1,720,995 research outputs found
Regulation of endosomal and phagosomal transport
Full text linkEndocytosis can be conceptually broken down into two stages: The formation of the nascent vesicle containing the ingested proteins and lipids, and its subsequent maturation into a degradative compartment. During this maturation process proteins can be recycled back to the plasma membrane to prevent degradation. The vesicular membrane and the luminal contents undergo considerable remodeling to transform this compartment into a degradative one. This stops signal transduction by degrading the receptors within the vesicle and also generates antigens for presentation on MHC class II molecules in order to elicit an adaptive immune response. The engulfment of large particles and microorganisms (e.g. pathogens) is referred to as phagocytosis. The vesicle created by phagocytosis is called the phagosome. Phagosome maturation is considered to involve a complex sequence of fusion and fission events with the sub compartments of the endocytic pathway. In many ways, phagosome maturation recapitulates the progression of cargo along the endocytic pathway. In this thesis we have described molecular mechanisms that regulate the endocytic route and how pathogens explore this endocytic route to ensure survival.Nederlands Kanker Instituut Nederlandse Vereniging voor Microscopie Koningin Wilhelmina FondsUBL - phd migration 201
Novel insights in MHC class II antigen presentation
Full text linkMajor Histocompatibility Complex class II (MHC-II) antigen presentation is involved in the regulation of immune responses against infections and tumours and strongly linked to autoimmune diseases. To better understand this process we performed a genome-wide RNAi screen. The effect of downregulation of each human gene on MHC class II expression and antigen loading was analyzed. Genes affecting MHC-II antigen presentation were tested for their involvement in MHC-II transcription and transport. Some genes might function in repositioning MHC-II towards the plasmamembrane upon Dendritic Cell maturation. This was confirmed by silencing these genes in immature Dendritic Cells. The novel GTPase ARF7, ARF7EP and MYO1E play a central role in this transport process. MHC-II antigen loading on intraluminal vesicles of the MHC-II compartment is facilitated by chaperone DM. In a confocal FRET study we investigated whether microdomain-forming tetraspanins CD63 and CD82, interacting with MHC-II and DM, could facilitate the MHC-II/DM interaction. MHC-II and DM preferably interacted with CD63 on the intraluminal vesicles, likely facilitating the MHC-II/DM interaction and antigen loading. MHC-II favoured interactions with CD82 on the limiting- and plasmamembrane suggesting a sorting role for CD82. This knowledge will contribute to development of new methodologies to influence MHC class II presentation in patients.UBL - phd migration 201
Manipulations of the ubiquitin proteasome system and their effects on antigen presentation
Full text linkSurgery 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.LEI Universiteit LeidenThe Netherlands Organization for Scientific Research (NWO) and the Dutch Cancer Society (KWF).
Publication of this thesis was financially supported by the Dutch Cancer Society (KWF)Moleculaire basis van immuunregulatie
A genome-wide cell biological analysis of genes involved in MHC class II antigen presentation
Full text linkThe human genome comprises of more than 22.000 genes. These genes encode for proteins playing a role in many general or tissue-specific biological processes, but for many of them the function is not yet unraveled yet. A way to study all the different genes at once in a certain biological process is by genome-wide screening. By considering all genes in a pathway, new players can be identified that would otherwise not have been linked to the studied process. In this thesis, I describe a siRNA-based genome-wide screen used to identify new proteins involved in MHC class II antigen presentation, expression and transport. The data obtained from this screen not only led to the identification of proteins involved in the MHC class II pathway specifically, but we also identified a regulator of a more general process namely intracellular endosome localization. This shows the importance of genome-wide screening in the identification of new players and regulators of certain biological pathways which may be considered as new drug targets to cure diseases.NKI/AvLUBL - phd migration 201
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Transport of Lysosome-Related Organelles
Full text linkMany intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is largely unclear how these motor proteins are targeted to specific compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments and therefore are ideal candidates for controlling motor protein recruitment. This work shows that dynein-dynactin motor recruitment to lysosomal compartments requires activation of the GTPase Rab7 that subsequently associates with its effector protein, RILP (for Rab7-Interacting Lysosomal Protein). RILP maintains Rab7 in the vesicle-bound, activated state and transmits a signal for specific recruitment of the dynein-dynactin motor. As a consequence, lysosomes are transported towards the minus-end of microtubules. This signalling cascade thus regulates lysosomal transport. In addition, we showed that this pathway also regulates transport of several other lysosomal compartments, including Salmonella-containing vacuoles and melanosomes.The work descripted in this thesis was performed at the Division of Tumor Biology of the Netherlands Cancer Institute, Amsterdam, The Netherlands and sponsored by the Koningin Wilhelmina Fonds and the Netherlands Organisation for Scientific Research NWO. KWF; NWOUBL - phd migration 201
The systems biology of MHC class II antigen presentation
Full text linkMajor histocompatibility class II molecules (MHC class II) are one of the key regulators of adaptive immunity because of their specific expression by professional antigen presenting cells (APC). They present peptides derived from endocytosed material to T helper lymphocytes. Consequently, MHC class II is fundamental in orchestrating both cellular and humoral immune responses. A genetic association of certain MHC class II alleles with autoimmunity has long been established. The molecular mechanisms underlying this association are only poorly understood. An in depth understanding of the antigen presentation pathway by MHC class II is essential for the improvement of current therapies. In this thesis, the tools to arrive at a systems understanding of MHC class II antigen presentation are discussed. What are the advantages and disadvantages of a genome-wide screen? And how can a multi-dimensional, data-integrating approach increase the understanding of the systems biology of MHC class II?UBL - phd migration 201
Protein complexes and cholesterol in the control of late endosomal dynamics : Cholesterol and multi-protein complexes in the control of late endosomal dynamics
Full text linkLate endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer__s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the direction of late endosomal transport by dynein-motors, and that this process is disrupted in Niemann-Pick disease. Furthermore cholesterol also regulates tethering via the multi-subunit HOPS complex. Homologues of this complex are mutated in ARC syndrome patients and the cellular consequences are described in this thesis. We find that the cholesterol sensing proteins ORP1L and MLN64 are major regulators of late endosomal transport, and that this transport is further regulated by the endoplasmatic reticulum.UBL - phd migration 201
Molecular basis for the control of motor-based transport of MHC class II compartments
Full text linkAntigen presentation by MHC class II is critical for immune responses against pathogens and tumors. Antigen loading occurs primarily in lysosomal-related organelles (LROs) known as MIICs. Ultimately, the MHC II-peptide complexes are transported for cell surface display. Here, we study intracellular transport of MIICs, a poorly understood process in MHC II antigen presentation. We propose that Rab7 lies at the heart of transport by assembling a specific receptor (Rab7-RILP-ORP1L) for the minus end-directed dynein-dynactin motor on the cytosolic face of MIICs/LROs. Full activation of transport requires a second receptor, _III spectrin. Whereas this model explains how Rab7 controls minus end-directed transport, it does not suffice to explain the characteristic pattern of bidirectional motility exhibited by MIICs/LROs. Here, we propose that cholesterol dictates ORP1L conformation which acts as a switch controlling access of dynein-dynactin to Rab7-RILP, thereby regulating LRO positioning, as observed in NPC disease. Rab7-RILP-ORP1L may also integrate transport and tethering of MIICs/LROs, consecutive processes within the endocytic pathway.UBL - phd migration 201
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