Transcript Preparation of enzymatically active recombinant class III

Preparation of enzymatically
active recombinant
class III protein deacetylases
Brian J.North, Bjoern Schwer, Nidhi Ahuja,
Brett Marshall, Eric Verdin
By:
Importance of Review
• Reversible acetylation of histone and
nonhistone proteins is emerging as a major
mechanism for regulating protein function.
• Acetylation is a pervasive modification
involved in most biological functions.
• Class III Histone Deacetylases (HDACs) have
generally proven easier to manipulate than
class I and II HDACs, hence are being
employed in the present review.
Introduction
• The review focusses on protocols for
the preparation and purification of
enzymatically active class III protein
deacetylases (human sirtuin 1, 2, and
3) and their activities on histone and
nonhistone substrates.
Classes of Histone Deacetylases
(HDACs)
• There are three classes of HDACs which
include eighteen distinct human protein
deacetylases which are grouped on the
basis of their primary homology to three
protein deacetylases in Saccharomyces
cerevisiae.
• Class I histone deacetylases
• Class II histone deacetylases
• Class III histone deacetylases
Class I Histone Deacetylases
• These are homologous to yRPD3,
sharing a compact structure.
• Includes HDAC1, -2, -3, -8, and –11.
• They are predominantly nuclear
proteins expressed in most tissues and
cell lines (1).
Class II Histone Deacetylases
• Class II HDACs are homologous to yHDA1.
• They are subdivided in two subclasses based
on sequence homology and domain
organization .
IIa (HDAC4, -5, -7, and -9 and its splice
variant MITR).
IIb (HDAC6 and HDAC10) based on
sequence homology and domain.
Class III Histone Deacetylases
• Class III HDACs are homologous to
ySIR2.
• They show no homology to class I
and II proteins [2].
• The class III enzymes are
characterized by their dependence
on nicotinamide adenine
dinucleotide (NAD+).
Purification of enzymatically
active class III HDACs
Class III HDACs with high enzymatic
activity
can be purified by two ways:
A. Immunoprecipitation after transient
or
stable transfection in
mammalian cells.
B. Expression of recombinant proteins in
Escherichia coli in an enzymatically
active form.
Mammalian cell culture
systems
• This method allows the purification of
enzymatically active sirtuins directly from
mammalian cells.
• It is safely assumed that proteins purified
from
mammalian cells more faithfully represent the
sirtuins in their native environment because
of the presence of associated cofactors and
proper post-translational modifications which
could contribute to enhanced activities of
sirtuins from mammalian cells.
Plasmid
• Each of the class III human HDACs has
been cloned into the pcDNA3.1 vector
as a C-terminal fusion protein with the
FLAG epitope.
• For purification, 293T cells are
transiently
transfected with the expression vector
by the calcium phosphate DNA
precipitation method.
• Using this protocol, significant HDAC
activity is detected with SIRT1, 2, 3, and
5, while the other SIRT proteins, SIRT4,
6, and 7 did not show activity.
• SIRT2 is the sole sirtuin to show tubulin
deacetylation activity.
The experiments suggest that the
SIRT
proteins 4, 6 and 7 with no
activity,might target other substrates
for deacetylation
or that other cofactors, proteins, or
small molecules are needed for their
enzymatic activity.
These enzymes may also function as
mono-ADP ribosyltransferases, as
recently demonstrated for another
sirtuin.
Expression in E. coli
• Class III HDACs can be readily purified
as enzymatically active protein after
overexpression in E. coli, generating a
large amount of enzymatic deacetylase
activity.
• It has some limitations including the
possible absence of regulatory
cofactors and post-translation
modifications.
Procedure
There are certain methods of overexpression
of HDACs in E. coil expression system.
GST-SIRT1
6X His-SIRT2
GST-tagged SIRT2
6X His-SIRT3
GST-SIRT1
• A DNA fragment encompassing the full SIRT1
open-reading frame was amplified by PCR
from a plasmid with primers containing
BamHI (forward
primer) and SalI (reverse primer) restriction
sites. The digested DNA fragment was
inserted into pGEX4T-1 cleaved with BamHI
and SalI by ligation and confirmed by
sequencing.
6X His-SIRT2
• Full-length human SIRT2 cDNA was cloned
into pHEX, a modified version of pGEX-2T
(Pharmacia) in which the GST-encoding
sequence was replaced with a hexahistidineencoding sequence (6X His).
• This vector was transformed in DH5α F’IQ
bacteria (Gibco) for expression.
• This protocol yields a recombinant SIRT2
protein with high enzymatic activity and
>90% purity as determined by SDS–PAGE
GST-tagged SIRT2
• SIRT2 has also been expressed using a GST
fusion protein (N-terminal) using the same
vector (pGEX-4T3, Amersham) as for SIRT1.
• The construct was transformed in E. coli
BL21 (DE3) cells.
• High-level expression of SIRT2 has been
achieved. The GST-SIRT2 fusion protein
• purified by this procedure is >90% pure as
determined by SDS–PAGE
6X His-SIRT3
• SIRT3 is proteolytically processed after its
import into the mitochondrial matrix [4,5] and
the proteolytic processing of SIRT3 occurs
between amino acids Ser101 and Ile102
• Based on the above results and the finding
that in vitro-synthesized SIRT3 can be
activated by proteolytic processing, an
expression vector was constructed that
expresses a truncated SIRT3 protein.
• The region comprising the coding
sequence for SIRT3 amino acids 101–
399 was cloned into the bacterial
expression vector pTrcHis (Invitrogen)
to yield an N-terminal 6X His-tagged
SIRT3, pTrcHis- SIRT3 101–399 and
transformed into DH5α cells.
• This protocol yields an enzymatically
active deacetylase with similar NAD2+
dependency and pH sensitivity as
recombinant SIRT2 and is inhibited by
nicotinamide as class III HDACs.
Measurement of HDAC activity
associated with SIRT1, 2, and
3 expressed in E. coli
• The enzymatic activity
of recombinant
GSTSIRT1,
6X His-SIRT2 and 6X
His-SIRT3 on a 3Hacetylated histone H4
peptide was measured
in the presence 1mM
NAD+ at different
concentrations of
recombinant protein (rU
D relative units).
• The enzymatic activity
of recombinant 6X HisSIRT2 and 6X HisSIRT3 on a 3Hacetylated histone H4
peptide was measured
in the presence of
increasing
concentrations of NAD+
(0, 1, 10, 100, 1000
_M).
• The activity of 6X
His-SIRT2 and 6X
His-SIRT3 on a 3Hacetylated histone
H4 peptide was
measured in a range
of pH [4–11] in the
presence of 1mM
NAD+.
In vitro translated protein
• As discussed above, SIRT3 after import into
the mitochondrial matrix, is proteolytically
processed and becomes activated as an
NAD2+-dependent protein deacetylase.
• The activation process of SIRT3 can be
reconstituted in vitro by incubation of in vitro
synthesized full-length SIRT3 with
recombinant yeast mitochondrial processing
peptidase (MPP) [3,5].
• For in vitro synthesis, TNT Coupled
Reticulocyte Lysate System (Promega) is
used.
Enzymatic substrates and
reaction
• Methods used for the assay:
• Histone substrate procedure
Purified histone acetyltransferase is used to
acetylate isolated nucleosomes [6] in vitro with high
specific activity. This method offers the significant
advantage of a physiologically relevant substrate
(chromatin) and should prove useful for examining
the activity of HDACs on their natural substrates
• Nonhistone protein enzymatic assays
for class III HDACs
» Pulse labelling of acetylated proteins
» Western blotting with antibodies for
acetylated state
References
[1] W. Fischle, V. Kiermer, F. Dequiedt, E. Verdin,
Biochem. Cell Biol. 79 (2001) 337–348.
[2] R.A. Frye, Biochem. Biophys. Res. Commun.
260 (1999) 273–279.
[3] B. Schwer, B.J. North, R.A. Frye, M. Ott, E.
Verdin, J. Cell Biol.158 (2002) 647–657
[4] P. Onyango, I. Celic, J.M. McCaVery, J.D. Boeke,
A.P. Feinberg, Proc. Natl. Acad. Sci. USA 99
(2002) 13653–13658.
[5] P. Luciano, S. GeoVroy, A. Brandt, J.F.
Hernandez, V. Geli, J. Mol. Biol. 272 (1997) 213–
225.
[6] P.A. Wade, P.L. Jones, D. Vermaak, A.P. WolVe,
Methods Enzymol. 304 (1999) 715–725.
Thanks