IPAH9.8 [GST-tagged]


Catalogue Number
63-0018-025
Product Size
25 µg
Price £
£130
Accession Number
AAP79029.1
Residues Expressed
1-545
Certificate of Analysis Size
25 µg
Species
Shigella flexneri
Source
E. coli expression
Quantity
25 µg
Storage
-70°C
Concentration
0.5 mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
~88.8 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: AAP79029.1. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

E3 ligase assay: The ubiquitin conjugating activity of GST-IpaH9.8 was validated through its ability to catalyse the generation of polyubiquitin chains in the presence of the E1 activating enzyme His-UBE1, the E2 conjugating enzyme His-UBE2D2 (UbcH5b) (several E2s were tested, data generated with this E2 is provided by way of example) and ubiquitin. Incubation of GST-IpaH9.8 for 30 minutes at 30°C in the presence of ubiquitin, His-UBE1, His-UBE2D2 and ATP (Lane 1) was compared alongside two control reactions with either ATP (Lane 2) or GST-IpaH9.8 (Lane 3) excluded from the reaction. Ubiquitin conjugates were identified by Western blotting using an anti-ubiquitin conjugate antibody and these were observed only in the presence of both ATP and GST-IpaH9.8.


Background

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including the regulated and targeted proteasome-dependent degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). IpaH9.8 is a bacterial E3 ligase and cloning of the gene from Shigella flexineri was first described by Demers et al. (1998). The IpaH family of proteins contain highly conserved leucine-rich repeats (LRR) in the amino-terminal region while the conserved carboxy-terminal regions possess E3 ligase activity (Rohde et al., 2007). The crystal structure of the IpaH protein has recently been described and based on this the IpaH family has been identified as a new class of E3 ubiquitin ligases found in pathogenic and symbiotic bacteria (Singer et al., 2008; Zhu, et al., 2008). IpaH9.8 has been shown to regulate the pheromone response by ubiquitylation and degradation of the MAPK kinase Ste7 (Rohde et al., 2007). IpaH9.8 has also been shown to inhibit NF-κB-dependent gene expression and the activity of U2AF35, an alternative splicing factor involved in host inflammatory responses (Haraga and Miller 2003).


References

Demers B, Sansonetti PJ, Parsot C (1998) Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins. EMBO J 17, 2894-903.

Haraga A, Miller SI (2003) A Salmonella enterica serovar typhimurium translocated leucine-rich repeat effector protein inhibits NF-kappa B-dependent gene expression. Infect Immun 71, 4052-8.

Rohde JR, Breitkreutz A, Chenal A, Sansonetti PJ, Parsot C (2007) Type III secretion effectors of the IpaH family are E3 ubiquitin ligases. Cell Host Microbe 1, 77-83.

Singer AU, Rohde JR, et al. (2008) Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases. Nat Struct Mol Biol 15, 1293-301.

Zhu Y, Li H, Hu L, Wang J, Zhou Y, Pang Z, Liu L, Shao F (2008) Structure of a Shigella effector reveals a new class of ubiquitin ligases. Nat Struct Mol Biol 15, 1302-8.