CG020 Genomika
Přednáška 7
Proteinové interakce v genových regulacích
Jan Hejátko
Funkční genomika a proteomika rostlin,
Středoevropský technologický institut (CEITEC)
a
Národní centrum pro výzkum biomolekul,
Přírodovědecká fakulta,
Masarykova univerzita, Brno
hejatko@sci.muni.cz, www.ceitec.eu
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 Zdrojová literatura
 Wilt, F.H., and Hake, S. (2004). Principles of Developmental Biology. (New York ;
London: W. W. Norton).
 Ainger, K., Avossa, D., Morgan, F., Hill, S.J., Barry, C., Barbarese, E., and Carson, J.H.
(1993). Transport and localization of exogenous myelin basic protein mRNA microinjected
into oligodendrocytes. J Cell Biol 123, 431-441.
 Alberts, B. (1998). The cell as a collection of protein machines: preparing the next
generation of molecular biologists. Cell 92, 291-294.
 Grefen, C., Stadele, K., Ruzicka, K., Obrdlik, P., Harter, K., and Horak, J. (2008).
Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene
receptor family members. Molecular Plant 1, 308-320.
 Hu, C.D., and Kerppola, T.K. (2003). Simultaneous visualization of multiple protein
interactions in living cells using multicolor fluorescence complementation analysis. Nat.
Biotechnol. 21, 539-545.
 Shahbabian, K., and Chartrand, P. (2012). Control of cytoplasmic mRNA localization.
Cellular and molecular life sciences : CMLS 69, 535-552.
 Van Leene, J., Witters, E., Inze, D., and De Jaeger, G. (2008). Boosting tandem affinity
purification of plant protein complexes. Trends Plant Sci 13, 517-520.
 Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D.,
Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. (2004). Visualization of
protein interactions in living plant cells using bimolecular fluorescence complementation.
Plant J 40, 428-438.
Genomika 07
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 Funkční význam specifických interakcí proteinů v
regulaci genové exprese
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Stabilita proteinů
 Přenos signálu
 Metody analýzy proteinových interakcí in vivo
 Koimunoprecipitace
 Tandemová afinitní purifikace (TAP-tag)
 Kvasinkový dvouhybridní test (Y2H)
 Bimolekulární fluorescenční komplementace (BiFC)
 Analýza zprostředkované membránové vazby (MeRA)
Osnova
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 Funkční význam specifických interakcí proteinů
 Většina proteinů v buňce existuje ve
formě komplexů, které mohou dále
navzájem interagovat
 Proteazom
 proteinový komplex zodpovědný za
degradaci nepotřebných proteinů v
buňce
Význam interakcí
proteinů
1fnt
Once obsolete proteins are tagged with at least four ubiquitin molecules,
they are destroyed by proteasomes. Proteasomes are voracious protein
shredders, but the destructive machinery is carefully protected so that it
can't attack all of the normal proteins in the cell. The proteasome, shown
here from PDB entry 1fnt, is shaped like a cylinder, with its active sites
sheltered inside the tube. The caps on the ends regulate entry into the
destructive chamber, where the protein is chopped into pieces 3 to 23
amino acids long.
Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is
performed by a nonspecific and abundant barrel-shaped complex called
the 20S proteasome. Substrates access the active sites, which are
sequestered in an internal chamber, by traversing a narrow opening
(alpha-annulus) that is blocked in the unliganded 20S proteasome by
amino-terminal sequences of alpha-subunits. Peptide products probably
exit the 20S proteasome through the same opening. 11S regulators (also
called PA26, PA28 and REG) are heptamers that stimulate 20S
proteasome peptidase activity in vitro and may facilitate product release in
vivo. (PDB, Whitby et al., (2000) Nature 408: 115-120,
http://www.rcsb.org/pdb/explore/explore.do?structureId=1fnt).
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Proteazom
 Skládá se z centrálního komplexu
označovaného jako 20S a regulačních
částí (19S, někdy také 11S)
 Umožňuje cílenou degradaci proteinů
označených specifickou značkou,
malým proteinem (76 aa) - ubiquitinem
Význam interakcí
proteinů
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By Rogerdodd, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=1291009
Ubiqutinace proteinů
„polibek smrti“
ubiquitin aktivující enzym
(ubiquitin activating enzyme)
ubiquitin vázající enzym
(ubiquitin conjugating/ubiquitin carrier enzyme
ubiquitin ligáza
(ubiquitin ligase)
By Rogerdodd - Own work, CC BY-SA 3.0,
https://commons.wikimedia.org/w/index.php?curid=477
1409
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Proteasome –řízená proteolýza
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 Funkční význam specifických inteakcí proteinů
 Struktura chromatinu
Význam PI
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Regulation by histone
acetyl transferases or
histone deacteylases
Regulation of the chromatin structure represents one of the very basal gene
expression regulatory levels. Chromatin is a substrate for DNA-dependent RNA
polymerases that transcript the DNA encoded information into the “words and
sentences” of RNA.
Regulation of chromatin structure and its accessibility to DNA-dependent RNA
polymerases depends on many factors, one of the most important is the
regulation of chromatin binding to nucleosomes and chromatin methylation.
Regulation of chromatin interaction with histones, the positively charged proteins
forming the core of nucleosomes, is performed via modification of acetylation
status of the N-terminal portion of histones, especially histones H3 and H4. This
occurs via action of histone acetyl transferases or histone deacteylases.
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CpG or CpNpG
CpNpNp
CpG
DNA methylation in animals vs. in plants
methylation status methylation status
Cell-specific methylation allows maintain of
tissue-specific gene expression profiles
Mechanism of transcriptional regulation by
DNA methylation mostly unknownImprinting and “cell memory”
Modification of the chromatin methylation is performed via DNA methyltransferases.
Interestingly, there is difference in the methylation in animals and in plants.
In animals, the methylation takes place mostly on the cytosine that occurs next to
guanosine (the sequence is denoted as CpG). In mammals, 60-90% of all CpGs are
methylated.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and
asymmetrically (CpNpNp), where N is any nucleotide.
Methylation status is usually “reset” in the zygote and is reconstituted during
development again. E.g. the methylation is very low in the mouse embryo at the blastula
stage, however, DNA derived from later stages when organogenesis is initiated is
substantially more modified by methylation.
DNA methylation also stably alters the gene expression pattern in cells such that cells
can "remember where they have been"; in other words, cells programmed to be
pancreatic islets during embryonic development remain pancreatic islets throughout the
life of the organism without continuing signals telling them that they need to remain
islets.
DNA methylation is involved in the genomic imprinting, i.e. the genes originating from
both parents are often diversely methylated, which results into differential expression of
parental genomes (for the importance of the imprinting in the parental conflict and
epigenetics, see the lecture “Bi0580 Developmental genetics” by prof. Vyskot).
Up to know it is not clear how methylation regulates transcription. Possibly, methylation
status affects chromatin configuration or binding general repressor factors.
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 Funkční význam specifických inteakcí proteinů
 Struktura chromatinu
 Regulace transkripce
Význam PI
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Iniciace Transkripce
Regulation of transcription occurs via specific interaction of both general and
tissue specific transcription factors (TFs) with promoter and/or enhancer
sequences.
The scheme above shows simplified subsequent formation of the complex of TFs
involved in the regulation of transcription. Interaction of general TFIID with the
TATA box induces distortion of the DNA structure (see the next slide).
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Induction of structural changes upon interaction of TFIID with DNA. This may be
important for the assembly of other TFs involved in the formation of transcription
initiation complex.
This change of confirmation provides a kind of “signature” that is recognized by
other proteins and NA polymerase to recognize the proper binding site. However,
there are also TATA box-less promoter, where probably other types of
“signatures” occur.
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Positive TFs
Negative TFs
Iniciace Transkripce
The scheme showing the formation of the transcription initiation complex and the
interaction of both positive (open symbols) and negative (solid symbols) factors.
These proteins bind to the regulatory sequences that might be hundreds or even
thousands of base pairs away from the promoter. These protein interact with
each other and with the RNA polymerase, integrating thus many signals into a
“yes” or “no” response of the basal promoter, i.e. the region adjacent to the TATA
box and recognized by the RNA polymerase.
The individual positive or negative factors are complex and their activity might be
regulated by their phosphorylation status or via their interaction with other
proteins (i.e. momomeric or dimeric) etc..
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Signal recognition
Dimerization
DNA binding
and
transcription
activation every 7th aa
Transcriční regulace
prostřednictvím TAFs
There is a whole family of transcription activating factors (TAFs) that interact with
signalling molecules, e.g. steroid hormones, thyroid hormones or retinoic acid
and in a response to the signal transfer to the nucleus where they regulate
transcription.
One of the type of TAF are leucine zipper or bZIP type TAFs. These TAFs are
dimeric, with leucine-rich hydrophobic face formed by the Leu that occurs every
7th aa.
That allows the factor to take the proper configuration, which provides the dimer
with the ability to bind DNA via charged aa.
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ProENDO16:REPORTER (sea urchin)
Deletion
mutagenesis Positive, interaction with
TAFs
Upregulation in the
presence of A and B
Developmental specificity
Combinatorial control
Midgut
Primary or skeletogenic
mesenchyme cells
Vegetal plate
Multifaktoriání kontrola promotorů
An example of the “microprocessor”-like acting promoter is a promoter of the endo16
gene from the sea urchin.
There have been identified several gene regulatory modules in the endo16 gene that
have positive or negative regulatory role. These modules were identified via formation of
deletion mutants of the transcriptional fusions with reporter gene.
The analysis has revealed that the module A has a positive function and must interact
with its cognate TAFs for transcription to occur.
Module G enhances the expression when the A and B are active.
C, D, E and F are responsible for the specificity of the expression of endo16 during sea
urchin development.
Each of the modules has several protein interaction sites, some of them general, other
unique. Site for the protein SpGCF1 is present in many modules and is probably
responsible for looping of chromatin, allowing thus bringing of distal regulatory modules
close to the basal promoter.
This type of regulation, i.e. based on the different activities of diverse regulatory
sequences is sometimes called combinatorial and is common for development of many
living creatures.
In the combinatorial type of regulations, some modules may act synergistically, some of
them antagonistically, some may have both positive and negative roles (e.g. the module
B, see the figure). This variability allows very precise and responsive regulations towards
changing environmental conditions.
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Regulation of β-globin type of
hemoglobin chains expression
Locus control region
Development-dependent
activation by LCR
•Acetylation of H3?
•Involvement of other
genes?
Cca 50 kbp
Multifaktoriání kontrola promotorů
An example of the combinatorial gene regulation is the regulation of β-globin type of hemoglobin
chains of humans.
As discussed in the Lesson 5 (course Bi8940 Developmental biology), the type of hemoglobin
produced by the fetus changes during development. The hemoglobin present in the erythrocytes is
composed of two α- and two β-type chains. The β-type hemoglobin chains are of several
developmental types, produced by ε, γ1, γ2 and β (in this order). In addition, there is minor adult
type of β-type hemoglobin, called δ globin.
The genes for the β-type chains are aligned on the chromosome in the order, in which they are
expressed during development (see the figure).
For the expression of individual cell types is distinctive an upstream regulatory sequence called
locus control region (LCR). LCR is located about 50 kbp away from the most proximal ε gene.
The LCR structure is different in erytrocyte precursor cells in comparison to other cells that could
be demonstrated by the changes in the sensitivity to low concentrations of DNase, suggesting low
amount of nucleosomes bound.
For the expression of the particular genes, the interaction of their regulatory sequences with LCRs
is necessary. Because of LCR can interact only with one regulatory sequence at a time, only one
type of genes for the particular β-type chain is activated (the first interaction of LCR with ε gene,
which is later in development replaced by the other one, is shown by the double-headed arrow).
The underlying molecular mechanisms of the specific pattern of the LCR movement from the most
proximal towards the most distal gene cannot be satisfactory explained.
Probably, acetylation of H3 histones might play a role and possibly, other genes outside of the βtype
chain family are involved in the regulation of LCR activity. That seems to be confirmed by the
identification of other human genes with similar structure, suggesting common regulatory
mechanisms via LCRs.
For
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 Funkční význam specifických inteakcí proteinů
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
Význam PI
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BICOID mRNA NANOS mRNA
 Význam lokalizace mRNA
 Lokalizace proteinového produktu
genu v čase a místě
 asymetrické dělení během
vývoje
 polarizace embrya
Lokalizace mRNA
Shahbabian and Chartrand, 2012
ASH1 mRNA
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 Role lokalizace mRNA
 Omezení exprese potenciálně
toxických proteinů
 lokalizace exprese
mRNA pro MYELIN
BASIC PROTEIN (MBP)
do oblasti myelinizace
nervových buněk
Lokalizace mRNA
Ainger et al., 1993
MBP mRNA
Myelin basic protein (MBP) is a protein believed to be important in the process of
myelination of nerves in the nervous system.
The images show localization of mRNA for MBP. Digoxigenin-labeled MBP RNA
was microinjected into mouse oligodendrocytes growing in primary culture. The
injected RNA appeared as small granules which were present throughout the
cytoplasm and processes, and was also found dispersed in the peripheral
membranes of the cell.
To analyze the three dimensional distribution of microinjected labeled MBP mRNA
throughout the cell, consecutive optical sections through a single oligodendrocyte
were collected, reconstructed, and visualized using volume rendering (Fig. A) or
isosurface rendering (Fig. B) techniques. An oligodendrocyte microinjected with
MBP mRNA, visualized by volume rendering is shown in Fig. A. RNA granules
were observed throughout the perikaryon and in some, but not all, processes.
The granules in the perikaryon and in the processes appeared to be equivalent in
size. In some regions the granules in the processes were aligned in tracks.
Although not apparent from this image, the nucleus was devoid of granules.
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Shahbabian and Chartrand, 2012
 Difůze a ukotvení mRNA
 Během ranné oogeneze u drápatky je
Xcat-2 mRNA lokalizována do tzv.
mitochondriálního oblaku (MO,
Balbianiho tělísko)
 Pohyb MO je částečně závislý na
depolymerizaci mikrotubulů (tzv.
molekulární motor)
 Ukotvení na vegetálním pólu je dáno
interakcíá MO s ER
Lokalizace mRNA
Mechanizmy
Another well studied example of the diffusion-entrapment mechanism is the
Xenopus Xcat-2 mRNA, which encodes a Nos related zinc-finger RNA-binding
protein.
During the early stages of Xenopus oogenesis, Xcat-2 mRNA is restricted to a
specific structure in the cytoplasm called the mitochondrial cloud (MC). The
mitochondrial cloud, also called Balbiani body, consists mostly of mitochondria
and small vesicles, and is the source of germinal granule material [32]. The
movement of the MC in the cytoplasm results in the localization of the Xcat-2
mRNA at the vegetal cortex (Shahbabian and Chartrand, 2012).
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 Lokalizovaná degradace mRNA
 V embryogenezi u Drosophila m.
dochází k polární lokalizaci Hsp83
mRNA, podobně jako NANOS
mRNA
 Hsp83 mRNA je lokalizována v
celém embryu, zde je však
destabilizována prostřednictvím cis
elementů jak v 3’UTR (HDE), tak v
kódující oblasti (HIE)
Lokalizace mRNA
Mechanizmy
 HIE elementy jsou rozpoznávány proteinem SMAUG, který
zprostředkováva vazbu degradačního komplexu
CCR4/POP2/NOT
 V oblasti posteriorního pólu je Hsp83 mRNA chráněna před
účinkem SMAUG tzv. HPE elementem v 3’UTR; mechanismus
této ochrany je dosud neznámý
Shahbabian and Chartrand, 2012
Localized stabilization of a transcript is another mechanism by which an mRNA can be
subcellularly targeted. In this case, an mRNA is rapidly degraded in most parts of the cell, but it is
protected from degradation at a specific location. The hsp83 mRNA, which encodes a heat shock
protein in Drosophila, is a well-characterized example of this kind of localization (Fig. 2b). This
transcript is localized at the posterior pole of the early Drosophila embryo by the selective
stabilization of the mRNA at the posterior pole and degradation of the transcript elsewhere in the
cytoplasm.
The level of hsp83 mRNA, which is a maternally encoded transcript, decreases more rapidly in
embryos than in unfertilized eggs, which suggests that two separate mechanisms control the
stability of this transcript [38]. These two independent pathways, which are called ‘‘maternal’’ and
‘‘zygotic’’ pathways, use maternally and embryonic encoded proteins, respectively, to degrade the
hsp83 transcript [38]. By analyzing the 3’UTR of hsp83 mRNA, a region from nucleotides 253–349
was identified as the Hsp83 degradation element (HDE), which directs the destabilization of this
mRNA in unfertilized eggs. However, this region has no effect in the zygotic degradation pathway,
and transcripts without the HDE domain are subject to degradation by the embryonic degradation
machinery [38]. The hsp83 ORF has also been shown to affect the stability of the transcript. A
region at the 3’ end of the ORF, which comprises 615 nucleotides, has been found to be
responsible for this destabilization, and was consequently called Hsp38 instability element (HIE)
[39]. This region, which has the major effect in the destabilization of the transcript, functions
together with the HDE for complete degradation. The HIE domain contains six stem-loop
structures that are recognized by the maternally encoded RNA-binding protein Smaug [39, 40]. It
was shown that in Smaug mutants, degradation and thus localization of hsp83 mRNA are
impaired. Smaug recruits the CCR4/POP2/NOT deadenylase complex, triggering deadenylation
and thus degradation of the hsp83 transcript [40]. Although Smaug is present throughout the pole
plasm, the hsp83 mRNA is protected from Smaug action at the posterior pole. This protection is
related to a 57 nt region in the 3’UTR (nucleotides 351–407) downstream of HDE, which is called
HPE (Hsp83 protection element). HPE is sufficient to confer stability to an unstable transcript at
the pole plasm [40]. The mechanism by which this domain functions is not clear, and may include
interaction of trans-acting factors that block the availability of the transcript to Smaug (Shahbabian
and Chartrand, 2012).
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Shahbabian and Chartrand, 2012
 Aktivní transport mRNA
 Asymmetric Synthesis of HO1
(ASH1) je represor HO u S.
cereviseae; inhibice HO
endonukleázy v dceřinných buňkách
zabraňuje změně párovacího typu
 ASH1 mRNA je aktivně
transportována prostřednictvím
„molekulárních motorů“
asociovaných s aktinem
Lokalizace mRNA
Mechanizmy
 ASH1 mRNA obsahuje 4 cis
elementy (3 v CDS a 1 ve
3’UTR), které jsou
rozpoznávány RNA vazebným
proteinem SHE2
 SHE2 umožňuje
prostřednictvím SHE3 vazbu
na „molekulární motor“,
MYO4, který se váže na aktin
a umožňuje transport ASH1
mRNA do dceřinné buňky
Shahbabian and Chartrand, 2012
ASH1 mRNA
Localization of ASH1 mRNA is essential for the asymmetric distribution of Ash1, which acts as a
transcriptional repressor of the HO endonuclease and results in inhibition of mating-type switching in
daughter cells [88, 89]. The ASH1 mRNA contains four localization elements, three in the coding sequence
(E1, E2A, and E2B) and one overlapping the end of the coding sequence and the 3’UTR (E3) [25, 90]. While
the presence of these four elements leads to an optimal localization, deletion analysis revealed that each
element is sufficient for localization of a reporter mRNA to the bud.
When each of these elements was inserted in multiple copies in the 30UTR, the new constructs showed
nearly normal localization. However, for these mRNAs, the asymmetric distribution of Ash1 was impaired,
suggesting that the position of these elements is important for Ash1 sorting but not for ASH1 mRNA
localization [91]. Although the primary sequences of the four ASH1 localization elements are different, they
all fold into a stem-loop structure that contains a few conserved nucleotides [92, 93]. All four elements
interact with the same RNA binding protein called She2, which is involved in the localization of bud-localized
mRNAs in S. cerevisiae.
She2 forms a tetramer under physiological conditions, and mutations that disrupt this tetrameric state
abolish its RNA-binding capacity and impair She2-dependent localization to the bud tip [94]. She2 interacts
directly with the C-terminal domain of She3, an adaptor protein that links the She2–mRNA complex to the
molecular motor Myo4 (Fig. 2c) [55, 95, 96]. Recent evidence also suggests that She3, besides its role in
connecting the She2–RNA complex to Myo4, is itself able to bind RNA and acts synergistically with She2 to
increase the affinity and specificity of RNA binding [97].
Recent studies on Myo4 helped to explain why multiple localization elements are required for proper ASH1
mRNA localization. Myo4 is a class V myosin whose main function is the transport of mRNAs to the bud tip
using actin filaments [98–100]. Myo4, unlike other type V myosins, is a nonprocessive monomer in vivo, but
it becomes processive when present in the form of oligomers [101, 102]. Purification of the localization
complex associated with a single localization element revealed that multiple copies of Myo4 are associated
with this RNA [103]. Moreover, increasing the number of Myo4 attached to the ASH1 mRNA increased the
efficiency of localization of this transcript. These results suggest that each localization element interacts with
higher order protein complexes in which a She2 tetramer may recruit multiple copies of Myo4, thus ensuring
a continuous and processive movement of the mRNP complex into the bud. Moreover, it is possible that a
She2 tetramer binds simultaneously to the localization elements of a single transcript or, alternatively, to
those of different mRNAs. This would bring multiple mRNAs together within a single complex in which
several Myo4 molecules modulate their transport to the bud tip (Shahbabian and Chartrand, 2012).
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 Funkční význam specifických inteakcí proteinů
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Sestřih hnRNA
Význam PI
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 Funkční význam specifických inteakcí proteinů
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Sestřih hnRNA
 Stabilita proteinů
Význam PI
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Jing and Strader, Plant Structural Biology, Hormonal Regulations (2018)
Auxinová signalizace
Scheme of the auxin signaling pathway as an example of the role of protein
stabilization leading to regulation of gene expression.
Under low intracellulr auxin concentrations, the transcription activators of auxinregulated
genes, which are called auxin responsive factors (ARFs), are in a
complex with negative regulators of transcription, so called AUX/|IAA proteins. In
the complex, Arfs can not activate transcription.
After auxin is imported into the cell, it binds to the TIR1 protein, that allows
interaction with AUX/IAA-ARF complex and targets AUX/IAA protein for the
degradation via proteosome.
That allows ARFs to enter nucleus and activate trancription of auxin-induced
genes.
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 Funkční význam specifických inteakcí proteinů
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Sestřih hnRNA
 Stabilita proteinů
 Přenos signálu
Význam PI
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 PI a přenos signálu
 prostřednictvím G proteinu a
fosfolipasy C
 Signální kaskády využívající cAMP
PI a přenos signálu
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Přenos signálu a
fosfolipáza C
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Receptory aktivované G
proteiny
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 Funkční význam specifických interakcí proteinů v
regulaci genové exprese
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Stabilita mRNA
 Stabilita proteinů
 Přenos signálu
 Metody analýzy proteinových interakcí in vivo
 Koimunoprecipitace
Osnova
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PI in vivo
Koimunoprecipitace
 založena na izolaci proteinových komplexů
pomocí protilátek rozpoznávajících jeden z
interagujících proteinů
CKI1
MY
C
CKI1
HA
αMYC
αHA
CKI1
MY
C
AHK3
HA
αMYC
CKI1-MYC
CKI1-HA
AHK3/4-HA
CKI1-HA
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 Funkční význam specifických interakcí proteinů v
regulaci genové exprese
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Stabilita mRNA
 Stabilita proteinů
 Přenos signálu
 Metody analýzy proteinových interakcí in vivo
 Koimunoprecipitace
 Tandemová afinitní purifikace (TAP-tag)
Osnova
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PI in vivo
Tandemová afinitní purifikace (TAP-tag)
 izolace proteinových komplexů pomocí
rekombinantních proteinů, fúzovaných s dvěma
různými vazebnými doménami
 proteiny izolovaných komplexů jsou po rozdělení
na 1D ELFO identifikovány pomocí MS
 calmodulin-binding protein (CBP)
 IgG vázající domény proteinu A (ProtA)
 místo rozpoznávané specifickou
proteázou z TEV viru (tobacco etch
virus)
POI
 výhodou je použití dvou nezávislých proteinových
domén pro afinitiní purifikaci a tedy velká
specificita
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 Funkční význam specifických interakcí proteinů v
regulaci genové exprese
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Stabilita mRNA
 Stabilita proteinů
 Přenos signálu
 Metody analýzy proteinových interakcí in vivo
 Koimunoprecipitace
 Tandemová afinitní purifikace (TAP-tag)
 Kvasinkový dvouhybridní test (Y2H)
Osnova
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36
PI in vivo
Dvouhybridní kvasinkový test (Y2H)
 izolace proteinových komplexů pomocí
rekombinantních proteinů, každý z nich fúzovaný
s částí transkripčního faktoru Gal4
 jeden z proteinů (návnada, bait)
fúzovaný s DNA vazebnou doménou
Gal4 (Gal4-BD)
 druhý z proteinů (kořist, prey) fúzovaný
s aktivační doménou Gal4 (Gal4-AD)
 Interakce proteinů umožní rekonstituci vazebné
domény s aktivační doménou a spuštění
reportérového genu
 vizuální detekce (modré zbarvení, LacZ)
 auxotrofní selekce (růst na médiu bez
histidinu, His)
 umožňuje vyhledávání interakčních partnerů v
expresních knihovnách jednotlivých organismů
37
Y2H
38
38
 Funkční význam specifických interakcí proteinů v
regulaci genové exprese
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Stabilita mRNA
 Stabilita proteinů
 Přenos signálu
 Metody analýzy proteinových interakcí in vivo
 Koimunoprecipitace
 Tandemová afinitní purifikace (TAP-tag)
 Kvasinkový dvouhybridní test (Y2H)
 Bimolekulární fluorescenční komplementace (BiFC)
Osnova
39
39
 Proteinová interakce je detekována na
základě reasociace fluoreskujícího proteinu
 každý z potenciálních interakčních
partnerů je fúzován s jednou z
podjednotek fluoreskujícího
proteinu, např. YFP
 při interakci dojde ke
znovuobnovení fluorescence
 Kromě identifikace vlastní interakce
umožňuje i lokalizovat interakci v buňce
PI in vivo
bimolekulární fluorescenční
komplementace (BiFC)
40
40
 Funkční význam specifických interakcí proteinů v
regulaci genové exprese
 Struktura chromatinu
 Regulace transkripce
 Lokalizace mRNA
 Stabilita mRNA
 Stabilita proteinů
 Přenos signálu
 Metody analýzy proteinových interakcí in vivo
 Koimunoprecipitace
 Tandemová afinitní purifikace (TAP-tag)
 Kvasinkový dvouhybridní test (Y2H)
 Bimolekulární fluorescenční komplementace (BiFC)
 Analýza zprostředkované membránové vazby (MeRA)
Osnova
41
41
PI in vivo
Analýza zprostředkované membránové
vazby (MeRA)
 Umožňuje identifikaci interakcí cytoplazmatických
proteinů s membránovými proteiny
 membránový protein je fúzován s
fluoreskujícím proteinem
 potenciální ineterakční partner je
fúzován s jimým fluoreskujícím
proteinem, lišícím se svým emisním
spektrem
 v případě interakce dojde ke změně
lokalizace cytoplazmatického proteinu
na membránu (kolokalizaci s
membránovým proteinem)
42
42

PI in vivo
Analýza zprostředkované membránové
vazby (MeRA)
43
43
GFP
GFPGFPGFP
P35S::ERS1:RFP + P35S::ΔTM-ETR2:GFP
PI in vivo
Analýza zprostředkované membránové
vazby (MeRA)
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44
 Proteiny a jejich interakce jsou zásadním
mechanismem regulace genové exprese
 Podílejí se na regulaci
 Struktury chromatinu
 Iniciace transkripce
 Lokalizace mRNA
 Zprostředkovávají regulaci genové exprese v odpovědi na různé
typy signáĺů
 Proteinové interakce lze detekovat in vivo např.
pomocí
 Koimunoprecipitace
 Tandemové afinitní purifikace (TAP-tag)
 Kvasinkového dvouhybridního testu (Y2H)
 Bimolekulární fluorescenční komplementace (BiFC)
 Analýzy zprostředkované membránové vazby (MeRA)
Klíčové koncepty
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