Lipid Complexation and Protein Deimination in Demyelinating Disease

Demyelinating diseases specifically multiple sclerosis (MS) present with detrimental neurological deficits that include impairment in visual function. MS has been associated with hyper-deimination of myelin basic protein isoform 5 (MBP) which positively correlates with an increase in MS severity. Deimination is a post-translational modification responsible for the conversion of protein bound arginine (positive charged) to protein bound citrulline (neutral charge). This modification has a direct effects on the tertiary structure of MBP, which significantly increases unfolding and susceptibility for proteolysis, generating immune reactive peptides. These peptides propagate the autoimmune response and are key component of MS pathogenesis. MS has also been documented to have deficiencies in phospholipids which are a major constituent of the cytosolic myelin membrane (side that interacts with MBP). Based on these key features we hypothesized that a) deficiencies of specific phospholipid species in the MS animal model (EAE) lead to differential complexation with MBP, b) restoring endogenous complexation can protect MBP against hyper-deimination and c) supplementation of deficient lipids in the optic nerve can improve visual function.</p

Lyso-Lipid-Induced Oligodendrocyte Maturation Underlies Restoration of Optic Nerve Function

Protein hyperdeimination and deficiency of lyso-phospholipids (LPC 18:1) has been associated with the pathology of demyelinating disease in both humans and mice. We uncovered interesting biology of LPC 18:1, in which LPC 18:1 induced optic nerve function restoration through oligodendrocyte maturation and remyelination in mouse model systems. Our studies show LPC 18:1 protection against neuron-ectopic hyperdeimination and stimulation of oligodendrocyte maturation, while investigations recorded optic nerve function improvement following optic nerve injections of LPC 18:1, in contrast with LPC 18:0. Thus, just a change in a single bond renders a dramatic alternation in biological function. The incorporation of isobaric C13-histidine in newly synthesized myelin proteins and quantitative proteome shifts are consistent with remyelination underlying restoration in optic nerve function. These results suggest that exogenous LPC 18:1 may provide a therapeutic avenue for stemming vision loss in demyelinating diseases

Nuclear prelamin a recognition factor and iron dysregulation in multiple sclerosis

Dysregulation of iron metabolism and aberrant iron deposition has been associated with multiple sclerosis. However, the factors that contribute to this pathological state remain to be understood. In this study, human multiple sclerosis and mice brain samples were analyzed through mass spectrometry as well as histological and immunoblot techniques, which demonstrated that iron deposition is associated with increased levels of nuclear prelamin A recognition factor (NARF). NARF is a protein associated with the mitochondria which has also been linked to mitochondrial defects in multiple sclerosis. We report NARF to be associated in multiple sclerosis pathology and aberrant iron deposition

Capillary Electrophoresis Plasma Fractionation for Lipids Analysis

Extraction of lipids has mainly used density liquid phase separation techniques; however, these methods are limited by their broad extraction and lack specificity. Complex mixtures like blood plasma contain multiple lipid classes, whose distribution in the body are mediated by protein-lipid interactions and integration of lipids in larger lipoprotein complexes. The capillary electrophoresis system separates complex mixtures by electrokinetic forces that preserve protein-lipid complexation and allow for the fractionation of samples. Here we present a methodology for fractionating plasma using the capillary electrophoresis system

Myelin Basic Protein Phospholipid Complexation Likely Competes with Deimination in Experimental Autoimmune Encephalomyelitis Mouse Model

Multiple sclerosis has complex pathogenesis encompassing a variety of components (immunologic, genetic, and environmental). The autoimmunogenicity against the host’s myelin basic protein is a major contributor. An increase in myelin basic protein deimination (a post-translational modification) and a change in phospholipid composition have been associated with multiple sclerosis. The interaction of myelin basic protein with phospholipids in the myelin membrane is an important contributor to the stability and maintenance of proper myelin sheath function. The study of this aspect of multiple sclerosis is an area that has yet to be fully explored and that the present study seeks to understand. Several biochemical methods, a capillary electrophoresis coupled system and mass spectrometry, were used in this study. These methods identified four specific phospholipids complexing with myelin basic protein. We show that lysophosphatidylcholine 18:1 provides a robust competitive effect against hyper-deimination. Our data suggest that lysophosphatidylcholine 18:1 has a different biochemical behavior when compared to other phospholipids and lysophosphatidylcholines 14:0, 16:0, and 18:0

Protein-Lipid Complex Separation Utilizing a Capillary Electrophoresis System

The separation and analysis of protein-lipid complexes has proven to be challenging due to the harsh conditions required by conventional methods of protein or lipid isolation, which disrupt the fine forces that govern the interactions between lipid head groups and protein side chains. The method described in this publication presents an alternative for the separation of protein-lipid complexes while maintaining the integrity of their interactions. The method exploits the specific electrophoretic forces that are unique to the geometry of the capillary system and allows purification of intact complexes and the systematic analysis of its constituents. This technique is specifically applied for the separation of native protein-lipid complexes found in the central nervous system

Capillary Electrophoresis Assessment of Plasma Protein Changes in an African Penguin (Spheniscus demersus) With Aspergillosis

A decrease of avian biodiversity in the African continent has been the result of anthropogenic pressure in the region. This has resulted in the African penguin (Spheniscus demersus) being placed on the endangered species list and requires conservation efforts to maintain its free-ranging population and placement under managed care. In the latter environment, infection by Aspergillus fumigatus can be common. The diagnosis and treatment of this fungal disease in birds has presented with many difficulties, largely due to the diversity and limited knowledge that exists about this species. In this study, we implement a high-resolution capillary electrophoresis system for the fractionation of African penguin plasma, followed by mass spectrometry analysis for the identification of proteins associated with aspergillosis. Several protein differences were revealed, including changes in acute phase proteins and lipid metabolism. In addition, our results demonstrated that fibrinogen β chain is a protein largely present during the inflammatory process in an African penguin infected with A. fumigatus. These findings present a new avenue for the measurement of plasma proteins as a potential method for identifying important biomarkers to aid in monitoring African penguin health

Isobaric Incorporation of C13-Histidine for the Assessment of Remyelination

Multiple sclerosis is a demyelinating disease of the central nervous system characterized by the loss of the myelin sheath-the nonconductive membrane surrounding neuronal axons. Demyelination interrupts neuronal transmission, which can impair neurological pathways and present a variety of neurological deficits. Prolonged demyelination can damage neuronal axons resulting in irreversible neuronal damage. Efforts have been made to identify agents that can promote remyelination. However, the assessment of remyelination that new therapies promote can be challenging. The method described in this chapter addresses this challenge by using isobaric C13-histidine as a tag for monitoring its incorporation into myelin proteins and thus monitoring the remyelination process