Periodic Reporting for period 1 - RHOMBOSMALPS (Enabling malaria rhomboid proteases as drug targets: usage of molecular cookie cutters to shape novel activity assays and inhibitors.)
Reporting period: 2017-10-01 to 2019-09-30
Intramembrane proteases (IMP) are proteolytic enzymes that are embedded in the lipid bilayer. Rhomboids a serine IMP subfamily which has been linked to important human diseases. For example, Plasmodium falciparum rhomboids 1 and 4, are thought to be key for the parasite invasion. In addition, human PARL and RHBDL-4 have a role in neurodegeneration. Nevertheless, despite their relevance in pathological processes, their specific role and druggability are unclear.
The transmembrane packing interactions of IMPs are responsible for their thermodynamic stability, which translates in a high dependence on the environment. Hence, IMPs protease activity is substantially influenced by membrane composition.
Unfortunately, the study of these IMPs in their native environment has rendered impractical to date. The bottleneck is that the current purification techniques use detergents that ravage the physiological membrane, yielding low enzyme stability and, in some cases, activity. In its turn, this rules out the use of activity assays and chemical probes to study their function. Encapsulating these proteins in their lipid environment will address these shortcomings.
Our objective was set to allow the study of Rhomboids in order to enable them to function as drug targets.
We have developed a detergent free purification method, based on maleic acid copolymers: SMA and DIBMA. Those function as a “molecular cookie cutter”, creating polymer-lipid-protein nanodiscs, which retain their biological properties upon purification. We have tested the suitability of these nanodiscs to maintain the activity and stability of rhomboids with promising results. Likewise, we have evaluated their suitability to undergo activity tests. Our results confirm that the nanodiscs are suitable to run inhibitor screening.
Our work enables the study of rhomboids. This is, it has opened a door to finding inhibitors against rhomboids which will serve to enable them as new therapeutic targets for malaria or Alzheimer's disease.
The transmembrane packing interactions of IMPs are responsible for their thermodynamic stability, which translates in a high dependence on the environment. Hence, IMPs protease activity is substantially influenced by membrane composition.
Unfortunately, the study of these IMPs in their native environment has rendered impractical to date. The bottleneck is that the current purification techniques use detergents that ravage the physiological membrane, yielding low enzyme stability and, in some cases, activity. In its turn, this rules out the use of activity assays and chemical probes to study their function. Encapsulating these proteins in their lipid environment will address these shortcomings.
Our objective was set to allow the study of Rhomboids in order to enable them to function as drug targets.
We have developed a detergent free purification method, based on maleic acid copolymers: SMA and DIBMA. Those function as a “molecular cookie cutter”, creating polymer-lipid-protein nanodiscs, which retain their biological properties upon purification. We have tested the suitability of these nanodiscs to maintain the activity and stability of rhomboids with promising results. Likewise, we have evaluated their suitability to undergo activity tests. Our results confirm that the nanodiscs are suitable to run inhibitor screening.
Our work enables the study of rhomboids. This is, it has opened a door to finding inhibitors against rhomboids which will serve to enable them as new therapeutic targets for malaria or Alzheimer's disease.
The summarized results follow:
1- Proof of principle that the xMALPs methodology can be successfully applied to ROMs.
2- Successful expression of PfROM4 in a bacterial system.
3- Successful expression of RHBDL4 in a bacterial system.
4- Stable native-lipid xMALP nanodiscs of activeRHBDL4 in a nanodisc. This is the first example of a stabilized active eukaryotic ROM protease in a nanodisc.
1- Proof of principle that the xMALPs methodology can be successfully applied to ROMs.
2- Successful expression of PfROM4 in a bacterial system.
3- Successful expression of RHBDL4 in a bacterial system.
4- Stable native-lipid xMALP nanodiscs of activeRHBDL4 in a nanodisc. This is the first example of a stabilized active eukaryotic ROM protease in a nanodisc.
In conclusion, during my Marie Curie IF I established a working method to solubilize stable and active prokaryotic and eukaryotic rhomboids. Meanwhile the work on the prokaryotic rhomboids has already been published, the research on the eukaryotic rhomboids did allow me to generate convincing data to continue researching rhomboids as novel therapeutic targets. In the future, I plan to use the nanodiscs that I developed to help identify novel targets for malaria and neurodegeneration.