gloss

Interazioni Proteina-proteina

•La modulazione dinamica delle interazioni proteiche è la

base fisica di molte reti biologiche che gestiscono i segnali.

La conoscenza dei meccanismi alla base, dei pattern e

regolazione di interazioni discrete, come dei network piu

grandi costruiti su di esse ci dicono come un organismo

funziona da entità omeostatica, in risposta a stimoli esterni,

variazioni ambientali, e come sia in grado di modificare il suo

fenotipo nel tempo o in risposta a input precisi

•Inoltre la caratterizzazione di interazioni P-P ci informa sulle

basi molecolari delle malattie e ci fornisce opportunità di

intervento per prevenire, rivelare e trattare le patologie

stesse

•

Punto di partenza è l'identificazione

delle interazioni, per poi progredire ai

pathway coinvolti

•

Ad es si parte da una proteina di

interesse e si cercano interattori non

noti finora

•

Approccio classico è preso dalle

tecniche di purificazione

opportunamente modulate

•

Una volta individuata l'interazione, si

procederà alla sua caratterizzazione in

vitro ed in vivo

•

Per ottenere una visione del

meccanismo molecolare, per capire la

sua rilevanza funzionale, per sviluppare

modalità per interferire o modulare

l'interazione

•

L'ultimo scopo è particolarmente

augurabile in caso di interazioni che

sottendono a patologie

Classificazione di tecniche 1

• termodinamici/energetici

–

Metodi quantitativi per misurare

parametri termodinamici e/o

energetici come costanti

d'equilibrio, costanti cinetiche,

energie di legame

–

Centrifugazione analitica, FTIR,

Calorimetria, SPR, AFM

Classificazione di tecniche 2

• Approcci strutturali

–

Tecniche per definire le basi

fisiche dell'interazione a livello

molecolare e costruirne un

modello

–

Cristallografia a raggi X. NMR,

ma anche mass spec MS,

cross linking

Classificazione di tecniche 3

• Approcci stuttura/funzione

–

Tecniche per definire il

rapporto tra struttura proteica e

caratteristiche dell'interazione

–

Coimmunoprecipitazione o Gst

fusion pull down in

combinazione con mutagenesi.

Phage display

Classificazione di tecniche 4

• Analisi in vivo

–

Per capire dove e quando una

interazione esiste, è rilevante e

come sia regolata

–

Coimmunoprecipitazione,

CHIP, Fusioni con GFP e sue

varianti, Complementazione di

frammenti proteici

Classificazione di tecniche 5

•

Interazione e funzione a livello

Genomico

– Riscalare l'informazione a

livello di genomi interi

–

Doppio ibrido, protein

microarray. TAP, affinity-MS.,

in parallelo integrazione e

cross validazione di database

di interazioni

Riconoscimento Molecolare

Metodiche sperimentali classiche

Cromatografia

Coimmunoprecipitazioni

Cross linking chimico

Doppio ibrido, lievito e batteri

Cromatografia di affinità

La purificazione tramite cromatografia d'affinità, a differenza degli altri tipi di cromatografia,

dell'elettroforesi e della centrifugazione, non si basa sulle differenze nelle proprietà fisiche delle

molecole da separare, ma sfrutta le interazioni altamente specifiche delle molecole biologiche.

Materiali

La matrice ideale per cromatografia d'affinità deve possedere le seguenti caratteristiche:

1. Deve contenere gruppi reattivi numerosi e adatti a legare covalentemente il ligando.

2. Deve essere stabile nelle condizioni d'interazione con la macromolecola e nella successiva eluizione.

3. Non deve interagire, se non debolmente, con altre macromolecole.

4. Deve possedere buone capacità di flusso.

Affinity co-purification

(pull down)

Analisi interazioni

DNA-proteine:

saggio di footprinting

Analisi interazioni

DNA-proteine:

saggio EMSA

Co-Immuno precipitazione

Coimmunoprecipitazione

Blue native

gels

Principle behind BN/SDS-PAGE. A) After

solubilization, the mixture of different protein

complexes is separated by BN-PAGE, according to

their molecular weight. B) Following the gel run, the

lane of the gel is excised and subjected to a

denaturation so that the native protein complexes

(underlayed in grey color) dissociate to their

constituent polypeptides.

The procedure: solubilization of the cellular membrane with a

nonionic detergents like digitonin, centrifugation, staining of

the supernatant with the anionic dye Coomassie blue (which

causes a charge shift and results in the migration of all protein

molecules to the anode, regardless of original charge, at pH

7.5), excision of specific bands and recovery of blue stained

native proteins by electroelution

Interference of coomassie blue with activity

measurements and fluorescent detection in general

cannot be excluded

Cross linking

Steps involved

1. the protein complex is allowed to react with a cleavable bifunctional reagent of the form RSSR’, and the R and R’ groups react

with susceptible amino acid side chains in the protein complex. This reaction forms adducts of the form RSSR’-P’. (Phizicky and

Fields 1995)

2. The proteins are fractionated on an SDS gel in the absence of reducing agents. The gel separates the proteins based on

molecular weight, and cross linked proteins migrate based on their greater molecular weights

3. A second dimension of the SDS-gel is run after treatment of the gel with a reducing agent to cleave the central S – S bond. The

un-cross-linked species align along the diagonal because their molecular weights do no change after the reduction. The cross –

linked proteins migrate off the diagonal because they migrated as P-RSSR’-P in the first dimension and as molecules of the form

P-RSH and P’R’SH in the second dimension. (Phizicky and Fields 1995) The cross-links are detected based on their size, which

matches that of the un-cross-linked species P and P’.

Cross linkers omo ed etero

funzionali

cross-linking reaction of two proteins with DSG (Disuccinimidyl glutarate)

EDC

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

Approaches

Cross linking

There are several approaches to cross-

linking. These approaches, as detailed by Phizicky

and Fields include

1. the proteins are prefractionated on urea

acrylamide gels or on CM Sepharose before

diagonal electrophoresis

2. Running two dimensional gels without cleaving

the cross-link, followed by elution of individual

species, cleavage of the cross-link, and resolution

of the resulting proteins on a third gel

3. the use of antibody to identify crosslinked

partners after running on the appropriate gels. The

gel can then be transferred followed by

immunoblotting to identify the cross-linked proteins.

The following crosslinking agents were used: F2DNB

(1,5-difluoro-2,4-dinitrobezene), used for forming a

bridge between amino groups and tyrosine phenolic

groups; the bifunctional imidoester, DMS

(dimethylsuberimidate dihydrochloride), used for

forming long bridges between amino groups, DTP

(dimethyl-3,3-dithiobispropionimidate

dihydrochloride), another bifunctional imidoester,

which contains a disulfide bond that is readily

cleaved; and H2O2 and CUP which were used to

facilitate the oxidation of free sulfhydryl groups on the

polypeptide chains

Cross linking

Advantages of Cross-linking

1. Cross-linking has the ability to enhance weak inteactions that would otherwise not be

visible by other methods.

2. Cross-linking can be used to detect transient contacts with different proteins at

various stages ina dynamic process such as glycosylation, by freezing the process at

different stages.

3. Cross-linking may be done in vivo with membrane-permeable cross-linking reagents.

Disadvantages of Cross-linking

1. Cross-linking detects nearest neighbors which may not be in direct contact. This

occurs when the cross-linking reagent reaches out to any protein in close vicinity. The

potential for this problem increases as the size of the cross-liking reagent increases.

2. Due to the propensity to detect False Negatives, the results from a cross-linking assay

must be verified by other protein – protein interaction assays.

Doppio ibrido

Sistema del doppio ibrido

Doppio ibrido

Basic schematic of two-hybrid

system to detect interactions

between two proteins. As shown,

a DNA-binding domain (DBD)

fused Bait protein of interest

interacts with an Activation-

domain (AD)-fused partner

protein (prey), either known or

selected from a cDNA library. The

interacting pair binds a specific

sequence motif, activating

transcription of at least two

separate reporter genes.

protein-RNA interactions.

In this strategy, the DNA-binding

domain is fused to a defined protein

with the capacity to bind either RNA

or a chemical ligand. This fusion

construct, termed "Hook" is either

coexpressed with a hybrid RNA

(Bait) containing the RNA binding

site for the Hook and a novel probe

sequence; or alternatively, yeast

containing the Hook are grown in the

presence of a chemically

synthesized molecule (Bait) that

fuses the Hook ligand and the actual

probe sequence. The Prey

constitutes an activation-domain

fused protein as in other two-hybrid

manifestations; in this case, the

protein has the property of binding

the probe RNA or chemical ligand.

Dual bait

These reagents allow the

discrimination of the interaction of

a single prey with two different

baits. A first DNA-binding domain

fusion (DBD1-B) directs the

expression of first set of reporters.

A second separate DNA-binding

domain fused to a distinct bait

(DBD2-C) directs expression of

the second set of reporters. This

reagent set can be used to select

preys that interact with the DBD1B

but not DBD2-C from a library.

Alternatively, if starting with a prey

that interacts with both DBD1-B

and DBD2-C, it can be used to

select for mutations or molecules

that selectively disrupt the

interaction with one of the two

baits

Reverse 2Hyb: modulare le interazioni

In the reverse two-hybrid system developed by

Vidal et al., reporter 1 (HIS3) is used for positive

selection, while reporter 2 (URA3) is used for

counterselection.

Top, (A): interaction between DBD-and AD-fused

proteins results in growth on medium lacking

histidine, but lethality on medium containing 5fluoroorotic

acid (5FOA), a toxic metabolite of the

URA pathway.

Below, (B): following mutagenesis of DBD- or AD-

fusion protein, missense mutations that weaken

the interaction of DBD- and AD-fusions can be

separated from nonsense mutants that result in

loss of either fusion by comparing profile on

histidine- and 5FOA medium: mutations which

weaken the interaction will display slow growth on

both media, while mutations or truncations which

completely abrogate the interaction will result in

moderate to strong growth on 5FOA medium, but

no growth on histidine- medium.

Doppio ibrido batterico

The central problem with two-

hybrid screening is that detection ofprotein–

protein interactions occurs in afixed context, the nucleus of

Saccharomycescerevisiae, and the results of a

screening must be validated asbiologically relevantusing other assays in appropriatecell, tissue, or organism models

The development of bacterial-based

systems analogous to the yeast one-

hybrid and two-hybrid methods could, in

principle, facilitate the rapid analysis of

larger libraries (due to the higher

transformation efficiency and faster

growth rate observed with Escherichia

coli)

It also provides an altenative cellurar

background in which to identify interaction

partners useful for proteins that are toxic

or that interact with endogenous proteins

in yeast.

Da controllare (per evitare falsi):

TAP: Tandem

Affinity Purification

2 tag di affinita

con un sito

proteolitico a

separarle

TAP: Tandem

Affinity

Purification

To utilize the TAP method discovered by

Rigaut et al, the TAP tag needs to be fused

with the protein of interest and introducing

the hybrid into host cells in order to keep the

protein of interest in a native condition. The

hybrid, as well as any interacting factors, is

then retrieved from the cell extract on an IgG

matrix. After washing, the TEV protease is

added to release the bound material. The

eluate is incubated with calmodulin-coated

beads in the presence of calcium. This

second affinity step is required to remove the

TEV protease as well as traces of

contaminants remaining after the first affinity

selection. After washing, the bound material

is released with EGTA.

TAP: Tandem Affinity

Purification

Optimal tags for protein complex purification and the analysis of protein

interaction should have the following characteristics:

1. High affinity for the cognate matrix for quantitative recovery of low-

abundance target proteins in dilute solutions.

2. Highly specific binding to increase the ratio of specifically tononspecifically bound materialto the affinity material.

3. Efficient and specific elution allowing high-level and specific recoveryof the target protein.

4. Mild conditions of elution to preserve protein interactions and proteincomplex structure.

TAP: Tandem Affinity

Purification

Although the TAP method is broadly applicable, there are some limitations.

First, a functional TAP-tagged protein must be produced.

For some proteins, tagging at both the amino and the carboxyl termini of the protein mayaffect its activity.

Even if the protein is functional, a similar problem may occur if the TAP tag is inaccessiblein the native protein complex. In this case,we suggest testing a construct expressing thetarget protein fused to the TAP tag at its other extremity.

Although some proteins also contain an endogenous TEV protease cleavage site that

interferes with the purification, this is not likely to be a verycommon problem.

One should remember, however, that the TEV protease is not a restriction enzyme and that

degenerate sites may be cleaved if well exposed, whereas perfect sites buried inthe interior of the protein will not be accessible to the protease.

The presence of a bona fide TEV protease cleavage site in the target protein remains to bedetermined by an experimentalapproach.

Far Western