Apoptosis: Cellular Suicidemcb.berkeley.edu/courses/mcb150/lecture2/Lecture2.pdf · Some microbes...
Transcript of Apoptosis: Cellular Suicidemcb.berkeley.edu/courses/mcb150/lecture2/Lecture2.pdf · Some microbes...
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Administrative issues:
Recommended text: Goldsby/Kuby Immunology, 6th edition(Note that Innate Immunity is not adequately covered in the 5th edition.)
Discussion sections start next week. The journal article Akira et al, andthe first problem set will be covered. Both are available on the website.
Office Hours: Questions about the lecture material are best addressedduring office hours (Tues 11-12). I will be holding extra office hours (dateand time TBA) before the first midterm.
Email: Please use email only for VERY simple yes/no questions or simpleadministrative matters.
Great questions, keep them coming!
Innate ImmunityInnate immune effector mechanisms
Physical and biochemical barriers (defensins)Phagocytosis and reactive oxygen speciesCell autonomous defenses
ApoptosisInterferons and PKR
Innate immune recognitiondiscovery of the Toll-like receptorsmammalian TLRs and their ligandsnon-TLR recognition of PAMPs
Connections between adaptive and innate immunity
Some microbes hijack cellular machinery to replicateand spread. Intracellular pathogens include viruses(influenza, HIV) and intracellular bacteria (listeria)and intracellular parasites (malaria, toxoplasma).
cell-autonomous defense:cell produces an immuneresponse that acts on itself
Apoptosis: Cellular Suicide
•Nuclear fragmentation•Proteolysis•Blebbing•Death
Remnantsundergophagocytosis
Apoptosis versus Necrosis•Tidy: contents of cellsdegraded from within,producing small cellular“blebs”
•Programmed from inside thecell
•Messy: contents of cellreleased.
•Induced by external insult
Cell death by necrosis is more likely to produce inflammation.
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Interferons are cytokines that areproduced in response to viral infection.
Produce an “anti-viral state” in targetcells.
Acts on cell that produces it, as well asneighboring cells.
Together with dsRNA, act to triggeringthe Protein Kinase R(PKR) pathway.
Shuts down protein synthesis machineryof cells, thus preventing viral replication.
Cells can avoid being hijacked by viruses by activating the ProteinKinase R (PKR) pathway. PKR is triggered by dsRNA and interferon. Protein Kinase R:
Interfering with Infection
RNA-bindingdomain
Kinasedomain
dsRNAor
interferon
PKR
eIF2a
PhosphorylatedPKR
(active)
PhosphorylatedeIF2a
(inactive)
Innate Immunity (finishing up)Innate immune effector mechanisms
Physical and biochemical barriers (defensins)Phagocytosis and reactive oxygenCell autonomous defenses
ApoptosisInterferons and PKR
Innate immune recognitiondiscovery of the Toll-like receptorsmammalian TLRs and their ligandsnon-TLR recognition of PAMPs
Connections between adaptive and innate immunity
Cell types of innate immunity
Components of the bacterial cell wall, such aslipopolysaccaride (LPS), peptidoglycan, lipoprotein cantrigger the innate immune system.
Cross section of the cellwall of a gram-negativebacteria.
Comparison of the adaptive and innate immune responses
innate adaptive
Response time hours days
Response to identical to primary stronger response uponrepeat infection (no memory) second exposure (memory)
Receptors that pattern recognition receptors antibodies and T cell antigen receptors (TCR)Mediate pathogen e.g. Toll-like receptors (TLR)Recognition limited diversity, unlimited diversity
fixed in germline generated by V(D)J recombination
Ligands Pathogen associated molecular virtually any component of pathogenpatterns (PAMPs)
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What triggers innate immune responses?( How do phagocytes know what to eat?)
• Long-known that bacterial cell wall components activatephagocytes
• Hypothesis-- microbes contains pathogen associatedmolecular patterns (PAMPs) which are recognized bypattern recognition receptors (PRRs)
• A series of disparate observations in transcriptionalregulation and drosophila development lead to thediscovery of PRRs
Genetic analysis ofearly embryonic
development in thefruitfly, Drosophila
(Nusslein-Volhard andcolleagues)
The Dorsal Signaling Pathway controlsdorsal/ventral polarity in the Drosophila early
embryo
Dorsal
Ventral
Toll
Cactus
Dorsal
(a receptor)
(a transcriptionfactor)
“Toll” is German slang for “weird”
NF-κB: a transcription factor whichbinds to antibody genes (Baltimore and
colleagues).
enhancer
Transcriptionfactor
Ig κ gene
Nuclear factor Igκ locus B (as in A, B, C, etc.)
NF-κB: A critical transcription factor forinnate immunity
p50
p65
IκB
p50
p65
Inactive,cytoplasmic
Active,nuclear
(cactus)(dorsal)
Mutant mice lacking NF-kB subunits have defects in innate immunity.
What is the mammalian receptor that leads to NF-kB activation inresponse to infection?
Discovery of the mammalian Toll-like receptors(TLR):
1997: Janeway and Medzhitov discovered a human proteinwith structural similarity to drosophila Toll that could activate
immune response genes in human cells (TLR4).
1998: Beutler discovered that a mouse strain with an alteredresponse to bacterial lipopolysaccharide (called LPS or
endotoxin) was due to a mutation in the TLR4 gene.
There are 11 TLR family members in human and 12 in mice.Each responds to a distinct set of microbial products.
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Insert Fig 3-11
Different mammalian Toll-like receptors (TLRs) are specificfor different classes of microbial products
Different mammalian Toll-like receptors (TLRs) are specificfor different classes of microbial products
Toll-like receptors(TLRs)
link microbialproducts (PAMPs) to
transcription factor activation
in a signalingpathway that is
conserved betweenmammals and insects
Toll-mutant drosophila aresusceptible to fungal infections
A more detailed look at the signaling pathway down-stream ofToll-like Receptors (TLRs)
A more detailed look at the signaling pathway down-stream ofToll-like Receptors (TLRs)
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Pathogen recognition receptors (PRR) in the innateimmune system: TLRs are not the whole story
scavenger receptorsNOD proteins (cytoplasmic PRR)
etc, etc
The benefits and hazards of TLR signaling
Septic shock can be caused by systemicinfection with gram negative bacteria
(Salmonella).
Shock is caused by overwhelmingproduction of cytokines and is induced by
presence of LPS in blood stream.
Mice lacking TLR4 are resistant to LPS-induced shock.
Mice lacking TLR4 are also more sensitiveto infection with gram negative bacteria.
(local vs. systemic effects of TLRsignaling?)
Triggering of PRRs on macrophage or dendritic cells can induce aLARGE variety of events including:
Increased phagocytosis
Production of cytokines and inflammatory mediators:Interferons to induce anti-viral stateChemokines to attract migrating cellsEtc, etc.
Increased cell migration
Changes in expression of molecules involved in T cellantigen presenting cell function.
Innate ImmunityInnate immune effector mechanisms
Physical and biochemical barriers (defensins)Phagocytosis and reactive oxygenCell autonomous defenses
ApoptosisInterferons and PKR
Innate immune recognitiondiscovery of the Toll-like receptorsTLRs and their ligandsnon-TLR recognition of PAMPs
Connections between adaptive and innate immunity
When innate immune signaling is insufficient to clear a pathogen, theadaptive immune system kicks in.Innate immune signaling turns on the adaptive immune response.
Macrophage and dendritic cells serve as antigen-presenting cells for adaptiveimmune cells (T cells).
T cell interacting with a macrophage(antigen presenting cell)
TLR-signaling activates innate immune cells (macrophage and dendritic cells).
Innate immune cells that have been activated by TLR-signaling are much moreeffective antigen presenting cells than resting immune cells.
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Dying infected cell(self, no TLR signaling)
Pathogen(non-self, TLR signaling)
Material inphagosome disposedof inside cell-nopresentation to T cells
Material in phagosomeenters antigenpresentation pathway-presentation to T cells
TLR signaling within phagosomes determines fate ofthat phagosome (destruction vs antigen presentation).
Blander and Medzhitov 2006 Nature v440 p808
Comparison of innate and adaptive immune recognition
Receptors that mediateinnate immune recognition: Toll-like receptors (TLR)
Receptors that mediate adaptive immune recognition:Antibody and the T cell receptor (TCR)
The genes encoding the T cell antigen receptor (TCR) are assembledby DNA rearrangement as T cells develop in the thymus
TCR ! locus:structure in germline
TCR ! locus:structure in T cells
DNA rearrangement(rag1, rag2)
transcriptionRNA splicingtranslation
T cell
!"
V segments D J C
The genes encoding the antigen receptors of T and B cells areassembled by DNA rearrangement as these cells develop. As aresult of V(D)J recombination, every B and T cells expresses aunique version of the antigen receptor.
A a result of V(D)J recombination every mature B cell expresses a unique antibody.Encounter with an antigen leads to clonal expansion of B cells with a particularspecificity.
What’s coming up in the next couple of weeks(Innate Immunity)
Antibodies and antigens I (emphasis on antibody structure)
Antibodies and antigens II(emphasis onantigen-antibodybinding interactions)
Techniques based onantibodies
V(D)J recombination
B cell development andfunction
Antigens & Antibodies IDiscovery of antibodies
Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins and the primary structure of antibodycrystal structure of antibody: the Ig domain
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes
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Early Observations of Acquired (adaptive)Immunity
•Thucydides, historian: 430 BC noted that those who hadsurvived plague could nurse the sick and not become sickagain.
•1600s, Turks and Chinese practiced “variolation”:intentional exposure to material from smallpox lesions toprovide protection against smallpox.
• Edward Jenner: ~1800 noted that exposure to vacciniavirus (cowpox) protects against smallpox. Tested firstvaccine using cowpox.
•Louis Pasteur: ~1880
•finds that exposureto attenuated bacteria(chicken cholera)protects against livebacteria.
•First vaccine againstrabies
•Immunize rabbits with tetanus bacteria.
•Isolate serum (non-cellular portion of blood) from immunizedrabbits, inject “immune serum” into naïve rabbit
•Challenge treated and untreated rabbits with lethal dose of livetetanus bacteria: only treated mice survived.
•Passive immunization with pooled human immunoglobulin iscurrently used to treat immunodeficiency and to providetemporary resistance to infection (hepatitis, rabies, measles,tetanus).
Von Behring & Kitasato: ~1890 discovered“passive immunization”
•Tiselius & Kabat: 1930s. Discovered that the γ-globulin fraction ofserum contains antibodies.
Immunize rabbits with chicken ovalbumin.Here a purified foreign protein (chicken ovalbumin), rather than a
pathogen, is the “antigen”, or substance that induces an antibody responseresponse. (“model antigen”)
A serum sample from an immunized rabbit contains a substance with the capacityto bind to ovalbumin, called “antibody”.
Serum contains many different proteins. Which serum fraction contains theantibody?
Blue: serumfrom immune rabbit
Black: serum with specificbinding activity depleted
•Tiselius & Kabat: 1930s. Discovered that the γ-globulin fraction ofserum contains antibodies.
Immunize rabbits with chicken ovalbumin. Isolate serum, divide into 2 parts.Separate one serum sample into different protein fractions (based on size, charge) usingelectrophoresis.Mix another serum sample with ovalbumin and remove the insoluble immune complexes(containing ovalbumin plus serum proteins that bind ovalbumin).Re-analyze depleted serum by electrophoresis and compare to the starting serum sample.
The γ-globulin fraction ofserum contains most antibody:Termed “immunoglobulin”
Antigens & Antibodies IDiscovery of antibodies
Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins and the primary structure of antibodycrystal structure of antibody: the Ig domain
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes
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Levels of Protein Structure Non-covalent forces that holdantigens and antibodies together
Energy of chemical interactionsinvolved in stability of protein
structure
Bond Length EnergyCovalent 0.15 nm 90 kcal/molIonic 0.25 nm 3 kcal/molHydrogen 0.30 nm 1 kcal/molVan der Waals 0.35 nm 0.1 kcal/molHydrophobic 0.35 nm 0.1 kcal/mol
The Alpha-Helix
The Beta-Sheet Ribbon Diagrams
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Antigens & Antibodies IDiscovery of antibodies
Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins, Ig domains, and hypervariable regions
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes
Basic Immunoglobulin (antibody) structure
Biochemical methods tocharacterize IgG structure
(Porter and Edelman 1960s)1972 Nobel Prize for medicine:
partial proteolysis chromatography
Fab: fragment antigen bindingFc: fragment crystalizes
cleavage of disulfide bondschromatography
Heavy chain ~50 KdaLight chain ~25 Kda
Basic Immunoglobulin (antibody) structure
2 identicalantigen bindingsites/molecule
Each antibody molecules consists of twoidentical heavy chains (50 kDa) and two
identical light chains (25 kDa).
Chains are held together by inter-chaindisulfide bonds.
Structure made up of repeating structuralunits of ~110 amino acids called Ig
domain (2/light chain and 4/heavy chain).
N-terminal Ig domain is variable (antigenbinding domain). The C-terminal Ig
domains are constant (effector functions).
Basic Immunoglobulin (antibody) structure Antibodies are bivalent (2 identical antigen binding sites).
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Membrane-associated vs.secreted immunoglobulin
Distinct carboxy-termini
Basic Immunoglobulin (antibody) structure
2 identicalantigen bindingsites/molecule
Each antibody molecules consists of twoidentical heavy chains (50 kDa) and two
identical light chains (25 kDa).
Chains are held together by inter-chaindisufide bonds.
Structure made up of repeating structuralunits of ~110 amino acids called Ig
domain (2/light chain and 4/heavy chain).
N-terminal Ig domain is variable (antigenbinding domain). The C-terminal Ig
domains are constant (effector functions).
Variations in the heavy chain lead todifferent antibody isotypes, and membrane
vs secreted forms of antibody.
Antigens & Antibodies IDiscovery of antibodies
Basic Antibody Structurebrief review of protein structuredisulfide linked tetramer: 2 heavy and 2 light chainsmyeloma proteins, Ig domains, and hypervariable regions
The antigen binding site of antibodies
Antibody isotypes: IgM, IgG, IgD, IgA, IgEThe advantages of multivalencyeffector functions of antibody isotypes
In a normal individual, antibodies are extremely heterogeneous.
Myeloma protein: key to determining Igstructure
• Heterogeneity of antibodies makes sequencingimpossible (each B cell clone produces a unique versionof antibody).
• Multiple myeloma: cancer derived from an antibodyproducing cells (plasma B cell).
• Myeloma patients have large amounts of one particularIg molecule in their serum (and urine)
• Many patients produce a large amount of one light chain,known as “Bence-Jones” proteins.
Antibodymolecules arecomposed ofrepeats of a singlestructural unitknown as the“immunoglobulindomain”
When the amino acid sequences of several differentBence-Jones proteins were compared, they were found toconsist of two repeating units of ~110 amino acids: onevariable and one constant.
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Protein homology• Identity or similarity between domains in two or more
proteins• Most easy to see at the level of primary amino acid
sequence (computer programs find it)• Sometimes no obvious primary sequence homology but
striking structural homology• Homology can sometimes predict structure and function
All Ig domains have asimilar 3D structure
known as an“Immunoglobulin Fold”.
2 β-pleated sheets cometogether to form a
sandwich, held togetherby disulfide bond and
hydrophobic interactions.
3 flexible loops at end:correspond to
hypervariable regions ofprimary sequence (HV).
The ImmunoglobulinFold is a very commonlyused structural motifamongst cell surface proteins
Ig domain: Genome Project Champion!
The variability of antibodies occurs within 3 discrete regionsof the primary sequence: hypervariable regions HV1-3
The hypervariable regions (HV1-3) are separated in primary structure,but come together in the tertiary structure where they form the antigenbinding site. Alias Complementary Determining Regions or CDR1-3.
The HV regions formloops at the end of the Ig
domain.
The intervening frame-work regions (FR1-4)
make up the rest of thestructure.
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The quaternary structure of immunoglobulin
Associations between Ig domains.
Interchain disulfide bonds
Hinge region allows flexible movementof Fc regions
6 CDR (3 from HC, 3 from LC) combineto make up antigen binding site
Because the CDR are highly variable, each antibody moleculehas a unique antigen binding site with its own dimensions and
complementarity.
Antibodies thatbind to large
proteins antigens
Antibodies thatbind to small
molecules