A variety of specific chemical damage occurs as a result of normal cellular senescence, as well as accelerated damage in the context of certain pathologies. One such chemical pathway is the degradation of aspartates into isoaspartyl residues through oxidant damage. As a repair mechanisms, PIMT1 is an enzymatic pathway that methylates isoaspartyl residues, creating an isoaspartyl methyl ester that is capable of then spontaneously reverting into aspartate, thus reversing isoaspartyl damage. Insufficient PIMT1 activity has been associated with increased oxidant stress and shorter cellular and organism lifespan in mice; however, a detailed metabolic and biochemical analysis of the role of PIMT1 has not been elucidated. In this application, we propose to study the role of PIMT1 in cellular aging. While multiple tissues will be analyzed to test general effects of PIMT1, this proposal mainly focusses on a specific central hypothesis regarding effects in red blood cells (RBCs). RBCs are essential to health, and dysfunction of RBCs plays a central role in multiple diseases. In addition, transfusion of RBCs is the single most common inpatient invasive therapy, being given to approximately 1 out of every 70 Americans, annually. RBCs that are transfused are stored (as a logistical necessity) for up to 42 days, during which time they undergo specific cellular and biochemical damage. RBCs are known to lose an essential regulatory function through a key gene product (AE1) in normal cellular aging and in RBC storage. However, the molecular mechanism by which AE1 dysfunction occurs has been unknown. In this application we provide novel data demonstrating that isoaspartyl damage occurs in AE1 of both human and murine RBCs in a domain of AE1 that requires aspartates to function. We likewise present data suggesting that failure of PIMT1 pathways accelerates this damage ? however whole animal modeling with deletion of PIMT1 is required to test a mechanistic role. In this context, we offer the following specific aims, designed to critically test hypotheses around the role of PIMT1 mediated repair of oxidant damage.
Specific Aim 1 : Mechanistic elucidation of the role of protein methylation by PIMT1 in the function and senescence of RBCs.
Specific Aim 2 : the interaction of increased oxidant stress on PIMT1 and its effects on RBCs aging in vivo and ex vivo (blood storage). PIMT1 null mice will be combined with additional strains designed to isolate metabolic pathways of functional relevance (e.g. G6PD deficient). Advanced experimental methodologies will be applied to these animals in order to isolate cells of particular age and physiological conditions. Finally, the controlled biologies generated from these approaches will be analyzed by cutting edge metabolomic and proteomic methodologies. In aggregate, these studies will advance our understanding of the role of specific pathways of biochemical cellular aging, of the mechanistic role of a conserved repair pathway (PIMT1), and in the context of advanced biochemical analysis and modeling to generate novel mechanistic understanding and critical testing of focused hypotheses.
Over time, animal cells undergo damage as a result of aging. A number of different natural pathways have evolved to mitigate, and in some cases reverse, cellular aging-related damage. This project proposes to study methylation of oxidized proteins as one of such pathways fixing this damage. We will focus on red blood cells, a simple cell type which is constantly exposed to oxidative damage while being devoid of de novo protein synthesis capacity, a peculiarity that makes red blood cells dependent on recycling of oxidatively damaged proteins. The goal of this proposal is to generate new mechanistic understanding of the effect(s) of this pathway and how such understanding can be used to generate new therapies and approaches to mitigate cellular aging in healthy people and to overcome diseases or conditions in which red cell-age related damage plays a role, such as during blood storage and transfusion ? the most common in-hospital medical procedure worldwide.
