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Rhomboids: at the cutting edge of the membrane

By Eliane Küttler (29.01.2014)

Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Unfortunately, substrates cannot be used universally. Therefore, during my PhD I developed a fluorescence polarization activity-based protein profiling (FluoPol ABPP) screen that uses small molecule probes and is compatible with membrane proteases. I identified new inhibitors for the E. coli rhomboid GlpG, among these was a structural class that has not yet been reported as rhomboid inhibitors. They form covalent and irreversible complexes with the active site serine of GlpG. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.

The proper functioning of enzymes is vitally important for every cell, which is why their activity is tightly regulated to prevent malfunctioning. Since such regulation problems are often the cause of severe diseases, enzymes are interesting drug targets. The majority of enzymes are soluble, but a couple of years ago some enzymes where discovered within the membrane. One class of intramembrane proteases are the rhomboids. With their α-helices they form a teacup-like structure with a water filled-cavity in the middle. Their job in nature is to cleave other membrane proteins so that a part is released. This so-called shedding is a fundamental biological process and explains why rhomboids can be found in almost all organisms ranging from weeds and fungi to humans. Consequently rhomboids are involved in countless processes such as EGF-signaling and quorum sensing. Although a lot is still unknown, they seem to play a role in the host cell infection process of Plasmodium, the parasite that causes Malaria, as well as in Parkinson's disease, cancer and wound healing. It remains unclear how their activity is regulated and which inhibitors can selectively inactivate them. The discovery of strong, selective and specific rhomboid inhibitors would provide new tools for their study and could potentially serve as lead structures for the future development of therapeutics. As rhomboids show great variations in their susceptibility towards inhibitors, each rhomboid must be screened separately. This is why in my PhD project, I aim to develop inhibitor screening assays for various rhomboids which will facilitate a fast and easy search. All available rhomboid assays so far require a protein or polypeptide substrate which is not available for many rhomboids. To overcome this caveat, I developed an assay that does not use substrates at all, but small chemical tools, so-called activity based probes (ABPs). ABPs are small molecules that can 

[Bildunterschrift / Subline]: Fig. 1: Scheme of the Rhomboid FluoPol ABPP. Rhomboid is pre-incubated with small molecules. Then the ABP is added and its binding observed in a fluorescence polarimeter in a 96-well plate format to identify hit structures.

covalently bind to the active site of active enzymes, but not to their inactive forms. A fluorophore is attached on one end of the ABP for visualization. ABPs are the basis for my Rhomboid Fluorescence Polarization Activity-Based Protein Profiling Assay (Rhomboid FluoPol ABPP). It is based on an existing polarization assay which has been succesfully used for inhibitor screening of soluble proteases and was now adapted by me for the rhomboid intramembrane proteases. The key aspect of this polarization assay is that binding of the ABP to the large rhomboid slows down the rotation of the small fluorophore. This difference can be measured in a fluorescence polarimeter. Once inhibited, the rhomboid cannot bind the ABP anymore and the fluorophore retains its original rotational speed. Using this assay, I have successfully screened a selection of small molecules for new inhibitors for the rhomboid

GlpG from E.coli. I have found a new structural class of rhomboid inhibitors not yet reported. In the future I will extend this assay to rhomboids that are poorly characterized, including several bacterial and eukaryotic rhomboids. I hope to be able to find novel and potent inhibitors that can be used as new valuable research tools and may even represent a first step towards drugs for treatment of some rhomboid-related diseases.

Scientific career
  • 2004-2008
  • B.Sc. in Molecular Biotechnology at the University of Technology in Dresden
  • 2008-2010
  • M.Sc. in Molecular Biotechnology (M.Sc.) at the Technical University Munich
  • since 2010
  • PhD in chemical proteomics at the Technical University Munich

  • * “Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay” Proc Natl Acad Sci U S A. 2013 Feb 12;110(7):2472-7. Vosyka O, Vinothkumar KR, Wolf EV, Brouwer AJ, Liskamp RM, Verhelst SH
  • * “Tuning probe selectivity for chemical proteomics applications” Curr Opin Chem Biol. 2012 Dec 27. S1367-5931(12)00159-7. Haedke U, Küttler EV, Vosyka O, Yang Y, Verhelst SH.