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

activitycan 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 frommammalian 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 transientlytransfected 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 SIRTproteins 4, 6 and 7 with no activity,might target other substrates for deacetylationor 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 SIRT26X 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 (forwardprimer) 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 hexahistidine-encoding 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, and3 expressed in E. coli

• The enzymatic activity of recombinant GSTSIRT1,6X His-SIRT2 and 6X His-SIRT3 on a 3H-acetylated 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 His-SIRT2 and 6X His-SIRT3 on a 3H-acetylated 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 3H-acetylated 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

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Thanks