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REGULATION OF CHOLESTEROL HOMEOSTASIS BY THE ARH-MEDIATED ENDOCYTOSIS OF THE LDL RECEPTOR

Final Report Summary - CHOLESTRUCTURE (REGULATION OF CHOLESTEROL HOMEOSTASIS BY THE ARH-MEDIATED ENDOCYTOSIS OF THE LDL RECEPTOR)

In order to develop the Structural Biology discipline on campus, a few years ago Technion had decided to recruit experts and establish a suitable infrastructure to enable modern and competitive macromolecular crystallography (MX) research. In alignment with this vision, the two main purposes of this fellowship were: (i) To establish the methodology and the infrastructure for MX at the Technion. (ii) To develop state-of-the-art research programs in Structural Biology, with a particular emphasis on LDL-cholesterol and its involvement atherosclerosis.
In 2012, I was recruited to Technion, as an experienced crystallographer, to establish and head the Technion Center for Structural Biology (TCSB).
Since no synchrotron facility exists in Israel, most structural projects require international traveling to be completed. Thus, it was clear that in order to make a significant difference for the local community of structural biologists, we need to establish a system that will allow us to perform high-resolution X-ray crystallography in house. Fortunately, we were able to purchase the most powerful home-source of X-rays, based on the Rigaku FR-X rotating anode, complemented with an ACTOR robot for automatic crystal mounting at cryogenic temperatures. Together with several other high-end robotic systems for crystallization and imaging, the TCSB includes a state-of-the-art facility for MX which is considered the best in the country.
Devoting enough of my attention emphasis to establish this superb infrastructure proved to be highly rewarding, since for the majority of structural biology projects currently performed at Technion we hardly need to travel abroad for synchrotron facilities, and our X-ray Diffractometer systems is powerful enough to yield high resolution structures even down to 1.5 Å resolution, and quite routinely we collect complete data sets at 2.0 Å resolution in just a few hours.
Hypercholesterolemia, i.e. excess blood levels of the low-density lipoprotein (LDL), is a factor profoundly associated with coronary heart disease due to the strong propensity of LDL to accumulate in the arterial walls and promote atherosclerosis. The level of plasma LDL is regulated by the ARH mediated endocytosis of the LDL-LDLR complex. The first year of this proposal was devoted to study the mechanism of ARH function by biochemical and structural tools. Towards this goal, ARH was expressed and isolated from bacteria together with its putative binding partner, the clathrin adaptor AP-2. AP-2 expresses very well and was isolated to high yield and purity, however ARH showed strong propensity for aggregation. Screening for stabilizing chemicals we identified a few stabilizing agents however these still didn't help in getting crystals of full length ARH or to get consistent binding data. Similarly, the phosphotyrosine-binding (PTB) domain of ARH still showed aggregation during binding studies with synthetic peptides from cytosolic portions of receptors. After re-assessment of the potential of this project to yield significant results within the time frame dedicated for this proposal, I decided to focus on related projects that appeared more promising.
The properties of oxidized LDL (OxLDL) has been a subject of a vast growing interest in the field of chronic inflammatory diseases since, rather than the associative factor LDL, OxLDL is considered as causative pathogenic factor directly triggering the inflammation process in the development of atherosclerosis. However, the chemical identity and structural properties of the many possible forms of OxLDL are not clear, since LDL is multipcomponent assembly of the protein apoB100 and many various lipids cholesterol and cholesteryl ester (CE) forms. The task of identifying a specific oxidized epitope of LDL that has atherogenic properties is therefore challenging and requires multidisciplinary approaches using in vivo animal models, samples from atherogenic plaques as well as in vitro biochemical characterization. There are many studies documenting the involvement of oxidized phospholipids, but the OxCE forms are less known and understood.
In collaboration with the Miller group at UCSD, we are using X-ray crystallography to characterize atherogenic OxCE using antibodies developed to recognize it. Recently we obtained crystals of the antibody mixed with a certain potent OxCE, which we are currently analyzing for their X-ray diffraction qaulity.
We anticipate that the high-resolution X-ray analysis, hopefully to become available within a few moths, will unravel the identity of this highly potent form of OxCE. The impact of such work on tracing inflammatory processes in atherosclerosis would be remarkable, but also for the rational development and improvement of neutralizing factors of therapeutic value.

Another area of investigation I conduct in collaboration with the Kornitzer group at Technion focuses on the acquisition of iron by pathogenic fungi. In this direction, we have made significant discovery by solving the first crystal structure of the CFEM protein. These fungal proteins are responsible for extracting iron from hemoglobin, in its heme-bound form, and for its delivery to downstream factors that shuttle it into the fungus cell. Since CFEM proteins are virulence factors of several fungi including candida albicans responsible for candidiasis, this work which is currently in the process of publication is expected to have great impact on the field, and will allow for structure-based drug design approaches to battle such infectious diseases.

In collaboration with the Glickman group at Technion, we study the mechanism of proteasomal degradation, a major pathway for selective protein destruction mechanism in eukaryotes. Efficient degradation of ubiquitin-labeled proteins by the massive multi-subunit proteasome is coupled to removal of the label before entering this proteasome. This is carried out by a de-ubiquitinate (DUB) enzyme composed of the Rpn11 and Rpn8 subunits of the proteasome. We have been able to isolate and crystallize the complex of Rpn11-Rpn8 in its active form, and solved the crystal structure of this hetero-dimeric complex at 2.35Å resolution (PDB ID 4OWP). Somewhat unfortunately for us, this important work has been a subject of stiff competition and at least two additional groups have solved similar structures and were able to publish them very recently before we did. We are currently focusing on testing some new structural hypothesis biochemically.

Together with the Landau lab at Technion, I have been studying the structure of an important tyrosine kinase from a pathogenic bacteria named bcef. The crystals we grew of bcef suffered from some crystallization pathology, therefore the structure determination process required my expertise. Excitingly, the structure solved at 2.1 Å resolution revealed an endogenous ADP molecule bound at the active site. This observation now serves as a basis for design of small molecule, by the Landau Lab, to modulate this enzyme in an attempt to target its pathogenic-involved activity.

Together with the Alian and Manor labs at Technion, we studied the involvement of Translin, an octameric protein of a barrel shape, in RNA metabolism. We crystallized translin in the presence of RNA and I determined the crystal structure at 3.0 Å resolution (PDB ID 4WYV). The structure revealed the barrel in a novel open conformation, and has thus hints on the potential entryway for RNA, published in JMB (Eliahoo et al, 2015).

The last project I was directly involved with was founded in collaboration with Dr. Benhar at Migal Institute, to study the crystal Structure of a bacterial serine palmitoyltransferase, which we were able to determine at 2.1 Å resolution. This data sheds light on the mechanism of this important protein modifying enzyme in a posttranslational manner.

Finally, as the head of the TCSB I supported, though less directly and more technically, a large number of other structural studies performed at TCSB during the period of this project, most of which are at various stages of analysis and scientific publication.

The transfer of knowledge elements from this fellowship are very significant not only through the research aspects as described above, but also via the unique dissemination of X-ray Crystallography education which our local community of structural biologists enjoy from. Of particular notice, is the establishment of the 1st Biostruct-X Macromolecular Crystallography Workshop, to be held for the first time in Israel and hosted by the TCSB in January 2016.

http://tcsb.technion.ac.il/Hay-Dvir