Protein motion in cancer signaling

Investigator: Matthew Coleman
UC Davis Medical School

Investigator's profile

Dr. Coleman's Lab is actively pursuing development of advance biochemical techniques using nanolipoproitein particles (NLPs) nanoparticles, made of apolipoproteins and phospholipids. NLPs closely mimic the cellular membrane bilayer, and represent an ideal platform for characterizing membrane proteins involved in signal transduction. Coleman Lab is also exploring the utility of NLPs in drug delivery, immuno-modulation and in-vivo imaging for cancer treatment.

Significance

Figure 1: Conformational dynamics of ErbB receptors. EGFR is a member of the ErbB family of receptors. Each EGF receptor protein (dark blue) consists of a large extracellular ligand binding domain (top), a single transmembrane segment, a juxtamembrane segment, a kinase domain (bottom), and a carboxy-terminal tail. By developing NLPs (phospholipid bilayer, light gray; belt, pink) and attaching fluorescent dyes (red, green stars), we can develop and apply single-molecule spectroscopic methods to explore the conformational dynamics upon ligand (dark blue) binding.

The goal of this collaborative project is to develop membrane systems and spectroscopic tools to study the mechanism behind receptor function. The four members of the ErbB family of mammalian receptor tyrosine kinases promote cellular proliferation, survival, transformation, invasion and migration. Overexpression has been observed in tumors and they are associated with worsened prognosis [1,2]. The extracellular domain is a target for therapeutics, yet often resistance to treatments. This suggests that more specific and efficient methods are required to target the receptors, requiring a mechanistic understanding of their function.

Studying the biochemical and structural changes upon activation of the ErbB family of receptor is an important focus of Dr. Coleman's laboratory. In ErbB activation, dimerization and ligand binding in the extracellular domain induces conformational changes in the intracellular kinase domain [3]. However, the structure and dynamics of these changes remain poorly understand. A major barrier to developing a mechanistic picture has been the inability to perform in vitro and high-resolution studies on the membrane-bound receptor without a background of extraneous cellular processes. Here, we overcome this barrier using a mixed biochemical (TRD4) and spectroscopic (TRD1) approach.

Approach

Studies of the mechanism behind receptor protein function require a near native environment to access the intrinsic behavior. Dr. Coleman's laboratory has developed novel nanolipoprotein particles (NLPs) that allow reconstitution of membrane proteins into a near native phosophilipid bilayer with an amphiphilic belt [4]. The LBRC will adopt this platform and will attempt to increase the membrane area two-fold and add easily functionalizable handles. Under native conditions, multiple proteins are inserted within the membrane and at least a dimer is required for activation. By extending this technology, multiple proteins can be inserted, replicating native conditions. Dr. Coleman has pioneered an approach to integrate polymer into the belt, improving the size tunability. By synthesizing a range of polymers (dendrimers), the membrane area and size homogeneity will be increased. Furthermore, the dendrimers will be functionalized to enable attachment of a quantum dot as an additional, spectrally separate fluorescent reporter. The quantum dot will monitor motion of the protein structure relative to the membrane.

Research plans

Dr. Coleman research requires methods that can monitor the receptor conformational changes, and the dynamics of these changes, in a physiological environment. To address this need, the LBRC will use single-molecule Förster Resonance Energy Transfer (smFRET) and develop new single-molecule methods (TRD1) to monitor the protein-protein association and large-scale conformational motions during the signaling process. Spectroscopic reporters will be attached to key points on the protein structure, as illustrated in Fig. 1. These experiments will read out the spatiotemporal dynamics, enabling an understanding of the receptor activation and drug interactions, which will facilitate the development of targeted cancer therapeutics.

Summary

In collaboration with Dr. Coleman's laboratory, the LBRC will develop novel nanolipoproteins membrane with two-fold area to add easily functionalizable handles. The LBRC will also develop smFRET to monitor the conformational dynamics of the ErbB receptors. The dvelopment of model membrane systems will enable us to carry out in vitro studies of these receptors.

References

  1. "The deaf and the dumb: the biology of ErbB-2 and ErbB-3," Experimental Cell Research, Vol. 284, Issue 1, March 2003. [ Pubmed ]
  2. "Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene," Science 09, Vol. 235, Issue 4785, pp. 177-182, Jan 1987. [ Pubmed ]
  3. "The EGFR family: not so prototypical receptor tyrosine kinases," Cold Spring Harbor Perpsectives in Biology, 2014. [ Pubmed ]
  4. "Controlling the diameter, monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry, Protein Science, Vol. 22, pp. 1078-1086, May 2013. [ Pubmed ]