Human Ras homolog gene family, member B

Target ID:

RHOBA

Aliases:

ARH6ARHBMST081MSTP081RHOH6

Deposition Date:

Monday 30th January 2006

Families:

GTPases

Related Diseases:

SignallingCancer

Structure type:

apo

Download Coordinates:

2FV8

Authors:

A.P.TURNBULL,M.SOUNDARARAJAN,C.SMEE,C.JOHANSSON,G.SCHOCH,F.GORREC,J.BRAY,E.PAPAGRIGORIOU,F.VON DELFT,J.WEIGELT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,D.DOYLE,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2FV8

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, amplifies 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.

The family of Rho GTPases are involved in cell motility and proliferation through the reorganisation of actin and vesicle trafficking. RHOB is localised mainly to early endosomes (Adamson et al, 1992) and, in contrast to its closely related family member RHOA, functions as a tumor suppressor (Adnane et al, 2002; Chen et al, 2000; Fritz & Kaina, 2001; Liu et al, 2001).

Post-translational modification (PTM) of the small GTPase family members is an important process that defines the cellular location and function of the enzymes. Targeting of the membrane localisation PTM via knockout of the lipid addition with particularly emphasis on the first step involving farnesyltransferase appears to be promising. Originally it was believed that the Ras family members of small GTPases were the targets however RHOB maybe the true target (Chen et al, 2000; Cox & Der, 2002; Du & Prendergast, 1999; Mazieres et al, 2005). Phase II and III clinical trials have shown activity of FTIs against selective malignancies (Sebti & Adjei, 2004).

Materials and Methods

RHOB

PDB:2FV8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RHOBA-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag followed by the TEV proteiease cleavable sequence
Host:BL-21(DE3)R3

Construct

Prelude:
Sequence:
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:2.5 µL of construct DNA was added to a well of a PCR plate on ice. 90µL of competent BL21(DE3)-R3/Rosetta bacteria were added, with the plate remaining on ice. The plate was left on ice for a further 25 minutes. The plate was transferred to a 42°C water bath for 45 seconds and then returned to sit on ice for 2 minutes. 100µL of SOC medium (pre-warmed to 42°C) was added and the plate incubated at 37°C for 60 minutes.180µL of the culture was plated out onto LB-agar containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol in a 5.5cm Petri dish. The plate were incubated at 37°C overnight.30mL of LB containing 50µg/mL Kanamycin and 34 µg/mL chloramphenicol was prepared in a 250 mL flask. Two colonies, from the transformation plate, were added to each starter culture. The flask was left in a shaker at 200 rpm and 37°C overnight. 19 mL of the starter culture was added to autoclaved 1litre TB medium that contained 50 µg/mL kanamycin 34 µg/mL chloramphenicol. The flasks were placed in a shaker at 180 rpm and 37°C. When the cell density reached an OD600 nm of approximately 1.0 the temperature was reduced to 20°C for 1 hr before induction with the addition of 1 mM IPTG. The cells were spun down (6500 rpm, 15 mins). The pellets were resuspended in 30 mL lysis/ binding buffer, transfered into 50 mL tubes and placed in a -80°C freezer.
Lysis buffer: 50 mM potassium phosphate pH 7.4; 10 mM Imidazole; 500 mM NaCl; 0.5 mM TCEP

Purification

Procedure
Column 1: Ni-affinity, HisTrap, 1 mL (GE/Amersham)
Buffers: Affinity Wash Buffer: 50 mM potassium phosphate pH 7.4, 30 mM Imidazole, 500 mM NaCl, 0.5 mM TCEP
Affinity Elution Buffer: 50 mM potassium phosphate pH 7.4, 250 mM Imidazole, 500 mM NaCl, 0.5 mM 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: 50 mM HEPES pH 8.0, 150 mM NaCl, 2 mM MgCl 2, 0.5 mM TCEP
Procedure: The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.0 mL/min. Eluted proteins were collected in 1 mL fractions.Using a Centricon 10 K cutoff concentrator the buffer was exchange to 10 mM HEPES pH 7.4, 500 mM NaCl, 2 mM MgCl 2 and 5 mM DTT. The volume was then brought to a total of 2 mL before adding 5 mM GDP and concetrating to concentrated to 17.9 mg/mL. Concentration was determined from the absorbance at 280 nm.

Extraction

Procedure
After thawing, the resuspended cells were lysed by passing through the Emusiflex C5 high pressure homogeniser. PEI (stock 5 %) was added to the homogenate to a final concentration of 0.15%. The cell debris, nuclei and DNA were spun down at 16,500 rpm for 45 min. The supernatant was collected .
Concentration:
Ligand
MassSpec:
Crystallization:Column 1: Ni-affinity, HisTrap, 1 mL (GE/Amersham)
Buffers: Affinity Wash Buffer: 50 mM potassium phosphate pH 7.4, 30 mM Imidazole, 500 mM NaCl, 0.5 mM TCEP
Affinity Elution Buffer: 50 mM potassium phosphate pH 7.4, 250 mM Imidazole, 500 mM NaCl, 0.5 mM 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.

References

Adamson P, Paterson HF, Hall A. (1992). Intracellular localization of the P21rho proteins. J. Cell. Biol. 119: 617-627.

Adnane J, Muro-Cacho C, Mathews L, Sebti SM, Munoz-Antonia T. (2002). Suppression of rho B expression in invasive carcinoma from head and neck cancer patients. Clin. Cancer Res. 8: 2225-2232.

Chen Z, Sun J, Pradines A, Favre G, Adnane J, Sebti SM. (2000). Both farnesylated and geranylgeranylated RhoB inhibit malignant transformation and suppress human tumor growth in nude mice. J. Biol. Chem. 275: 17974-17978.

Cox AD, Der CJ. (2002). Farnesyltransferase inhibitors: promises and realities. Curr. Opin. Pharmacol. 2: 388-393.

Du W, Prendergast GC. (1999). Geranylgeranylated RhoB mediates suppression of human tumor cell growth by farnesyltransferase inhibitors. Cancer Res. 59: 5492-5496.

Fritz G, Kaina B. (2001). Ras-related GTPase Rhob represses NF-kappaB signaling. J. Biol. Chem. 276: 3115-3122.

Liu AX, Rane N, Liu JP, Prendergast GC. (2001). RhoB is dispensable for mouse development, but it modifies susceptibility to tumor formation as well as cell adhesion and growth factor signaling in transformed cells. Mol. Cell. Biol. 21: 6906-6912.

Mazieres J, Tillement V, Allal C, Clanet C, Bobin L, Chen Z, Sebti SM, Favre G, Pradines A. (2005). Geranylgeranylated, but not farnesylated, RhoB suppresses Ras transformation of NIH-3T3 cells. Exp. Cell. Res. 304: 354-364.

Sebti SM, Adjei AA. (2004). Farnesyltransferase inhibitors. Semin. Oncol. 31(1 Suppl 1):28-39.