Surgery is the most effective cancer therapy, followed by radiotherapy. These techniques usually target tumour specific tissue only, unlike most forms of chemotherapy as is best illustrated by the... Show moreSurgery is the most effective cancer therapy, followed by radiotherapy. These techniques usually target tumour specific tissue only, unlike most forms of chemotherapy as is best illustrated by the relatively moderate side effects of such treatments. When the immune system could find and destroy tumour cells, they (and their metastases) would be selectively destroyed without to many side effects as well. But then tumour cells have to be recognized and this requires presentation of tumour specific proteins to the immune system. This process called antigen presentation by the MHC class I molecules is studied here. Chapter 1 and 2 form an introduction to the ubiquitin proteasome system and the MHC class I antigen presentation route, which is operational in most cell types and is involved in presentation of antigens derived from degraded intracellular proteins (of self, tumour or viral origin). Proteins are not randomly degraded, but targeted for degradation by ubiquitin or ubiquitin-like post-translational modifications and subsequently degraded by the major cellular protease, the proteasome. Proteins are not only targeted for degradation because they are old, they may also be targeted for example in a cell cycle specific way or just because they have not been folded correctly during protein synthesis. Further trimming to free amino acids by other proteases follows degradation of cellular proteins by the proteasome. Only a minor pool of peptides that meets the requirements for antigen presentation may circumvent further degradation by binding to proteins involved in MHC class I presentation, like the transporter associated with antigen presentation (TAP), and MHC class I itself. Once the peptide is loaded onto MHC class I, the MHC class I-peptide complex can be transported to the plasma membrane. Here, the peptide is presented to cytotoxic T-cells (CTLs), which can in this way examine the intracellular protein content in their search for foreign content. The first step in antigen presentation by MHC class I is the decoration of target proteins with a degradation signal. The first discovered and best-studied degradation signal is a polymer of ubiquitin proteins. A ubiquitin polymer of more than four ubiquitin proteins can be recognized by the proteasome and subsequently unfolded, de-ubiquitylated and degraded by the proteasome. Free ubiquitin and mono- ubiquitylated proteins are not targets for degradation, but serve other functions. Most studies on ubiquitin have been of biochemical nature, but the introduction of the green fluorescent protein (GFP) allowed the study of ubiquitin behaviour in living cells. It was shown before that a GFP-ubiquitin construct could be stably expressed in human cells. In chapter 4, we have used this chimeric protein to study ubiquitin in living cells under normal cell culture conditions and during proteotoxic cell stress as the result of proteasome inhibition, and heat shock. In untreated cells we were able to confirm previous biochemical experiments showing that a large pool of ubiquitin molecules is coupled to histone 2A and 2B in the nucleus, whereas a small pool of ubiquitin is present as free monomers in both nucleus and cytosol. A third pool of ubiquitin was present in the form of ubiquitin polymers in both the nucleus and the cytosol. Manipulation of the cells with different proteotoxic stress conditions revealed a rapid de-ubiquitylation of the histone-bound ubiquitin pool in favour of poly-ubiquitin chains, which may even reach a size similar to the proteasome complex, which is at least one hundred times bigger as a single ubiquitin molecule. These rapid changes in the ubiquitin equilibrium do not only affect proteasomal degradation, but also induce chromatin condensation and altered gene transcription, thus establishing cross talk between these, at first sight unrelated, cellular processes. Alterations in the UPS are correlated with a variety of human pathologies, like cancer, immunological disorders, inflammation and neurodegenerative diseases. The exact role of the UPS in the pathophysiology of these diseases however, remains poorly understood. Because ubiquitin and the ubiquitin proteasome system are involved in several neurodegenerative diseases like Parkinson__s disease, Alzheimer__s disease and polyglutamine diseases like Huntington__s disease we set out our hypothesis of a sensitive ubiquitin equilibrium in the cell in chapter 5. Besides surgery, radiotherapy is one of the most effective ways of anticancer treatment. The main effects of radiotherapy on cells are induction of double-stranded DNA breaks and the formation of reactive radical species, which may lead to protein modifications like amino acid side-chain oxidation and breakage of di-sulphide bonds. These modifications will hopefully lead to DNA and protein damage, sufficient for cells to enter apoptosis or cell arrest. In chapter 6 we have shown that following exposure to g-irradiation, cell surface MHC class I-peptide complex expression is dose dependently upregulated in two phases. In the first phase of upregulation, proteins are degraded and presented that were directly damaged by the radiation and subsequent radical formation. The second phase is caused by a radiation driven activation of the mTOR pathway, which results in enhanced protein synthesis. This leads to the formation of malformed proteins called rapidly degraded proteins (RDPs) or defective ribosomal products (DRiPs) that are subsequently degraded by the proteasome and presented by MHC class I. The second phase does not only quantitatively alter MHC class I expression, but because of the mTOR pathway-specific protein expression also qualitatively. In addition, proteins may be upregulated to g-irradiation especially DNA repair proteins, resulting in more specific peptides. CTLs directed against these radiation-specific peptides were found in peripheral blood, but appeared in an anergic state. The existence of these CTLs and the expression of radiation-specific peptides may explain the inhibition of distant tumours after local radiotherapy if these CTLs could be activated. This effect is known as the abscopal effect of local radiotherapy. If these CTLs could be activated prior to irradiation in a combination therapy, these could induce a potent immune response against the irradiated cells. We show that prior radiation of a local tumour strongly improves the response to immunotherapy (adoptively transferred CTLs), showing the feasibility of a novel combination therapy: radio-immuno therapy. The majority of MHC class I loaded peptides is derived from cytosolic proteins. But it has been shown that MHC class I also presents peptides derived from extracellular sources like bacteria and proteins from neighbouring cells. This phenomenon is called cross-presentation and many pathways have been postulated to explain how proteins from extracellular sources may intersect with the MHC class I loading machinery. Examples are endosome to cytosol relocation, intercellular peptide transport through gap-junctions, exosomes and ER-phagosome fusion. In chapter 3, we have evaluated the evidence for and against the ER-phagosome theory and concluded that cross-presentation via fusion of phagosomes with the ER is very inefficient if at all possible. Our evaluation of the ER-phagosome theory was a commentary on a study by Touret et al, 2005. This study attempted to validate previous results leading to the ER-phagosome fusion theory, but failed to do so. We have also tried to show ER-phagosomal fusion in dendritic cells, but the best near-fusion event of the ER we could find was a close encounter of ribosome containing ER membranes with a mitochondrion. Also our calculations on the odds of presentation of phagosome-derived peptides were not in favour of antigen presentation via ER-phagosome fusion events. We conclude that cross-presentation to support vaccination should find a different route. Show less
One of the mechanisms used by HCMV to downregulate cell surface expression of the MHC class I complex involves the dislocation of newly synthesized class I heavy chains into the cytosol, where they... Show moreOne of the mechanisms used by HCMV to downregulate cell surface expression of the MHC class I complex involves the dislocation of newly synthesized class I heavy chains into the cytosol, where they are degraded by the proteasome. Misfolded ER proteins have been found to be degraded via the same route that HCMV uses to dispose of MHC class I molecules. The ubiquitin system plays an important role in this process. In this thesis, the role of ubiquitin in the US2- and US11-dependent dislocation of MHC class I heavy chains has been studied. Show less
