In Search for Biomarkers of Aging : A Proteomics Approach

Abstract

Research on both higher and lower eukaryotic model systems have revealed that numerous aspects are involved in the course of the aging process. Although strides have been made to understand the role of these processes in the development of aging, a general consensus has not been reached on the relative importance of each of them. Mapping the changes that occur in the course of the aging process, and thereby define so called markers of aging, could provide the information necessary to understand the aging process and the contributions of the different mechanisms herein. Changes that occur during aging, such as increased cellular death and decreased tissue and organ functioning, could be reflected in the blood plasma protein composition. However, proteomic analysis of plasma is hampered by the presence of several highly abundant bulk proteins. In chapter 2 of this thesis, the selection of highly specific single domain Llama antibody fragments (VHH) for affinity chromatography purposes is described, which can be used to remove the highly abundant human plasma proteins, human serum albumin (HSA) and all subclasses of immunoglobulin G (IgG). This removal resulted in the visualization of previously masked protein spots and an increased visibility of previously non-detectable protein spots on 2D-gel. In chapter 3, the use of these affinity ligands, combined with two-dimensional difference gel electrophoresis (2D-DIGE), is shown in a human blood plasma proteomics study to reveal protein expression differences between young and old individuals. This revealed the importance of studying levels of protein isoforms next to total protein expression levels. Furthermore, this approach demonstrated that upon aging a slightly increased pro-coagulant and pro-inflammatory state is induced. The increase of oxidative stress observed during aging can damage various different cellular molecules. An organelle that seems especially vulnerable for oxidative stress is the ER. Increased oxidative stress might interfere with protein folding and lead to differences in expression of the proteins involved in proper protein folding in the ER. These proteins contain a C-terminal KDEL signal sequence, which determines their ER localization. In chapter 4, the selection and application of VHHs specific for the C-terminal KDEL sequence is described that can be used to study protein expression patterns in cellular ER stress models. In chapter 5 the application of the KDEL specific VHH provides preliminary data on the changed expression of several ER resident proteins in a HUVEC senescence model comparing young and senescent cells. Intriguingly, clear changes are observed. However, these changes do not resemble the expression differences observed during severe ER stress. Furthermore, the expression of the membrane protein endoglin, which is involved in cellular proliferation, is decreased upon replicative senescence. These data clearly show that during replicative senescence changes occur at the protein level that could influence cellular and consequently tissue functionality, which could provide an environment for the development of age-associated diseases like atherosclerosis.