High affinity ligands for NOTCH proteins

 

Lab rotation project description

The Notch proteins that span the cytoplasmic membrane are an evolutionarily conserved family of four different proteins at the head of cell-to-cell signalling cascades. These proteins have been shown to have an essential role in regulating development and homeostasis in many tissues.

They are involved in lineage commitment, cell cycle progression, differentiation and the maintenance and self-renewal of stem cells. Mutations in Notch pathway genes have been linked with many congenital disorders including Alagille, Hajdu-Cheney and Adam-Oliver syndromes as well as cerebral autosomal-dominant arteriopathy (CADASIL). Mutations have also been identified in a number of cancers (squamous cell carcinoma, small-cell carcinoma, B cell lymphoma).

A number of viral proteins from human herpes viruses, papillomavirus and Epstein-Barr viruses also target proteins in the Notch cascade. In this project, affibodies3 (small 58-residue, 6.5 kDa proteins derived from the Z domain triple helix of staphylococcal protein A) with a high affinity against the extracellular portion of a Notch protein will be selected from a phage display library. Affibodies are attractive alternatives to antibodies or their fragments as biological molecular recognition elements as they are considerably smaller than immunoglobulin G (150 kDa; 4 peptide chains linked by disulfides). Additionally, they do not contain cysteines, disulfide bonds or post-translational modifications allowing them to be expressed in a wide variety of hosts including E. coli. This makes them easy to engineer (randomization of 13 contiguous residues in the domain is well tolerated) and produce in large quantities. Affibodies with nM to pM affinities have been identified for EGFR, HER2 and interleukin receptors. The affibodies can also be easily modified with fluorophores or other imaging probes for use in vivo.

The project will involve the isolation of the extracellular portion of a Notch protein and its immobilization on magnetic beads. A phage display library of the affibody scaffold (M13 or T7 phage) will then be produced and panned against the Notch protein to select high affinity binders. After a number of rounds of panning, the highest affinity affibodies will be sequenced and re-expressed in soluble form for follow-on biological studies.

Fact file

Research theme

MCO

Location

TBC

Rotation

TBC

Contact

2nd supervisor

Kevin Gaston

BBSRC Doctoral Training Partnerships
 

Linked PhD Project Outline

Cholangiocarcinoma is cancer of the ducts that carry bile from the liver to the gall bladder and the small intestine.  The incidence of cholangiocarcinoma in the UK is increasing year by year as is the number of deaths caused by this disease.  Non-resectable disease is common and this has an average survival of around two months post-diagnosis.   Although serum alkaline phosphatase and CA 19-9 and CA-125 may be elevated at diagnosis, these markers are not specific to this disease.  Therefore, there is a pressing unmet clinical need for new treatment approaches and earlier diagnosis.  Recent work has shown that NOTCH proteins play an important role in Cholangiocarcinoma.  Cleavage of NOTCH proteins at the cell surface by g-secretase results in the production of a NOTCH Intracellular Domain (ICD) that regulates transcription and promotes tumour growth in mouse models. 

In other types of cancer the NOTCH ICD is known to interact with the hypoxia inducible transcription factor HIF1a resulting in increased ICD activity.  The aim of this project is to develop novel reagents that target NOTCH proteins and to use these reagents and existing drugs that inhibit NOTCH cleavage and HIF1a activity to probe the relationship between NOTCH signalling and hypoxia in cholangiocarcinoma cells.  Our overarching hypothesis is that the simultaneous inhibition of NOTCH cleavage and HIF activity will have synergistic inhibitory effects of the proliferation of cholangiocarcinoma cells and their ability to form tumours.  This could result in novel combinatorial approaches to the treatment of this disease.

To achieve these aims we will examine the expression of NOTCH and NOTCH ICD proteins in cholangiocarcinoma cells and use this information to produce affibodies that bind to the extracellular domain of the most relevant NOTCH proteins. Affibodies are attractive alternatives to antibodies or their fragments as they are considerably smaller than immunoglobulin G and they lack cysteines, disulfide bonds or post-translational modifications allowing them to be expressed in a wide variety of hosts and produce in large quantities. We will also examine the effects of these affibodies on NOTCH cleavage and on the proliferation of cholangiocarcinoma cells and other properties of these cells including cell migration and invasion of extracellular matrix. In addition, we will determine the genes that are altered in response to the inhibition of NOTCH cleavage using g-secretase inhibitors, the inhibition of HIF activity using small molecules, and the combination of these inhibitors genome-wide using RNA sequencing. We will also examine whether affibodies or g-secretase inhibitors act synergistically with inhibitors of HIF to block the proliferation of cholangiocarcinoma cells.

Together these experiments will further our understanding of the molecular mechanism that underpin cholangiocarcinoma and explore new approaches to the treatment of this disease.  In doing so they will provide a comprehensive training in a range of current techniques in molecular and cellular biology delivered by three research groups with complementary areas of expertise.

 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk