lezione 2

CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL
FARMACO
Adriana Maggi
BIOTECNOLOGIE FARMACOLOGICHE
2008/09
LEZIONE 2
I PROCESSI DI METILAZIONE GIOCANO UN RUOLO
NELLA PATOGENESI UMANA
methyl DNA binding protein 2 (MeCP2)
The mechanism(s) by which the native MeCP2 protein operates in the
cell are not well understood.
Historically, MeCP2 has been characterized as a proximal gene
silencer with 2 functional domains:
1. a methyl DNA binding domain and
2. a transcription repression domain.
However, several lines of new data indicate that MeCP2 structure and
function relationships are more complex: an analysis of cell-based
experiments suggesting MeCP2 is a regulator, rather than a strict
silencer, of transcription. The new data establish MeCP2 as a
multifunctional nuclear protein, with potentially important roles in
chromatin architecture, regulation of RNA splicing, and active
transcription..
SINDROME DI RETT
Andreas Rett, che aveva osservato un comportamento insolito, tragico e affascinante insieme,
in alcune bambine sedute nella sala d'aspetto del suo studio. Correva l'anno 1966.
Nel 1999, il team di Huda Zoghbi del Baylor College of Medicine di Houston scoprì che la
sindrome di Rett è associata a una mutazione del gene MeCP2, localizzato sul cromosoma X. Il
gene MeCP2 è un repressore della trascrizione genica.
La proteina MeCP2 è espressa in modo predominante nei neuroni maturi. MeCP2 modula le
connessioni sinaptiche e quindi la comunicazione cellulare attraverso la regolazione di alcuni
geni, tra cui il brain-derived neurotrophic factor (BDNF).
Guy et al., con un sistema murino molto sofisticato hanno silenziato la sintesi
della proteina MeCP2 causando la patologia, poi ne hanno reinstaurato la
sintesi con una remissione della sintomatologia
Più recentemente, si è visto che le MeCP2 interagisce con la proteina YB-1
che è coinvolta nella matutazione degli RNA e in particolare della subunità
NR1 dei recettori per il glutammato NMDA
Guy J, Gan J, Selfridge J, Cobb S, Bird A. Reversal of neurological defects in
a mouse model of Rett syndrome. Science 2007; 315:1143-1147
MeCP2 gene
METILAZIONE DEL DNA E CANCRO
The alterations of DNA methylation level and
patterns are a common feature of human cancer
cells. A global DNA hypomethylation has been
observed in many cancers, without obvious
sequence specificity
Recently, an extensive study of about 1200 CpG islands has
indicated that hypermethylated CpG islands are not randomly
distributed and the patterns of the hypermethylation might
be specific of subclasses of cancers.
The methylation status of tumor suppressor genes has been
extensively investigated and such alterations have been
reported in many human tumors (Robertson and Jones,
2000).
METILAZIONE DEL DNA E CANCRO
Several reports link genome hypomethylation to
genome instability. In particular, it was shown
recently that strongly reduced DNMT activity in a
transgenic mouse model caused demethylation of
centromeric satellite and other repeat sequences,
which resulted in a variety of chromosome defects
and concomitant tumorigenesis
Figure 1. Epigenetic cancer therapy. DNA methyltransferases (DNMTs) can
cause ectopic methylation and gene silencing. These events are called
epimutations and promote tumorigenesis if directed to tumor suppressor
genes. Importantly, the maintenance of epimutations requires the continuous
activity of DNMTs. This accounts for the principal reversibility of
epimutations by DNMT inhibitors.
Il mantenimento della metilazione del DNA
(da parte di DNMT1) è indispensabile nella
duplicazione del DNA per mantenere
metilati specifiche porzioni del DNA.
Nel topo la mutazione di DNMT1 è letale
Human DNA methyltransferases (DNMTs) and their functionally important
domains.
All known DNMTs share a highly conserved C-terminal catalytic domain.
The N-terminal domains differ strongly between DNMT1 and DNMT3 enzymes
and contain several motifs for regulatory functions: a PCNA binding domain
(PBD), a replication foci targeting domain (RFTD), a CXXC domain implicated in
DNA binding, a PWWP domain linked to protein targeting and an ATRX domain
implicated in histone deacetylase interactions.
The DNMT3A and DNMT3B proteins are similar and are probably the products
of a recent gene duplication event.
5-Azacytidine (Vidazae) has been approved
for the treatment of myelodysplastic
syndrome.
This has been a major milestone in the
field of cancer epigenetics and provides an
important validation for the concept of an
epigenetic cancer therapy
Deacetilasi istoniche: una nuova classe di farmaci
nella terapia antitumorale
Generalmente agiscono causando apoptosi di
cellule tumorali,
ma non di cellule non tumorali; le HDAC sono
generalmente associate a fattori di trascrizione
oncogeni.
The effects of HDAC inhibitors on gene expression are highly selective,
leading to transcriptional activation of certain genes such as the cyclindependent kinase inhibitor p21WAF1/CIP1 but repression of others.
HDAC inhibition not only results in acetylation of histones but also
transcription factors such as p53, GATA-1 and estrogen receptor-alpha.
The functional significance of acetylation of non-histone proteins and the
precise mechanisms whereby HDAC inhibitors induce tumor cell growth
arrest, differentiation and/or apoptosis are currently the focus of
intensive research.
Several HDAC inhibitors have shown impressive antitumor activity in vivo
with remarkably little toxicity in preclinical studies and are currently in
phase I clinical trial.
Classificazione delle deacetilasi istoniche
HDACs are classified in four groups based on their homology to
yeast histone deacetylases:
Class I which includes HDAC1, -2, -3 and -8 are related to yeast
RPD3 gene;
Class II which includes HDAC4, -5, -6, -7, -9 and -10 are related
to yeast Hda1 gene;
Class III, also known as the sirtuins are related to the Sir2 gene
and include SIRT1-7, and
Class IV which contains only HDAC11 has features of both Class I
and II.
Classificazione degli inibitori delle istone deacetilasi
The “classical” HDIs act exclusively on Class I and Class II HDACs by
binding to the zinc containing catalytic domain of the HDACs. These
classical HDIs fall into several groupings, in order of decreasing
potency:
(i) hyroxamic acids, such as Tricostatine A,
(ii) cyclic tetrapeptides (such as trapoxin B), and the depsipeptides,
(iii) benzamides,
(iv) electrophilic ketones, and
(v) the alophatic acid compounds such as phenylbutyrate and valproic
acid.
"Second generation" HDIs include SAHA/Vorinostat,
Belinostat/PXD101, MS275, LAQ824/LBH589, CI994, and
MGCD0103.
The sirtuin Class III HDACs are NAD+ dependent and are therefore
inhibited by nicotinamides, as well derivatives of NAD,
dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.[4]
O
N
H
N
N-hydroxy-N'-phenyl-octanediamide
O
H
OH
VORINOSTAT (Zolinza) è il primo inibitore delle acetilasi
istoniche approvato per il trattamento di neoplasie
Nel 2006 il VORINOSTAT è stato approvato per il
trattamento del linfoma a cellule T cutaneo
Esistono studi preclinici che indicano una attività
antinfiammatoria del vorinostat
Nel 2007 ricerche presso la Mayo Clinics hanno dimostrato
che il Vorinostat è efficace nel glioblastoma ricorrente
HDAC inhibitors can activate both the deathreceptor and intrinsic apoptotic pathways
Histone deacetylase inhibitors as therapeutics for polyglutamine
disorders Rachel Butler and Gillian P. Bates
Nature Reviews Neuroscience 7, 784-796 (October 2006)
P53
P53 puo’ agire a diversi livelli:
Puo’ attivare proteine per il riparo
del DNA
Puo’ regolar e la duplicazione
cellulare a livello G1/S
Puo’ iniziare processi apoptotici
MODULAZIONE DELLA TRASCRIZIONE DA PARTE DI RECETTORI NUCLEARI
Il ruolo degli INSULATORS nella regolazione della
espressione genica
Es. i geni globinici e LCR HS4,
scs di Drosophila che isolano hsp70)
In genere sono sequenze ricche di isole CpG non metilate
Ci sono proteine che legano gli insulators
Transcription factors are associated with the nuclear matrix. It
has been proposed that the nuclear matrix recruits
transcription factors, facilitating their interaction with
regulatory DNA elements.
ESPRESSIONE GENICA IN EUCARIOTE: SEQUENZA DI EVENTI
3’ TRASCRIZIONE
RNA, trascritto primario
Capping e poliadenilazione
MODIFICAZ.
TP
Splicing o maturazione
Fuoriuscita dal nucleo
riconoscimento da parte dei ribosomi
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