Transcript J club 12-8-04 - KSU Faculty Member websites

Direct Binding of Fas-associated Death
Domain (FADD) to the Tumor Necrosis
Factor-related Apoptosis-inducing
Ligand Receptor DR5 Is Regulated by
the Death Effector Domain of FADD
Lance R. Thomas , Adrianna Henson , John C. Reed , Freddie
R. Salsbury, and Andrew Thorburn
J. Biol. Chem., Vol. 279, Issue 31, 32780-32785, July 30, 2004
Intrinsic and Extrinsic Pathways of Apoptosis
http://arthritis-research.com/content/4/Suppl+3/S243/figure/F2?highres=y
Death receptors
 Death receptors (DRs) are cell surface receptors that transmit
apoptosis signals initiated by specific ligands.
 DRs can activate a caspase cascade within seconds of ligand binding.
Induction of apoptosis via this mechanism is therefore very rapid.
 DRs belong to the tumor necrosis factor (TNF) gene superfamily and
can have several functions other than initiating apoptosis.
 The best characterized of the DRs are CD95 (or Fas), TNFR1 (TNF
receptor-1) and the TRAIL receptors DR4 and DR5.
Tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL)
 TRAIL (Apo2L) is member of the TNF ligand family of cytokines.
 TRAIL is known to trigger apoptosis in many malignant cells.
 Whereas cancer cells are responsive to TRAIL-induced cell death,
normal cells are known to be relatively less sensitive to the ligand,
making it a desirable therapeutic agent to target a variety of cancers.
TRAIL receptors
• TRAIL binds four major different receptors:
- DR4 and DR5, which can induce apoptosis;
- decoy receptors, DcR1 and DcR2, which do not have the
intracytoplasmic death domain necessary to transduce apoptotic death
signals. They protect cells from TRAIL-mediated cell death by
interfering with signaling through DR4 and DR5.
 Transformed tumor cells are generally more susceptible to TRAILmediated cell death due to the absence of decoy receptors.
TRAIL receptors, mechanism
1
F
Nat Med. 1999 Feb;5(2):146-7.
Activation of apoptosis through CD95 / Fas
http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html
Induction of apoptosis by TRAIL vs TNF
http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html
FADD domains
 The adapter protein FADD is an essential component of the death
inducing signaling complex (DISC). It consists of two protein
interaction domains (DD and DED).
 The solution structures of the DD (amino acids 96-208) and the DED
(amino acids 1-81) of FADD have been solved.
 Both domains are globular structures consisting of six a-helices that
are tethered together by a linker.
FADD binding.
 The binding between DDs has been suggest to occur through charge
interactions. By contrast, binding between the DED of FADD and
procaspase 8 is the result of hydrophobic interactions.
 The FADD DD binds to activated receptors such as Fas or other
adapters such as TRADD, whereas the FADD DED binds to
procaspase 8. Each domain can interact with its target in the absence
of the other domain, and this has led to the idea that the two domains
function independently.
 The recruitment of procaspase 8 to the DISC is thought to result in the
autoactivation of the caspase. FADD binds directly to Fas to activate
caspase 8 in response to Fas ligand and binds the adapter protein
TRADD to activate caspase 8 in response to TNFa.
Yeast Hybrid Systems
The Yeast One-Hybrid System
 It is an application of the two-hybrid system that utilizes cis-acting
sequences to identify proteins, usually DNA-binding proteins, that can
initiate transcriptional activation.
The yeast Two-Hybrid System
 It is a genetic method that uses transcriptional activity as a measure of
protein-protein interaction.
The Yeast Three-Hybrid System
 It is a modification of the two-hybrid system for the detection of RNAprotein interactions. In this system, the association of the DNA-binding
and transcription activation domains is dependent on an RNA-protein
interaction.
The Yeast Two-Hybrid System
 The two-hybrid system is a genetic method that uses transcriptional
activity as a measure of protein-protein interaction.
 It relies on the modular nature of many site-specific transcriptional
activators, which consist of a DNA-binding domain (DBD) and a
transcriptional activation domain (AD).
 The DBD serves to target the activator to the specific genes that will
be expressed, and the AD contacts other proteins of the transcriptional
machinery to enable transcription to occur.
http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/
The Yeast Two-Hybrid System,
two-hybrid transcription
 A DBD fused to the protein of interest, X, and a transcription AD
fused to some protein, Y are constructed. These two hybrids are
expressed in a cell containing one or more reporter genes.
 If the X and Y proteins interact, they create a functional activator by
bringing the AD into close proximity with the DBD; this can be
detected by expression of the reporter genes.
http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay
The Yeast Two-Hybrid System,
Plasmid
construction
 A variety of versions of the two-hybrid system exist, commonly involving
DBDs that derive from the yeast Gal4 protein or the E. coli LexA protein.
 ADs are commonly derived from the Gal4 protein or the herpes simplex
virus VP16 protein. Reporter genes include the E. coli lacZ gene and
selectable yeast genes such as HIS3 and LEU2.
LacZ
http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay
Advantages of the Yeast Two-Hybrid System
 It is highly sensitive, detecting interactions that are not detected by
other methods.
 The interactions are detected within the native environment of the cell
and hence that no biochemical purification is required.
 The use of genetic-based organisms like yeast cells as the hosts for
studying interactions allows both a direct selection for interacting
proteins and the screening of a large number of variants to detect
those that might interact either more or less strongly.
 With a reporter gene such as the yeast HIS3 gene, the competitive
inhibitor 3-aminotriazole can be used to directly select for constructs
which yield increased affinity.
Limitations of the Yeast Two-Hybrid System
 Proteins must be able to fold and exist stably in yeast cells and to
retain activity as fusion proteins.
 The use of protein fusions also means that the site of interaction may
be occluded by one of the transcription factor domains.
 Interactions dependent on a posttranslational modification that does
not occur in yeast cells will not be detected.
 Many proteins, including those not normally involved in transcription,
will activate transcription when fused to a DBD, and this activation
prevents a library screen from being performed.
 However, it is often possible to delete a small region of a protein that
activates transcription and hence to remove the activation function
while retaining other properties of the protein.
Reverse Two-Hybrid Assay
 It identifies mutations in proteins that result in a loss of protein-protein
interactions.
 in vitro mutagenesis to create a library of mutants of one of the components
in a two-hybrid screen, either the DBD fusion plasmid or the AD fusion
plasmid screen for the loss of two-hybrid interaction.
 The major problem with current reverse two-hybrid methods is that one
commonly identifies mutations that prevent stable expression of the twohybrid protein or that affect gross protein folding.
 Thomas et al. developed a reverse two-hybrid system that identifies
mutations, which specifically abolish interactions among particular partner
proteins while requiring that the mutated protein still interacts with a
different protein partner; thus, demanding that the mutant protein is stably
expressed in its native conformation.
 Using this method, Thomas et al. identified mutations in FADD, which
suggest that in contrast to current models, the FADD DED regulates the
interaction between FADD and Fas.
Modified Reverse Two-hybrid Screening
 Thomas et al. modified the yeast two-hybrid system to include reporters for two
DBD fusion proteins (sometimes called "baits").
 The first bait fused to the Gal4 DBD is used to detect loss of interaction via a dual
reporter system. A two-hybrid interaction between the Gal4-DBD fusion and the AD
fusion results in the expression of the Tn10 Tet repressor, which blocks transcription
of ADE2 from the TetO-ADE2 reporter.
 Thus, the two-hybrid interaction with the Gal4-DBD fusion results in no ADE2
expression and an Ade-phenotype. A mutation that disrupts this interaction removes
ADE2 inhibition, and the yeast are able to grow in the absence of adenine. Thus, we
can select for the loss of two-hybrid interaction by selecting for Ade+ yeast.
 The second bait protein, a LexA-DBD fusion, is used to eliminate mutations in the
AD fusion plasmid that affect expression or stability of the AD protein fusion. Twohybrid activation between the prey and the LexA-DBD fusions will activate the
LexA(op)-HIS3 reporter, resulting in an His+ phenotype.
 Thus, specific mutations in the AD fusion that block interaction with partner 1 (the
Gal4-DBD fusion) but maintain overall protein integrity, allowing interaction with
partner 2 (the LexA-DBD fusion), can be selected as Ade+ His+ transformants.
Modified Reverse Two-hybrid Screening
 D, yeast expressing Gal4-DBD-FAS (partner 1) and LexA-DBD-TRADD (partner 2) were
transformed with empty vector (pACT3), wild type FADD, or FADD (R117A). Yeast
expressing the pACT3 and the R117A mutant fail to interact with Fas and grow on Ademedium, yeast expressing wild type FADD, and FADD (R117A) grow on His- media. Only
yeast expressing FADD (R117A) can grow in the absence of both adenine and histidine.
Direct Binding of Fas-associated Death
Domain (FADD) to the Tumor Necrosis
Factor-related Apoptosis-inducing
Ligand Receptor DR5 Is Regulated by
the Death Effector Domain of FADD
Lance R. Thomas , Adrianna Henson , John C. Reed , Freddie
R. Salsbury, and Andrew Thorburn
J. Biol. Chem., Vol. 279, Issue 31, 32780-32785, July 30, 2004
Hypothesis
 FADD binds directly to DR5, and the FADD-DED regulates binding
of FAD-DD to DR5.
Question
 Some reports indicate that an adaptor protein such as TRADD or
DAP3 might be involved in recruiting FADD to the DR5 DISC.
 Does FADD bind directly to DR5?
FADD binds directly to DR5
FIG. 1.
Figure 1. A
BJAB cells were stimulated
with nonspecific IgG or
aDR5  DR5 DISC
precipitated  blot for FADD
& procaspase8
Conclusion
 Both FADD and processed caspase-8 co-precipitate in cells stimulated with DR5
but not in cells stimulated with IgG.
Figure 1. B
 A directed yeast two-hybrid
assay to test for interaction
between the cytoplasmic
domain of DR5 and fulllength FADD.
 Fas/CD95 was used as a
positive control.
Conclusion
 Both Fas/CD95 and DR5 interacted with FADD in yeast, suggesting that DR5 is
recruited directly to the activated TRAIL receptor complex
Figure 1. C
 Empty FLAG vector or
FLAG-tagged FADD was
transfected into HeLa cells
with GFP-tagged Fas/CD95
or DR5 cyt. domains.
 FLAG complexes were
immunoprecipitated, and
interaction was detected by
immunoblotting for GFP.
 Whole cell lysates were
blotted with anti-FLAG and
anti-GFP to show equal
transfection.
C
Conclusion
 Both GFP-DR5 and GFP-Fas co-precipitated with FLAG-FADD, indicating a direct
interaction.
Conclusion
 FADD binds directly to DR5.
The DED of FADD regulates binding to DR5
FIG. 2.
Figure 2. A
 A directed yeast two-hybrid
assay was used to test for
interaction of DR5, Fas,
• and caspase-8 with
TRADD,
empty vector (pACT3), wild
type FADD, or FADD DED
mutations.
 A mutation in FADD at Arg 71, which is located in the loop between helices 5 and 6
of the DED, to either Trp or Ala prevented binding to DR5 and Fas/CD95 while
retaining interaction with TRADD and caspase-8.
Conclusion
 Similar to Fas/CD95, the DED of FADD participates in binding to DR5.
Figure 2. B & C
 B. FADD-deficient Jurkat cells
were stably transfected with
GFP vector, wild type FADD, or
FADD mutants. The level of
FADD protein was determined
by immunoblot.
 C. Stable Jurkat cells were left
untreated or stimulated with
TRAIL, and caspase-8 and
caspase-3 processing was
measured by immunoblot.
 The cells expressing wild type FADD showed cleavage of caspase-8 and caspase-3 in
response to TRAIL, whereas cells expressing GFP or the FADD DED mutations did not.
Conclusion
 FADD proteins containing the DED point mutations that prevent binding to DR5 cannot
rescue the phenotype associated with FADD deficiency.
Conclusions
 The binding phenotype observed in yeast correlates with signaling ability in
mammalian cells.
 The DED of FADD modulates binding to DR5.
Helix 5 of the DED regulates binding of FADD to
DR5 and Fas/CD95
FIG. 3.
Figure 3. A
 Using a forward two-hybrid
approach, a second round of
random mutagenesis on FADD
(R71A) has been performed.
 Screening for second site
mutations that restore the binding
activity of FADD (R71A)
Conclusion
 The second site mutations were located in helix 5 of the FADD DED: glutamate 61
to lysine, leucine 62 to phenylalanine, and glutamate 65 to lysine.
 These mutations restored binding of FADD (R71A) to both DR5 and Fas/CD95
Figure 3. B, C, and D
 FADD molecules with the
double mutations were
introduced into FADD-deficient
Jurkat cells  FADD expression
was determined by immunoblot.
 Jurkat cells expressing the
second site FADD mutations
were treated with TRAIL or
FasL.
 caspase-8 and caspase-3
processing was etected by
immunoblot.
 Each of the second site mutations rescued DR5 and Fas/CD95-induced caspase processing.
Conclusion
 DR5 and Fas-induced processing of caspase-8 and caspase-3 is prevented by mutations in the
DED of FADD and second site mutations that are also in the DED restored processing.
Conclusion
 The second site mutations in FADD that rescued binding to DR5 also rescued
binding to Fas/CD95, suggesting that FADD uses the same surface of the DED, ,
specifically helix 5, for binding to both receptors.
DR5 binds to full-length FADD better than the death
domain alone
FIG. 4.
Figure 4. A
 The interaction of DR5 with
full-length FADD or the DD
alone was tested in yeast using
quantitative b-galactosidase
assays.
 There is about a 20% increase in binding of DR5 to full-length FADD compared with the DD
alone.
Conclusion
 DR5 binds to full-length FADD better than the death domain alone.
Figure 4. B
 HeLa cells were transfected
with FLAG-DR5 along with
GFP, GFPFADD-DD, or GFPFADD FLAG complexes
precipitated.
 The interaction was measured
by immunoblotting for GFP.
 Full-length FADD co-precipitated with DR5 to a much greater extent than the DD alone.
Conclusion
 Both the DD and the DED of FADD contribute to the interaction with DR5.
Question
 which residues in FADD are required for binding to one receptor but
not the other (i.e., DR5 vs Fas)?
FADD (V108E) is able to bind DR5 and transduce TRAIL
signaling but is unable to bind Fas/CD95 or transduce
signaling through FasL.
FIG. 5.
Figure 5.A
 A reverse two-hybrid screen has
been performed to identify mutations
in FADD that prevent binding to
Fas/CD95 but retain binding to DR5.
 Interaction with other DD-containing
proteins was determined by a
directed yeast two-hybrid assay.
 A change in Val 108 to Glu (V108E) in the DD of FADD prevents binding to Fas/CD95 but
does not alter binding to DR5, TRADD, or caspase-8.
Conclusion
 Other than Val 108, the same residues that are required for FADD binding to DR5 are also
required for Fas/CD95 binding.
Figure 5. B & C
 we introduced this FADD mutation
into FADD-deficient Jurkat cells.
 The expression level of FADD
(V108E) along with cells
expressing wild type FADD or GFP
has determined.
 Jurkat cells expressing GFP,
FADD, or FADD (V108E) were
stimulated with TRAIL or FasL,
and caspase processing was
measured by immunoblot.
 Cells expressing FADD showed both caspase-8 and caspase-3 processing. Cells expressing FADD
(V108E) underwent caspase processing in response to TRAIL but not when treated with FasL.
Conclusion
 FADD (V108E) is able to bind DR5 and transduce TRAIL signaling but is unable to
bind Fas/CD95 or transduce signaling through FasL.
In the context of the full-length FADD, helix 5 of the
DED comes into direct contact with DR5.
FIG. 6. A
 The effect of each mutation on the
overall structure of FADD was
determined by energy minimizations.
Conclusion
 The mutations do not disrupt the overall protein structure because the effect on free
energy for most mutations was small.
 FADD R71A L62F is the only mutation with a significant change in free energy, but
this mutation actually leads to a more stable structure.
Figure 6. B & C
 FADD DED mutations were
modeled onto the solved structure of
the FADD DED.
 Arg 71, which is required for the
FADD-DR5 interaction, flanks helix
5, and the compensating mutations
were in helix 5.
 This suggests a direct role for helix
5 in the FADD-DR5 interaction
 Residues shown to be important for
the FADD-Fas/CD95 interaction
(red and blue) along with valine 108
(green) were modeled onto the
solved structure of the FADD DD
Conclusion
 In the context of the full-length FADD, helix 5 of the DED comes into direct contact
with DR5.
Summary
 Both immunoprecipitation and two-hybrid experiments indicate a
direct interaction between FADD and DR5.
The DED of FADD regulates binding to DR5.
 Helix 5 of the DED regulates binding of FADD to DR5 and Fas/CD95
 DR5 binds to full-length FADD better than the DD alone.
 FADD (V108E) is able to bind DR5 and transduce TRAIL signaling
but is unable to bind Fas/CD95 or transduce signaling through FasL.
 Other than valine 108, the same residues that are required for FADD
binding to DR5 are also required for Fas/CD95 binding.
 In the context of the full-length FADD, helix 5 of the DED comes into
direct contact with DR5 and Fas/CD95.
Critique
 Well organized paper.
 For the most part, conclusions are supported by the data presented.
 Cells from FADD-deficient mice, which are resistant to apoptosis induction
by CD95, TNFR1, show full responsiveness to DR4, confirming the
existence of a FADD-independent pathway that couples TRAIL to caspases.
 Some reports suggest that FADD and TRADD were not involved in TRAILinduced apoptosis, whereas others have demonstrated direct binding of FADD
and TRADD to the TRAIL receptor.
 Fibroblasts from FADD knockout mice were shown to undergo TRAIL-induced
apoptosis, suggesting that FADD was not essential in TRAIL signaling.
 Jurkat cells express very little DR4 so almost all TRAIL signaling is through
DR5. So, what about DR4?
 In Fig.1C, HeLa cells were used? What about FADD-deficient Jurkat cells?
Critique, contd.
Changes in arginine 71 to either alanine or tryptophan prevented
interaction with both DR5 and Fas/CD95 while retaining interaction with
TRADD and caspase-8 ?
Fig. 2
Critique, contd.
A mutation in FADD at
Arg71, which is located
in the loop between
helices 5 and 6 of the
DED, to either Trp or Ala
prevented binding to DR5
and Fas/CD95 while
retaining interaction with
TRADD and caspase-8.
Future Directions
 Test whether FADD binds DR5 directly in several different cell lines.
 Test whether FADD binds DR4 directly in several different cell lines.
 The C-terminal tails of Tumor Necrosis Factor-related apoptosisinducing ligand (TRAIL) and Fas receptors have opposing functions
in Fas Associated Death Domain (FADD) recruitment and can
regulate agonist-specific mechanisms of receptor activation.
J Biol Chem. 2004 Sep 27; [Epub ahead of print].
 It may be possible to design drugs that specifically interfere with some
but not all FADD interactions by searching for molecules that disrupt DD
interactions through an effect on the DED.
 The yeast system might be a useful screening method for such molecules,
which could be used to selectively inhibit signaling by some DRs without
affecting signaling from the other receptors that use FADD.
The Yeast Three-Hybrid System
 It is a modification of the two-hybrid system for the detection of RNA-protein interactions. In
this system, the association of the DNA-binding and transcription activation domains is
dependent on an RNA-protein interaction.
 This system uses a transactivator protein in yeast, such as Gal4p, that is able to recruit the
transcriptional machinery and trigger transcription of a gene. It consists of a DBD and an AD
and, importantly, these two domains are functionally independent, meaning that they can be
inserted into other molecules.
 The DBD (LexADB or Gal4DB) is fused to an RNA binding protein (MS2-coat protein or
Hiv-1 RevM10). The second fusion protein contains on one hand the Gal4AD and on the
other hand the RNA binding protein ‘Y’ of interest. The two fusion proteins are bridged by a
third hybrid RNA molecule containing the binding site for the first RNA binding protein
(MS2 or RRE) and the binding site ‘X’ for the RNA binding protein ‘Y’ studied.
 Binding of protein ‘Y’ to the RNA binding site ‘X’ will create a functional transactivator,
which is tethered at the upstream activating sequence of two reporter genes (HIS3 and lacZ)
that will be transcribed and expressed by yeast cells.
 The expression level of lacZ gene can be determined in vitro by measuring the bgalactosidase activity, or visualized in vivo by plating the yeast transformants on media
supplemented with X-Gal.
 On the other hand, HIS3 is the gene encoding imidazoleglycerol-phosphate dehydratase
(His3p) and its expression confers the ability to grow on a medium lacking histidine. 3amino-1,2,4-triazole (3-AT) is a competitive inhibitor of HIS3 gene product, and therefore
cells containing more His3p can survive at higher concentrations of 3-AT in the medium.
Thus, the level of 3-AT resistance of the yeast cells reflects the HIS3 expression level and
consequently the strength of the RNA–protein interaction in the yeast three-hybrid context.
•
The basic strategy of the tri-hybrid method. ( A )
Schematically shows the components. The first
hybrid-protein (I) contains the DNA-binding domain
of GAL4 (Ia) fused to the RRE-RNA-binding
protein RevM10 (Ib). A hybrid- RNA (II) containing
the RRE sequence (IIa) and a target RNA sequence
X (IIb). The second hybrid-protein (III) contains the
activation domain of GAL4 (IIIa) fused to a protein
Y (IIIb) capable of recognising the target RNA X on
the RNA-hybrid. ( B ) Upon productive interaction
of the three hybrids a reconstituted GAL4
transcription factor (I+II+III) bound to a GAL4
responsive promoter (IV) stimulates the basal
transcriptional machinery (V) of the lacZ gene and
the nutritional reporter gene HIS3 (VI).
Induction of apoptosis by TRAIL
Apoptosis signaling by DR4 and DR5 and its modulation by decoy receptors.
http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html
Science, Vol 281, Issue 5381, 1305-1308 , 28 August 1998
Science, Vol 281, Issue 5381, 1305-1308 , 28 August 1998
Proapoptotic and antiapoptotic signaling by TNFR1
and DR3.
Science, Vol 281, Issue 5381, 1305-1308 , 28 August 1998
http://www.genomicobject.net/member3/GONET/apoptosis.html
fbspcu01.leeds.ac.uk/.../ apop_diagram.html
http://biopathways.bu.edu/apoptosis/apoptosis_mechanism.html
Whether FADD could bind directly to DR5 in
mammalian cells?
FADD binds directly to DR5
•A. BJAB cells + nonspecific IgG or an
agonistic DR5 antibody (aDR5) DR5
DISC precipitated 
C
Clinical Cancer Research Vol. 10, 6650-6660, October 1, 2004
 Tumor necrosis factor (TNF)-related apoptosis-inducing ligand [TRAIL (Apo2L)] is a
member of the TNF family and, like TNF- and Fas ligand, is a type II membrane protein that
can induce apoptotic cell death in a variety of transformed cell types. However, unlike other
members of this family, TRAIL does not appear cytotoxic to normal cells in vitro. The
potential importance of TRAIL as an anticancer agent has been supported by studies in
animal models that demonstrate selective toxicity to transplanted human tumors but not to
normal tissues.
 TRAIL binds to the apoptosis-inducing receptors DR4 and DR5, which are type I
transmembrane receptors, expressed at the cell surface. TRAIL also binds to non-apoptosisinducing decoy receptors, which compete with death receptors for the ligand and suppress
apoptosis. These include DcR1, DcR2, and osteoprotegerin and may constitute one
mechanism by which normal cells can evade the induction of apoptosis by TRAIL. The
mechanism of induction of apoptosis by TRAIL is believed to be similar to that of TNF- and
Fas ligand and to be initiated by ligand-induced aggregation of DR4 and DR5 and their death
domains on the cytoplasmic side of the receptors. The death domains in turn orchestrate the
assembly of adaptor proteins such as Fas-associated death domain (FADD), which activate
caspases after interaction of caspase recruitment domains of the adaptor proteins with the
prodomains of the caspases. The adaptor proteins involved in TRAIL-induced apoptosis have
been controversial, with some reports suggesting that FADD and TNF receptor-associated
death domain were not involved, whereas others have demonstrated direct binding of FADD
and TNF receptor-associated death domain protein to the TRAIL receptor. Fibroblasts from
FADD knockout mice were shown to undergo TRAIL-induced apoptosis, suggesting that
FADD was not essential in TRAIL signaling. The caspases involved also appear to be similar
to those activated by Fas ligand, with activation of caspase-8 being an early event that
eventually leads to activation of effector caspases including caspase-3. Ectopic expression of
the cowpox virus gene cytokine response modifier A (CrmA) was also shown to inhibit
TRAIL-induced apoptosis, consistent with involvement of caspase-1 and/or -8.
• Site-directed mutagenesis experiments
suggest that the Fas-FADD and TRADDFADD interactions occur on the same
surface of the FADD death domain. Indeed,
the mutations in helices 2 and 3 of the
FADD death domain abolish interactions
with both Fas and TRADD, although one
mutation, FADD (R117A), seems to prevent
binding to Fas only (7).
• Current models are based on the idea that the two
domains function independently of each other (i.e.
that the death domain does not affect death effector
domain interactions and vice versa). This view is
supported by experiments showing that each domain
in isolation can interact with its partner. For
example, the isolated death domain can inhibit
apoptosis by binding to activated Fas.
TNF receptor signaling
http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html
THE YEAST TWO-HYBRID SYSTEM,
Normal Transcription
LacZ
http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/
The Yeast Two-Hybrid System,
Normal Transcription
http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/
The Yeast Two-Hybrid System,
Plasmid construction
The Yeast Two-Hybrid System,
Transfection
Significance of the Yeast Two-Hybrid Assay
 Generally the yeast two-hybrid assay can identify novel protein-protein
interactions. By using a number of different proteins as potential binding
partners, it is possible to detect interactions that were previously
uncharacterized.
 The yeast two-hybrid assay can be used to characterize interactions already
known to occur. Characterization could include determining which protein
domains are responsible for the interaction, by using truncated proteins, or
under what conditions interactions take place, by altering the intracellular
environment.
 the yeast two-hybrid can be used to manipulate protein-protein interactions
in an attempt to understand its biological relevance. For example, many
disorders arise due to mutations causing the protein to be non-functional, or
have altered function. Such is the case of some cancers; a mutation in a progrowth pathway does not allow for the binding of negative regulatory
proteins, resulting in the pro-growth pathway never turning 'off'.
-The yeast two-hybrid is one means of determining how mutation affects a
protein's interaction with other proteins. When a mutation is identified that
affects binding, the significance of this mutation can be studied further by
creating an organism that has this mutation and characterizing its phenotype.
•
The Yeast Two-Hybrid System
 The yeast two-hybrid system provides a relatively straight forward approach
to understanding protein function.
 The main application is to isolate proteins that interact with a target protein,
usually by screening a cDNA library.
 A protein is expressed in yeast as a fusion to the DNA-binding domain
(DBD) of a transcription factor lacking a transcription activation domain
(AD). The DNA-binding fusion protein is generally called the bait. The
yeast strain also contains one or more reporter genes with binding sites for
the DBD.
 To identify proteins that interact with the bait, a plasmid library that
expresses cDNA-encoded proteins fused to a transcription AD is introduced
into the strain. Interaction of a cDNA-encoded protein with the bait results
in activation of the reporter genes, allowing cells containing the interactors
to be identified.
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 The second bait protein, a LexA-DBD fusion, is used to eliminate mutations in the
AD fusion plasmid that affect expression or stability of the AD protein fusion. Twohybrid activation between the prey and the LexA-DBD fusions will activate the
LexA(op)-HIS3 reporter, resulting in an His+ phenotype. Thus, specific mutations in
the AD fusion that block interaction with partner 1 (the Gal4-DBD fusion) but
maintain overall protein integrity, allowing interaction with partner 2 (the LexADBD fusion), can be selected as Ade+ His+ transformants.
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Modified Reverse Two-hybrid Screening
 Thomas et al. modified the yeast two-hybrid system to include reporters for two
DBD fusion proteins (sometimes called "baits").
 The first bait fused to the Gal4 DBD is used to detect loss of interaction via a dual
reporter system. A two-hybrid interaction between the Gal4-DBD fusion and the AD
fusion results in the expression of the Tn10 Tet repressor, which blocks transcription
of ADE2 from the TetO-ADE2 reporter.
 Thus, the two-hybrid interaction with the Gal4-DBD fusion results in no ADE2
expression and an Ade-phenotype. A mutation that disrupts this interaction removes
ADE2 inhibition, and the yeast are able to grow in the absence of adenine. Thus, we
can select for the loss of two-hybrid interaction by selecting for Ade+ yeast.