Biomedical Engineering Reference
In-Depth Information
GAL4
as a transcription factor and
lacZ
as a gene reporter. Expression of the
lacZ
gene is observable thanks to the enzymatic ability of the gene product to metabolize
the substrate X-gal (which can be added to the culture medium) to a blue compound
[51]. The two-hybrid assay, hypothesized in a yeast cellular background, has been
extended to bacterial cells to discover protein-protein interactions [52].
Nevertheless, the use of such methodology in the
S. cerevisiae
system guarantees
several advantages. Indeed, the two-hybrid assay can be used in the budding yeast
to assess the interactions occurring between both yeast-specific and heterologous
proteins (such as mammalian proteins). Whereas the bacterial cells do not bear the
mechanisms to posttranscriptionally modify the expression product (i.e., to eliminate
eventual introns), yeast cells do. One of the main limitations to use of the yeast
two-hybrid assay is that such a method is useless when one of the proteins whose
interactions is to be studied is a receptor or, in general, a membrane-anchored protein.
Indeed, a protein bound to the membrane is stationary, enabling translocation of the
complex studied and of the newly assembled transcription factor to the nucleus and
to the promoter of the reporter gene.
Aiming at the reverse chemical genetics scope, Y2H can be used when the target
protein is known and has been selected. As an example, when the protein induc-
ing a disease is known and new molecules able to inhibit its function have to be
screened, Y2H acquires a fundamental role in high-throughput screenings [53,54].
The Y2H assay can be applied with a forward chemical approach as well. Indeed,
thanks to advancements in genomic sequencing and genetic handling techniques, it
is now possible to screen the effects of sets of molecules on all the combinations
of protein couples interactions. With an evolved version of this approach, Chidley
et al. revealed that the tetrahydrobiopterin biosynthesis was the target of the known
anti-inflammatory drug sulfasalazine [55]. In another study, the Y2H assay was used
to verify the antiviral activity of a novel peptide selected previously by an in vitro
assay [56]. In this work, the NDFRSKT heptapeptide was selected for its ability
to bind purified proteins of the AIV subtype H9N2 (avian influenza virus H9N2).
Thanks to the Y2H assay, it was possible to assume that the antiviral activity of
the peptide selected was given by the inhibition of the viral replication achieved by
binding of the peptide to the hemagglutinin (HA) protein, which is involved in viral
linking to host cells before infection. An elegant evolution of the Y2H methodology
has been developed to detect interactions between proteins and small molecules, the
yeast three-hybrid assay (Y3H, Figure 14.7) [57]. In this case, three chimeric entities
are generated, the first composed of the BD of the transcription factor and a protein
(A), the second composed of the AD of the transcription factor and another protein
(B). The third chimera is composed of two bounded small molecules, one ligand for
the A protein (sm) and a second ligand for the B protein (lig). The binding of the two
small molecules to their corresponding proteins allows for generation of the func-
tional transcription factor, thus resulting in the induction of the reporter phenotypic
effect. The utility of the Y3H assay as a tool for identifying the binding proteins of a
compound was demonstrated by Henthorn et al. by screening a mouse cDNA library
for clones encoding unknown proteins able to bind methotrexate [58]. Furthermore,
the suitability of the Y3H method for the de novo identification of small-molecule
