Human Ras-like estrogen regulated growth inhibitor

Target ID:

RERGA

Aliases:

MGC15754

Deposition Date:

Friday 26th August 2005

Families:

GTPases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2ATV

Authors:

A.P.TURNBULL,E.SALAH,G.SCHOCH,J.ELKINS,N.BURGESS,O.GILEADI,F.VON DELFT,J.WEIGEL,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,D.DOYLE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2ATV

tabs

Structure Details

Cells respond in many ways to environmental changes including altering gene expression,
physically moving locations, changes to cell-cell interactions, differentiation and even altering their life spans. This is achieved through a signalling network that consists of several signalling pathways.
The network receives multiple signals, interprets the signals, amplified and then transmits the signals.
One of the best characterised signalling pathways involves the ras GTPase family.
The importance of this family of small GTPases to human health was first
recognised through the identification of the human H-Ras,
N-Ras and K-Ras genes as being oncogenic.

Generally, the small GTPase family of proteins exist in two forms:
an inactive GDP bound form and an active GTP bound state.
An increased conversion of the active to inactive state occurs through the interaction
of GTPase activating proteins (GAPs) while guanine nucleotide exchange factors (GEFs)
enhances the exchange of GDP for GTP.
The mutant forms of the H-, N- and K-Ras GTPases knocks out the hydrolytic activity of the enzymes
hence the signalling pathway is always on as the GTPase is in the GTP bound active state
(Bos et al, 1985; Capon et al, 1983; Feinberg et al, 1983; Sekiya et al, 1984)
ultimately leading to uncontrolled cell proliferation.
Another feature that is common to most small GTPase is the localisation of the proteins to the membrane. This interaction with the membrane occurs mainly at a C-terminal site on the protein via lipid modification (Takai et al, 2001).

Not all small GTPases are onocogenic.
A study to identify high level expressing proteins that correlate with a positive outcome for patients with breast tumors identified one as a small GTPase (Perou et al, 2000).
This enzyme was later characterised as a Ras-related, Estrogen-Regulated GTPase (RERG)
and shown to slow the rate of tumor cell proliferation (Finlin et al, 2001).
The lack of a lipid modification motif on the C-terminus of RERG corresponds with its mainly cytosolic cellular distribution. Finlin et al. (2001) also showed that the signal pathway was not related to the Ras signalling pathway.

As RERG is a small GTPase that functions as a breast tumor suppressor protein, it along with the proteins involved in its unresolved signalling pathway, are potentially significant pharmaceutical targets against this disease.

Materials and Methods

RERG

PDB:2ATV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RERGA-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag
Host:BL-21(DE3)R3

Construct

Prelude:
Sequence:
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformed 50 µl competent BL-21 (DE3) phage resistant cells with 10 µl of the plasmid DNA and plated out onto LB plate plus 50 mg/ml kanamycin. The next day colonies were picked out into fresh deep well blocks containing 1 ml TB + 50 mg/ml kanamycin. These were grown overnight and glycerol stocks prepared by adding 333 ml of 60 % glycerol to 1 ml of cell suspension, mixing and then storing in a -80°C. freezer. The glycerol stock was used to innoculate 10 mls of TB + 50 mg/ml kanamycin which was grown overnight at 37°C as a starter culture for a 1 litre growth. The large scale growth was grown at 37°C until approximately 30 mins before induction when the temperature was lowered to 25°C. Protein production was induced with the addition of 1mM IPTG. The next day cells were harvested by centrifugation at 4000 rpm for 15 minutes. The pellet was then stored in the -80°C freezer.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham)

Buffers: Affinity Binding Buffer: 10mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4, 0.5mM TCEP. Affinity Wash Buffer: 50mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4, 0.5mM TCEP. Affinity Elution Buffer: 250mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4, 0.5mM TCEP.

Procedure: The cell extract was loaded on the column at 0.8 ml/min on an AKTA-express system (GE/Amersham). The column was then washed with 10 column volumes of Affinity Binding Buffer, 10 column volumes of Affinity Wash Buffer, and then eluted with Affinity Elution Buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2 : Gel filtration, Hiload 16/60, S75 16/60 - 120 ml

Buffers : GF buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP.

Extraction

Procedure
Lysis buffer: 10mM Imidazole, 300mM NaCl, 50mM pH8.0 NaH2PO4, 0.5mM TCEP, 1x complete PI EDTA free tablet/50mls. The pellet (14.78 gms) was resuspended with 3x volume of lysis buffer (approximately 130 mls final) by intermitently placing the pellet in a 37°C water bath and vortexing. Once resuspended the cells were (1) broken by one passage through the Constant Systems cell breaker; (2) sonicating; (3) DNA precipitation with the addition of PEI to a final oncentration of 0.15 % for 30 mins on ice followed by a 17,000 rpm at 4°C to remove precipitation; (4) the supernatant was filtered through a GF/0.2 µM serum acrodiscs.
Concentration:
Ligand
MassSpec:
Crystallization:Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham)

Column 2 : Gel filtration, Hiload 16/60, S75 16/60 - 120 ml

Buffers : GF buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Bos JL, Toksoz D, Marshall CJ, Verlaan-de Vries M, Veeneman GH, van der Eb AJ, van Boom JH, Janssen JW, Steenvoorden AC. (1985). Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia. Nature 315: 726-730.

Capon DJ, Chen EY, Levinson AD, Seeburg PH, Goeddel DV. (1983). Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature. 302: 33-37.

Feinberg AP, Vogelstein B, Droller MJ, Baylin SB, Nelkin BD. (1983). Mutation affecting the 12th amino acid of the c-Ha-ras oncogene product occurs infrequently in human cancer. Science. 220: 1175-1177.