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Research

Protein complexes and cholesterol in the control of late endsosomal dynamics

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Title Protein complexes and cholesterol in the control of late endsosomal dynamics
Period 10 / 2007 - 09 / 2013
Status Completed
Dissertation Yes
Research number OND1325375
Data Supplier Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)

Abstract

MHC class II molecules are expressed on specialized immune cells to present a fragment of a pathogen to other cells of the immune system(1-5). This process is essential for proper antibody responses following infection, for efficient activation of immune cells and for recognition of phagocytosed pathogens. On the other hand, MHC class II molecules are also involved in most situations of ?overactivation? of the immune response leading to auto-immune diseases like diabetes, rheuma and multiple sclerosis(6). Antigen presentation by MHC class II should therefore be tightly controlled. As a result of our and others work, the cell biology and biochemistry of the process of MHC class II antigen presentation is understood in detail. MHC class II molecules are assembled in the ER and transported by the invariant chain to a late-endosomal structure called MIIC(7, 8). Here the invariant chain is degraded by resident proteases(9), leaving a small fragment (called CLIP) in the peptide-binding groove of MHC class II. This CLIP fragment can be exchanged for fragments from pathogenic proteins (10, 11). Some half-one hour after entry in MIIC, MHC class II molecules are transported to the plasma membrane to present CLIP (when the process was unsuccessful) or antigenic peptides (when successful) to the immune system. Many intracellular proteins, including a kinase called PKB/Akt(13), are probably involved in controlling antigen presentation by MHC class II(14), but most are undefined. Still these should be attractive targets for compounds controlling auto-immune responses. The NKI has established a robotics facility that includes automated microscopy and automated FACS. We have used these to identify kinases inhibiting intracellular pathogens by following a strict order of experiments: 1. Use chemical compounds to identify a protein family affecting intracellular growth of Salmonella; 2. Synthesize and test a series of variations of the most successful compound for chemical profiling; 3. We identified kinase inhibitors preventing intracellular growth of Salmonella and other pathogens. We then combined screening with siRNA libraries representing the human kinome with analyses by automated microscopy to identify kinases with similar biological effects; 4. Test in in vitro assays which kinase activity followed the chemical profile; 5. Test compound under in vivo conditions. Within 1.5 years, we identified targets and lead compounds as new antibiotics (Kuijl et al., submitted). Now we intend to follow a similar approach for MHC class II molecules. We will use cells expressing GFP-tagged MHC class II molecules. These cells will be exposed to siRNAi (first representing the kinome, then the full human genome) and stained with antibodies recognizing the unsuccessfully loaded (CerCLIP) and successfully loaded (L243) MHC class II molecules. The antibodies are directly labelled with two different colours not overlapping with GFP fluorescence. The cells will be labelled with these antibodies and the ratio of the two colours will be determined by automated 2color-FACS. Candidates will be re-analyzed for the effect on MHC class II distribution by the GFP signal. The analysis will also exclude kinases affecting cell proliferation. In collaboration with Prof. Dr H. Overkleeft, a set of about 60 kinase inhibitors have been synthesized that will be tested, new kinase inhibitors may be synthesized on the bases of published (patent) literature. In addition, we will merge with the activities of Organon bv to identify interesting compounds inhibiting the candidates identified in our screen. Compounds will be tested for the effect on antigen presentation in vitro and in vivo (mice). Once identified, we will continue on identifying the mechanism of action of the kinases. We have already identified a few candidates from the original antibiotic screen, and these apparently affected intracellular trafficking to lysosomes. Obviously a direct data base comparison (with the candidates from two different screens that centre around the control of the endo/lyso/phagosomal pathway) may yield further information on the underlying biological mechanism. We have ample experience and running systems to visualize all known steps in MHC class II antigen presentation. However, the control of MIIC transport (ie motor proteins) is the least understood step in the process, and we proposed to study this item next to our screening approach, since various targets may affect this pathway. And the pathway should be better understood in order to understand the action of our hits. We have identified a novel controlling protein that appears to use cholesterol to time dynein motor binding(17, 18). In addition, (a) kinase should be involved in controlling intracellular transport of MHC class II molecules (detected with the broad kinase inhibitor staurosporine)(16). This will be identified and further analysed in this proposal since it is certainly a target for control of MHC class II antigen presentation. With our ?chemical genetics? approach we intend to identify new candidates controlling a critical step in the immune system; antigen presentation by MHC class II molecules. At the same time, we will identify the mechanism of action of these targets that manipulate MHC class II antigen presentation. The major undefined step is the mechanisms of control of transport of MHC class II molecules and we will study these in more detail in this proposal to also understand the action of potential targets. We combine the activities of a pharmaceutical industry (Organon bv) and the bio-organic chemistry group in Leiden to identify compounds for manipulation of MHC class II molecules. These will first be kinase inhibitors but will be expanded to other protein families when the results from larger screens become available. The inhibitors will be tested in in vitro and in mouse models to determine the feasibility of these targets and inhibitors for manipulation of immune responses. The use of such inhibitors may find application for patients with auto-immune diseases, especially the more life-threatening ones such as multiple sclerosis.

Related organisations

Related people

Supervisor Prof.dr. J.J. Neefjes
Doctoral/PhD student Dr. R.H.N. van der Kant

Classification

D21800 Immunology, serology
D23100 Pathology, pathological anatomy

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