PPAD
PPAD (Protein Production and Assay Development in the ubiquitylation system)

The PPAD team generates for all MRC-PPU researchers and their collaborators recombinant proteins of the Ubiquitin, SUMO, NEDD8, ISG15, autophagy and FAT10 systems. The also have a remit in helping setup robust quantitative assays to enable activity of ubiquitin enzymes to be rigorously assessed. These reagents are being deployed to discover and validate functions of ubiquitin components in various signalling pathways, where they play pivotal roles in such diverse biological processes as transcriptional regulation, DNA-repair, signalling in the innate immune response, mitochondrial homeostasis, cell cycle regulation, metabolism and ion-channel regulation in the kidney. The thorough understanding of these processes may provide keys for the development of cures to diseases, such as cancer, inflammatory diseases, Parkinsons, Alzheimers, ALS and hypertension. A major remit of this service is to produce proteins that will be used in our DSTT collaborations with Pharmaceutical collaborators and help accelerate drug discovery in the ubiquitin system.

The PPAD team has so far generated:

imageProtein Modifiers:
Ubiquitin, SUMO1, SUMO2, NEDD8, ISG1, LC3 isoforms, GABARAP, FAT10

E1 activating enzymes
UBE1, SAE1/SAE2, NAE1/NAE2, UBA7, ATG7, UBA6

E2 conjugating enzymes
35 different E2s with various tags

E3 ligases
36 E3 ligases, including HECT, RING, RBR-type and bacterial ligases. We have, for example, produced a range of active full length Cullin/RING complexes and several of the exciting new drug targets that were discovered in the last couple of years. We are also heavily involved with the production of RBR-type ligases and many of their mutants.

DUBs
Our ultimative aim is to produce all of the DUBs from all the systems. So far we have expressed and characterised over 45 DUBs, representing members of all the DUB families. We have developed a good understanding of their activities and specificities in vitro.

Ub-receptors
A broad portfolio of Ub-receptors to be used as tools for the analysis and isolation of ubiquitylated proteins in cells.

Substrates
A large number of substrates for DUBs and E3-ligases including poly-Ubiquitins for testing DUBs and a range of proteins that can be readily ubiquitylated in vitro.

 

The PPAD Team:

image

from the left:
Clare Johnson
       Expert in protein expression and purification from insect and bacterial systems.

Dr Richard A. Ewan
       Expert for Deubiquitylating enzymes (DUBs) and assay development, now working with Matthias Trost

Daniel Fountaine
       Highly experienced in bacterial recombinant protein expression and purification, now working in DSTT

Dr Axel Knebel (team leader)
Expert in the purification of tagged and untagged proteins. Highly experienced in kinase and ubiquitin signalling. Inventor of the KESTREL method to identify protein kinase substrates and the Dac-tag for high performance recombinant protein purification.

 

Contact
For all enquires on proteins and assays developed by the PPAD team please contact Axel Knebel who directs this team (a.knebel@dundee.ac.uk  or Tel: +44 (0)1382 384994)

 

References:

Madrzak J., Fiedler M., Johnson C.M., Ewan R., Knebel A., Bienz M., Chin J.W. (2015) Ubiquitination of the Dishevelled DIX domain blocks its head-to-tail polymerization.Nat Commun. 6:6718

Kristariyanto Y.A., Abdul Rehman S.A., Campbell D.G., Morrice N.A., Johnson C., Toth R., Kulathu Y. (2015) K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of k29 polyubiquitin. Mol. Cell. 58:83-94.

Stanley M., Han C., Knebel A., Murphy P., Shpiro N., Virdee S. (2015) Orthogonal Thiol Functionalization at a Single Atomic Center for Profiling Transthiolation Activity of E1 Activating Enzymes.  ACS Chem Biol. (in press).

Bulatov E., Martin E.M., Chatterjee S., Knebel A., Shimamura S., Konijnenberg A., Johnson C., Zinn N., Grandi P., Sobott F., Ciulli A. (2015)  Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2). J. Biol. Chem. 290:4178-41 91.

Bett J.S., Ritorto M.S., Ewan R., Jaffray E.G., Virdee S., Chin J.W., Knebel A., Kurz T., Trost M., Tatham M.H., Hay R.T. (2015)  Ubiquitin C-terminal hydrolases cleave isopeptide- and peptide-linked ubiquitin from structured proteins but do not edit ubiquitin homopolymers. Biochem J. 466:489-498.  

Ritorto M.S, Ewan R., Perez-Oliva A.B., Knebel A., Buhrlage S.J., Wightman M., Kelly S.M., Wood N.T., Virdee S., Gray N.S., Morrice N.A., Alessi D.R., Trost M. (2014)  Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nat Commun. 5:4763.

Baron Y., Pedrioli P.G., Tyagi K., Johnson C., Wood N.T., Fountaine D., Wightman M., Alexandru G. (2014)  VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase. BMC Biol. 12:39.  

Kazlauskaite A., Kondapalli C., Gourlay R., Campbell D.G., Ritorto M.S., Hofmann K., Alessi D.R., Knebel A., Trost M., Muqit M.M. (2014) Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem J. 460:127-39.  

Kelsall I.R., Duda D.M., Olszewski J.L., Hofmann K., Knebel A., Langevin F., Wood N., Wightman M., Schulman B.A., Alpi A.F. (2013)  TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes.  EMBO J. 32:2848-2860.

Zemla A., Thomas Y., Kedziora S., Knebel A., Wood N.T., Rabut G., Kurz T. (2013)  CSN- and CAND1-dependent remodelling of the budding yeast SCF complex. Nat Commun. 4:1641

Strickson S., Campbell D.G., Emmerich C.H., Knebel A., Plater L., Ritorto M.S., Shpiro N., Cohen P. (2013)  The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system. Biochem J. 2013 451:427-437.

Ohta A., Schumacher F.R., Mehellou Y., Johnson C., Knebel A., MacCartney T.J., Wood N.T., Alessi D.R., Kurz T. (2013) The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms; disease-causing mutations in KLHL3 and WNK4 disrupt interaction. Biochem. J 451:111-122

Lee D.W., Peggie M., Deak M., Toth R., Gage Z.O., Wood N., Schilde C., Kurz T., Knebel A. (2012) The Dac-tag, an affinity tag based on penicillin binding protein 5. Analytical Biochemistry 428:64-72 .

Kondapalli C., Kazlauskaite A., Zhang N., Woodroof H., Campbell D., Gourlay R., Burchell L., Walden H., Macartney T.J., Deak M., Knebel A., Alessi D.R., Muqit M.M.K. (2012) PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biology May 5: 120080 .

Bandau S., Knebel A., Gage Z.O., Wood N.T., Alexandru G. (2012) UBXN7 docks on neddylated cullin complexes using its UIM motif and causes HIF1a accumulation BMC Biol. 26:10-36.

Rabut G., Le Dez G., Verma R., Makhnevych T., Knebel A., Kurz T., Boone C., Deshaies R.J. Peter M. (2011) The TFIIH subunit Tfb3 regulates cullin neddylation. Mol. Cell 43:488-495.