、第一信使 4、第二信使 5、G 蛋白 6、蛋白激酶A 二、填空题 1、细胞膜表面受体主要有三类即、、和。 2、在细胞的信号转导中，第二信使主要有、、、和。 3、硝酸甘油之所以能治疗心绞痛是因为它在体内能转化为，引起血管，从而减轻的负荷和的需氧量。 三、选择题 1、能与胞外信号特异识别和结合，介导胞内信使生成，引起细胞产生效应的是( )。 A、载体蛋白 B、通道蛋白 C、受体 D、配体 2、下列不属于第二信使的是（）。 A、cAMP B、cGMP C、DG D、CO 3、下列关于信号分子的描述中，不正确的一项是（）。 A、本身不参与催化反应 B、本身不具有酶的活性 C、能够传递信息 D、可作为酶作用的底物 4、生长因子是细胞内的（）。 A、结构物质 B、能源物质 C、信息分子 D、酶 5、肾上腺素可诱导一些酶将储藏在肝细胞和肌细胞中的糖原水解，第一个被激活的酶是（）。 A、蛋白激酶A B、糖原合成酶 C、糖原磷酸化酶 D、腺苷酸环化酶 6、（）不是细胞表面受体。 A、离子通道 B、酶连受体 C、G蛋白偶联受体 D、核受体 7、动物细胞中cAMP的主要生物学功能是活化（）。 A、蛋白激酶C B、蛋白激酶A C、蛋白激酶K D、Ca2+激酶 8、在G蛋白中，α亚基的活性状态是（）。 A、与GTP结合，与βγ分离 B、与GTP结合，与βγ聚合 C、与GDP结合，与βγ分离 D、与GDP结合，与βγ聚合
9、下面关于受体酪氨酸激酶的说法哪一个是错误的 A、是一种生长因子类受体 B、受体蛋白只有一次跨膜 C、与配体结合后两个受体相互靠近，相互激活 D、具有SH2结构域 10、在与配体结合后直接行使酶功能的受体是 A、生长因子受体 B、配体闸门离子通道 C、G蛋白偶联受体 D、细胞核受体 11、硝酸甘油治疗心脏病的原理在于 A、激活腺苷酸环化酶，生成cAMP B、激活细胞膜上的GC，生成cGMP C、分解生成NO，生成cGMP D、激活PLC，生成DAG 12、霍乱杆菌引起急性腹泻是由于 A、G蛋白持续激活 B、G蛋白不能被激活 C、受体封闭 D、蛋白激酶PKC功能异常 13下面由cAMP激活的酶是 A、PTK B、PKA C、PKC D、PKG 14下列物质是第二信使的是 A、G蛋白 B、NO C、GTP D、PKC 15下面关于钙调蛋白（CaM）的说法错误的是 A、是Ca2+信号系统中起重要作用 B、必须与Ca2+结合才能发挥作用 C、能使蛋白磷酸化 D、CaM激酶是它的靶酶之一16间接激活或抑制细胞膜表面结合的酶或离子通道的受体是 A、生长因子受体 B、配体闸门离子通道 C、G蛋白偶联受体 D、细胞核受体 17重症肌无力是由于 A、G蛋白功能下降
细胞受体类型、特点 及重要的细胞信号转导途径 学院：动物科学技术学院 专业：动物遗传育种与繁殖 姓名：李波
学号：2015050509
目录 1、细胞受体类型及特点 (4) 1.1离子通道型受体 (4) 1.2 G蛋白耦联型受体 (4) 1.3 酶耦联型受体 (5) 2、重要的细胞信号转导途径 (5) 2.1细胞内受体介导的信号传递 (5) 2.2 G蛋白偶联受体介导的信号转导 (6) 2.2.1激活离子通道的G蛋白偶联受体所介导的信号通路 (7) 2.2.2激活或抑制腺苷酸环化酶的G蛋白偶联受体 (7) 2.2.3 激活磷脂酶C、以lP3和DAG作为双信使 G蛋白偶联受体介导的信号通 路 (8) 2.2 酶联受体介导的信号转导 (9) 2.2.1 受体酪氨酸激酶及RTK-Ras蛋白信号通路 (10) 2.2.2 P13K-PKB(Akt)信号通路 (10) 2.2.3 TGF-p—Smad信号通 (11) 2.2.4 JAK—STAT信号通路 (12)
1、细胞受体类型及特点 受体(receptor)是一种能够识别和选择性结合某种配体(信号分子)的大分子物质，多为糖蛋白，一般至少包括两个功能区域，与配体结合的区域和产生效应的区域，当受体与配体结合后，构象改变而产生活性，启动一系列过程，最终表现为生物学效应。受体与配体问的作用具有3个主要特征：①特异性；②饱和性；③高度的亲和力。 根据靶细胞上受体存在的部位，可将受体分为细胞内受体(intracellular receptor)和细胞表面受体(cell surface receptor)。细胞内受体介导亲脂性信号分子的信息传递，如胞内的甾体类激素受体。细胞表面受体介导亲水性信号分子的信息传递，膜表面受体主要有三类：①离子通道型受体(ion—channel—linked receptor)；②G蛋白耦联型受体(G—protein —linked receptor)；③酶耦联的受体(enzyme—linked recep—tor)。第一类存在于可兴奋细胞。后两类存在于大多数细胞，在信号转导的早期表现为激酶级联事件，即为一系列蛋白质的逐级磷酸化，借此使信号逐级传送和放大。 1.1离子通道型受体 离子通道型受体是一类自身为离子通道的受体，即配体门通道(1igand—gated channel)，主要存在于神经、肌肉等可兴奋细胞，其信号分子为神经递质。神经递质通过与受体的结合而改变通道蛋白的构象，导致离子通道的开启或关闭，改变质膜的离子通透性，在瞬间将胞外化学信号转换为电信号，继而改变突触后细胞的兴奋性。如：乙酰胆碱受体以三种构象存在，两分子乙酰胆碱的结合可以使之处于通道开放构象，但该受体处于通道开放构象状态的时限仍十分短暂，在几十毫微秒内又回到关闭状态。然后乙酰胆碱与之解离，受体则恢复到初始状态，做好重新接受配体的准备。离子通道型受体分为阳离子通道，如乙酰胆碱、谷氨酸和五羟色胺的受体，和阴离子通道。 1.2 G蛋白耦联型受体 三聚体GTP结合调节蛋白(trimeric GTP—binding regulatory protein)简称G蛋白，位于质膜胞质侧，由a、p、-／三个亚基组成，a和7亚基通过共价结合的脂肪酸链尾结合在膜上，G蛋白在信号转导过程中起着分子开关的作用，当a亚基与GDP结合时处于关闭状态，与GTP结合时处于开启状态，“亚基具有GTP酶活性，能催化所结合的ATP 水解，恢复无活性的三聚体状态，其GTP酶的活性能被RGS(regulator of G protein signaling)增强。RGS也属于GAP(GTPase activating protein)。 G蛋白耦联型受体为7次跨膜蛋白(图10—6)，受体胞外结构域识别胞外信号分子并与之结合，胞内结构域与G蛋白耦联。通过与G蛋白耦联，调节相关酶活性，在细胞内
南京信息工程大学2012年研究生招生入学考试 《信号与系统》考试大纲 科目代码：814 科目名称：信号与系统 第一部分课程目标与基本要求 一、课程目标 “信号与系统”课程是电子信息学科、通信学科、网络学科以及信号和信息分析与处理等专业的技术基础课。本课程考查考生对信号、系统的基本概念的理解，对信号分析和系统特性的基本分析方法掌握的程度；考查考生基本知识的运用能力。 二、基本要求 “信号与系统”课程的任务是研究信号与系统理论的基本概念和基本分析方法，使学生认识如何建立系统的数学模型，掌握基本分析、求解方法，并对所得结果赋予物理意义。通过本课程的学习，学生能运用数学工具正确分析典型的物理问题，使学生具备进一步学习后续课程的理论基础。 第二部分课程内容与考核目标 第一章绪论 1、理解信号、系统的概念及分类； 2、掌握典型信号的定义及其波形表达； 3、理解和掌握阶跃信号与冲激信号的定义、特点（性质）及两者的关系； 4、了解信号的不同分解形式； 5、理解和掌握系统的线性性、时不变性、因果性含义，并能做出正确判断； 6、熟练掌握信号的时域运算，理解运算对信号的影响结果； 7、了解系统模型的意义，掌握由线性系统微分方程绘出系统模拟框图或系统模拟框图写出系统微分方程的方法。 第二章连续时间系统的时域分析 1、理解0 －和0 ＋ 时刻系统状态的含义； 2、理解冲激响应、阶跃响应的意义，至少掌握一种时域求解方法； 3、掌握系统全响应的两种求解方式：自由响应和强迫响应、零输入响应和零状态响应； 4、会分辨全响应中的瞬态响应分量和稳态响应分量； 5、掌握卷积积分的定义、代数运算规律和主要性质、会用卷积积分法求解线性时不变系统的零状态响应。 6、了解系统微分方程的算子表示。 第三章傅立叶变换 1、掌握周期信号的频谱分析方法； 2、理解非周期信号的频谱密度函数的概念、周期信号与非周期信号的频谱特点与区别；
细胞信号转导 第一套 一、选择题（共10题，每题1分） 1、Ca2+在细胞信号通路中是（） A. 胞外信号分子 C. 第二信使 B. 第一信使 D. 第三信使 2、动员细胞内源性Ca2+释放的第二信使分子是（）。 A. cAMP C. IP3 B. DAG D. cGMP 3、细胞通讯是通过（）进行的。 A. 分泌化学信号分子 C. 间隙连接或胞间连丝 B. 与质膜相结合的信号分子 D. 三种都包括在内 4、Ras蛋白由活化态转变为失活态需要( )的帮助。 A. GTP酶活化蛋白（GAP） C. 生长因子受体结合蛋白2（GRB2） B. 鸟苷酸交换因子（GEF） D. 磷脂酶C-γ(PLCγ) 5、PKC在没有被激活时，游离于细胞质中，一旦被激活就成为膜结合蛋白，这种变化依赖于（）。 A. 磷脂和Ca2+ C. DAG和 Ca2+ B. IP3和 Ca2+ D. DAG和磷脂 6、鸟苷酸交换因子（GEF）的作用是（）。 A. 抑制Ras蛋白 C. 抑制G蛋白 B. 激活Ras蛋白 D. 激活G蛋白 7、cAMP依赖的蛋白激酶是（）。 A. 蛋白激酶G（PKG） C. 蛋白激酶C（PKC） B. 蛋白激酶A（PKA） D. MAPK 8、NO信号分子进行的信号转导通路中的第二信使分子是（）。 A. cAMP C. IP3 B. DAG D. cGMP 9、在下列蛋白激酶中，受第二信使DAG激活的是（）。 A. PKA C. MAPK B. PKC D. 受体酪氨酸激酶 10、在RTK－Ras蛋白信号通路中，磷酸化的（）残基可被细胞内的含有SH2结构域的信号蛋 白所识别并与之结合。 A. Tyr C. Ser B. Thr D. Pro 二、判断题（共10题，每题1分） 11、生成NO的细胞是血管平滑肌细胞。（） 12、上皮生长因子（EGF）受体分子具酪氨酸激酶活性位点。（） 13、Ras蛋白在cAMP信号通路中起着分子开关的作用。（）
2. 双组分调节系统的功能 细菌的生存环境中存在各种环境变化。包括感应pH，养分，氧化还原状态，渗透压力和抗生素等，因此细菌含有多套双组分系统，来应对各种环境的变化。此外，一些双组分系统还能控制基因簇，该基因簇对细胞生长、毒力、生物膜和群体感应有重要作用。 2.1大肠杆菌双组分调节系统 在大肠杆菌中，EnvZ-OmpR系统是研究比较清楚的一个双组分系统，是一种渗透胁迫相关的双组分系统，该系统是通过调节OmpF和OmpC的基因表达，能影响OmpF和OmpC在外膜上数量的多少，跨膜运输物质最终受到影响，调控者细胞对渗透胁迫的响应过程。OmpF和OmpC是大肠杆菌细胞膜上的两个主要孔道蛋白，其表达是由磷酸化的OmpR调节，小分子量的亲水性物质可以通过它们进入细胞。它们对环境渗透势的大小极为敏感。二者在外膜上数量的多少都受渗透势改变的影响，渗透势低时，外膜中有较多的OmpF，渗透势高时，外膜中有较多的OmpC。OmpF和OmpC基因的表达是由EnvZ-OmpR双组系统调控的。渗透感应器是EnvZ，属于HPK，能进行自身磷酸化是在感受到外界渗透势变化下，然后将其高能His-Pi基团传递到RR的OmpR上，磷酸位点接受模件的Asp残基。随后，磷酸化的OmpR与OmpF和OmpC的上游序列结合，调节这两个基因的表达[10]。 2.2 病原性细菌 在病原性细菌中，双组分信号系统转导系统经常控制基因簇对细胞生长和致病过程发生的作用。因此，可以通过引入双组分系统的抑制子作用于传感器的感官结构域，阻止群体感应系统，或者作用于必要的RR，通过特异性抑制双组分系统的信号转导控制病原性细菌的致病性，对医学研究新药品提供一定的理论依据[11]。 2.3结核分枝杆菌双组分调节系统 在结核分制杆菌中，PhoPR 双组分系统对结核分枝杆菌的毒力及持留
第三节主要的信号转导途径 一、膜受体介导的信号传导 （一）cAMP-蛋白激酶A途径 述：该途径以靶细胞内cAMP浓度改变和激活蛋白激酶A（PKA）为主要特征，是激素调节物质代谢的主要途径。 1．cAMP的合成与分解 ⑴引起cAMP水平增高的胞外信号分子：胰高血糖素、肾上腺素、 促肾上腺皮质激素、促甲状腺素、甲状旁腺素和加压素等。 α-GDP-βγ（Gs蛋白）激素+受体→激素-受体→↓ α-GTP + βγ ↓ AC激活 ↓ ATP →cAMP 述：当信号分子（胰高血糖素、肾上腺素和促肾上腺皮质激素）与靶细胞质膜上的特异性受体结合，形成激素一受体复合物 而激活受体。活化的受体可催化Gs的GDP与GTP交换，导 致Gs的α亚基与βγ解离，蛋白释放出αs-GTP。αs-GTP能激 活腺苷酸环化酶，催化ATP转化成cAMP，使细胞内cAMP 浓度增高。过去认为G蛋白中只有α亚基发挥作用，现知βγ 复合体也可独立地作用于相应的效应物，与α亚基拮抗。 腺苷酸环化酶分布广泛，除成熟红细胞外，几乎存在于所有组织的细胞质膜上。cAMP经磷酸二酯酶（PDE）降解成 5'－AMP而失活。cAMP是分布广泛而重要的第二信使。
⑵AC活性的抑制与cAMP浓度降低 ◇Gα-GTP结合AC并使之激活后，同时激活自身的GTP酶活性，Gα-GTP→Gα-GDP，Gs、AC均失活。从而在细胞对cAMP浓度升高作出应答后AC活性迅速逆转。 ⑶少数激素，如生长激素抑制素、胰岛素和抗血管紧张素II 等，它们活化受体后可催化抑制性G蛋白解离，导致细胞内AC活性下降，从而降低细胞内cAMP水平。 ⑷正常细胞内cAMP的平均浓度为10－6mol/L。cAMP在细 胞中的浓度除与腺苷酸环化酶活性有关外，还与磷酸二酯酶的活性有关。举例如下： ①一些激素如胰岛素，能激活磷酸二酯酶，加速cAMP降解； ②某些药物如茶碱，则抑制磷酸二酯酶，促使细胞内cAMP 浓度升高。 2．cAMP的作用机制――cAMP激活PKA（幻灯64） ⑴cAMP对细胞的调节作用是通过激活cAMP依赖性蛋白激酶 或称蛋白激酶A （PKA）系统来实现的。 ⑵PKA的结构 2C（催化亚基）：蛋白丝/苏氨酸磷酸化酶活性四聚体蛋白 变构酶 2R（调节亚基）：各有2个cAMP结合位点述：催化亚基有催化底物蛋白质某些特定丝／苏氨酸残基磷酸化的功能。调节亚基与催化亚基相结合时，PKA呈无活性状态。当4分子cAMP与2个调节亚基结合后，调节亚基脱落，游离的催化亚基具有蛋白激酶活性。PKA的激活过程需要Mg2+。
信号与系统概念，公式集： 第一章：概论 1.信号：信号是消息的表现形式。（消息是信号的具体内容） 2.系统：由若干相互作用和相互依赖的事物组合而成的具有特定功能的整体。 第二章：信号的复数表示： 1.复数的两种表示方法：设C 为复数，a 、b 为实数。 常数形式的复数C=a+jb a 为实部，b 为虚部； 或C=|C|e j φ，其中，22||b a C +=为复数的模，tan φ=b/a ，φ为复数的辐角。（复平面） 2.欧拉公式： wt j wt e jwt sin cos +=（前加-，后变减） 第三章：正交函数集及信号在其上的分解 1.正交函数集的定义：设函数集合)}(),(),({21t f t f t f F n Λ= 如果满足： n i K dt t f j i dt t f t f i T T i T T j i Λ2,1)(0)()(2 1 2 12 ==≠=? ? 则称集合F 为正交函数集 如果n i K i Λ,2,11 ==，则称F 为标准正交函数集。 如果F 中的函数为复数函数
条件变为： n i K dt t f t f j i dt t f t f i T T i i T T j i Λ2,1)()(0)()(2 1 2 1* *==?≠=?? ? 其中)(*t f i 为)(t f i 的复共轭。 2.正交函数集的物理意义： 一个正交函数集可以类比成一个坐标系统； 正交函数集中的每个函数均类比成该坐标系统中的一个轴； 在该坐标系统中，一个函数可以类比成一个点； 点向这个坐标系统的投影（体现为该函数与构成坐标系的函数间的点积）就是该函数在这个坐标系统中的坐标。 3.正交函数集完备的概念和物理意义： 如果值空间中的任一元素均可以由某正交集中的元素准确的线性表出，我们就称该正交集是完备的，否则称该正交集是不完备的。 如果在正交函数集()()()()t g n Λ,t g ,t g ,t g 321之外，不存在函数x （t ） ()∞<