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.

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 is known to trigger apoptosis in many malignant cells.

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 TRAIL-mediated cell death due to the absence of decoy receptors.

TRAIL receptors, mechanism

F

Nat Med. 1999 Feb;5(2):146-7.

1

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 solution structures of the DD (amino acids 96-208) and the DED (amino acids 1-81) of FADD have been solved.

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).

Both domains are globular structures consisting of six -helices that are tethered together by a linker.

FADD binding.

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 TNF.

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.

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 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.

The yeast Two-Hybrid System It is a genetic method that uses transcriptional activity as a measure of

protein-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

http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay

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

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.

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.

Limitations of the Yeast Two-Hybrid System

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 two-hybrid 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. Two-hybrid 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 Ade-medium, 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

Does FADD bind directly to DR5?

Some reports indicate that an adaptor protein such as TRADD or DAP3 might be involved in recruiting FADD to the DR5 DISC.

FADD binds directly to DR5

FIG. 1.

Figure 1. A

BJAB cells were stimulated with nonspecific IgG or DR5 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.

A directed yeast two-hybrid assay to test for interaction between the cytoplasmic domain of DR5 and full-length 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. B

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.

Conclusion Both GFP-DR5 and GFP-Fas co-precipitated with FLAG-FADD, indicating a direct

interaction.

Figure 1. C

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, TRADD, and caspase-8 with 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.

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.

Each of the second site mutations rescued DR5 and Fas/CD95-induced caspase 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 aloneFIG. 4.

Figure 4. A

The interaction of DR5 with full-length FADD or the DD alone was tested in yeast using quantitative -galactosidase assays.

Conclusion DR5 binds to full-length FADD better than the death domain alone.

There is about a 20% increase in binding of DR5 to full-length FADD compared with the DD alone.

HeLa cells were transfected with FLAG-DR5 along with GFP, GFPFADD-DD, or GFP-FADD FLAG complexes precipitated.

The interaction was measured by immunoblotting for GFP.

Figure 4. B

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.

which residues in FADD are required for binding to one receptor but not the other (i.e., DR5 vs Fas)?

Question

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.

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.

Figure 5.A

Conclusion Other than Val 108, the same residues that are required for FADD binding to DR5 are also

required for Fas/CD95 binding.

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 FADD (V108E) is able to bind DR5 and transduce TRAIL signaling but is unable to

bind Fas/CD95 or transduce signaling through FasL.

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.

Figure 5. B & C

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.

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

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.

Jurkat cells express very little DR4 so almost all TRAIL signaling is through DR5. So, what about DR4?

Some reports suggest that FADD and TRADD were not involved in TRAIL-induced 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.

In Fig.1C, HeLa cells were used? What about FADD-deficient Jurkat cells?

Well organized paper. For the most part, conclusions are supported by the data presented.

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

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.

Critique, contd.

Future Directions

Test whether FADD binds DR5 directly in several different cell lines.

Test whether FADD binds DR4 directly in several different cell lines.

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 C-terminal tails of Tumor Necrosis Factor-related apoptosis-inducing 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].

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 -galactosidase 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. 3-amino-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

http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html

Apoptosis signaling by DR4 and DR5 and its modulation by decoy receptors.

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 (DR5) 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-apoptosis-inducing 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 TRADD-FADD 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

http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/

LacZ

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 pro-growth 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.

•

•

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. Two-hybrid 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 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.