Case Report
A Proteomics. 2015;2(1): 1007.
Proteomics Profiling of Heterozygous and Homozygous Patients with ABCA1 Gene Mutation: A Tangier Disease Molecular
Ucciferri N1, Rocchiccioli S1*, Puntoni M1,2 Bigazzi F2, Cecchettini A1,3, Citti L1 and Sampietro T2
1Institute of Clinical Physiology-CNR Pisa, Italy
2Fondazione Toscana Gabriele Monasterio Pisa, Italy
3Department of Clinical and Experimental Medicine, University of Pisa, Italy
*Corresponding author: Rocchiccioli S, Institute of Clinical Physiology-CNR, Via Moruzzi 1, 56124 Pisa, Italy
Received: November 18, 2014; Accepted: February 04, 2015; Published: February 10, 2015
Abstract
Tangier Disease (TD) is a rare inherited disorder with approximately 100 worldwide identified cases. Alpha-lipoprotein deficiency is the main characteristic of this disease, associated with a virtual absence of High Density Lipoproteins (HDL) in blood. Additional symptoms are mild hypertriglyceridemia, neuropathy and enlarged, orange-colored tonsils. Genetically TD is caused by mutations in the ABCA1 gene, which prevent the release of cholesterol and phospholipids from cells, leading to the accumulation of lipids within cells and body tissues.
In this work a TD patient and his parental heterozygous were examined from a proteomics point of view. Plasma as well as proteome and secretome of circulating monocytes were analyzed.
Plasma proteins underlined in TD the imbalance of lipid trafficking and metabolism, associated with the stimulation of pro-inflammatory pathways. Proteome and secretome of monocytes highlighted an extensive down regulation of mitochondrial enzymes and vesicular trafficking agents along with a substantial cytoskeletal rearrangement, suggesting a reduced activation state of monocytes from TD homozygous patient.
This work is the first proteomics profiling of heterozygous and homozygous TD phenotypes and it suggests a TD case as a model to understand general mechanisms of lipid transport and metabolism and their linkage to inflammatory processes.
Keywords: Tangier disease; Protemics profiling; Monocyte proteomics; Rare disease
Case Presentation
Tangier Disease (TD) is an autosomal recessive genetic disorder, described for the first time in 1961 [1] and characterized by impaired HDL-mediated cholesterol efflux and abnormal intracellular lipid trafficking and turnover. TD patients accumulate cholesterol in body tissues; show a reduced level of HDL, disturbances of nerve functions, premature atherosclerosis and a high incidence of Coronary Artery Disease (CAD). TD is a rare genetic disorder, with less than 100 cases reported in the literature, and patients present both alleles of ABCA1 gene mutated; this gene encodes a member of the ATPBinding Cassette (ABC) transporter family [2, 3]. Carriers of a single ABCA1 mutation (heterozygotes) display an intermediate phenotype with a 50% reduction in the ABCA1-mediated cell cholesterol efflux [4]. Despite the commitment of a unique documented gene, TD is characterized by high variability in phenotypic manifestations, either qualitatively or quantitatively, in terms of clinical severity and organ involvement. In brief there is not a direct correlation between the gene mutation and the numerous and various clinical features described in TD patients.
ABCA1 is the major responsible transporter for clearing cholesterol from macrophages and, since cholesterol accumulation in arterial macrophages is atherogenic, this pathway has a clear involvement in the progression and/or regression of cardiovascular diseases [5]. Due to its central role in the modulation of cholesterol homeostasis, ABCA1 is an attractive target for drug development, but the molecular actors of the ABCA1 lipid pathway are not completely revealed. Studies are needed to understand how lipids are translocated across the plasma membrane and to identify associated proteins that modulate this pathway. Proteomics studies would help to disclose intracellular and secreted factors involved in dysfunction of lipid trafficking and in its clinical manifestation suggesting new targets for therapeutic interventions that might modulate ABCA1 activity assisting the traffic of cholesterol from cells and tissues.
In this paper, proteomics analyses of monocytes and plasma of a TD patient compared with his heterozygous father are reported. The clinical characterization, proband’s kindred and plasma lipoprotein profiles of these two cases have been already published in Sampietro et al. [6] and in Puntoni et al. [7] (a brief description is also presented in the Supplementary Information).
In brief, molecular factors and activation pathways directly responsible of the pathological phenotype are highlighted and the TD case has been suggested as model to obtain insights in possible mechanisms responsible for lipid dysfunctions and eventually, as a consequence, for atherosclerosis initiation.
Proteomics Profiling
TD homozygous proteomics profile was compared with his parental heterozygous (for the ABCA1 mutation). This choice was suggested by the fact that genetic background is akin and both patients are subjected to the same clinical treatments for cardiovascular disease, even if the heterozygous is mildly affected. In this way it was possible to approximate differences in the proteomics profiles due mostly to the different expression of the pathology so as to extrapolate factors involved in dyslipidemia as a main cause of early onset of atherosclerosis.
Using a shot-gun proteomics strategy (for details see Supplementary Information), we were able to identify 197 proteins in depleted plasma samples of both patients (Table S1).
46 plasma proteins (Table 1) were found differentially expressed between TD patient and his heterozygous father. In particular 22 proteins resulted down-regulated and 24 up-regulated in homozygous with respect to heterozygous. Among the downregulated in the homozygous, 13 different apolipoproteins were particularly interesting. In addition, also Paraoxonase 1 has been found down-regulated in homozygous patient plasma. This enzyme has been suggested to be involved in the protection against oxidative modification of lipoproteins and consequently against pivotal events leading to atheroma formation [8,9].These results, although preliminary, confirm that plasma proteomics can evidence the imbalance of lipid trafficking and metabolism in TD, suggesting a distinctive characteristic fingerprint, provided with a panel of correlated elements. Among the differentially expressed proteins some are linked to inflammation, such as Orosomucoid 1 and 2 and Kininogen 1. Kininogen 1 has been found over expressed in plasma of homozygous patient and it seems to be a key mediator of inflammation [10]. On the other hand, Orosomucoid 1 and 2 resulted down-expressed in homozygous plasma and, besides being involved in pro-inflammatory responses [11,12], they also exert a possible role as lipid carrier in blood circulation [13,14]. Moreover and of note, 4 differentially expressed proteins are related to different growth factor pathways: Vasorin may act as inhibitor of TGFbeta signaling [15], Insulin like Growth Factor Binding Protein 4 (IGFBP4) and Insulin-like Growth Factor-Binding Protein Complex Acid Labile Subunits (IGFALS) are key elements of the IGF pathway and Hepatocyte Growth Factor Activator (HGFA) that seems to play role in inflammatory processes [16]. In conclusion, plasma proteome of TD patient proves a general dysregulation of lipid trafficking and metabolism, associated with the stimulation of growth factor and inflammatory pathways.
Although ABCA1 is expressed in many tissues, the accumulation of cholesterol in TD interests mostly macrophages [17]. An early event in atherogenesis is the recruitment of monocytes from the peripheral blood vessel intima as a consequence of high amounts of lipids and high levels of protein oxidation. Monocytes transmigrate into the vessel wall and differentiate into macrophages. Macrophages are well known to exert an important role in atheroprotection/ atheroformation, removing excess of cholesterol from tissues. Reverse Cholesterol Transport (RCT) is a pathway responsible of the transport of accumulated cholesterol from the vessel wall to the liver for excretion, this process preventing inflammation and atherosclerosis [18]. Many studies indicate that the inflammatory process impairs RCT [19]. ABCA1 is the major cholesterol efflux system in macrophages, it plays a crucial role in the modulation of inflammatory response [20] and in mice its knockout increases inflammatory cell infiltration [21]. Monocytes are the circulatory precursors of macrophages and play a central role under several pathophysiological conditions, particularly when inflammatory reactions are involved. For these reasons we were interested in analyzing the proteome and the secretome of monocytes from a TD patient and to compare these profiles with those of the heterozygous father.
Monocyte proteome analysis identified 198 proteins while 128 proteins were found in secretome. Proteins are reported in (Supplementary Data Table S2 and Table S3 respectively). They were classified based on their localization (Figure 1) using Uniprot database (www.uniprot.com), while the secretome proteins were also subdivided, according to the secretion pathway (Figure 2), in classically or non classically secreted, as predicted by the SecretomeP 2.0 Server (https://www.cbs.dtu.dk/services/SecretomeP/).
Monocyte whole proteome analysis brought to the identification of 198 proteins, of which, so much as 47 resulted differentially expressed (Table 2). Four principal functional groups may be underlined: a)- cytoskeleton organization (Coronin 1, Profilin 1, Tropomyosin 3, S100A6, Gelsolin, Thymosin beta4, Actin-related protein 3 and Macrophage-capping protein), b)- mitochondrial functions(Superoxide dismutase, Cytochrome C oxidase, ATP synthase beta, Thioredoxin-dependent peroxide reductase, Heat shock cognate 71 protein, Heat shock 60 kDa protein, Heat shock 10 kDa protein, Dihydroprolyl dehydrogenase, Hydroxyacylcoenzyme A dehydrogenase, Trifunctional enzyme subunit beta, Complement component 1 Q subcomponent-binding protein), c)- vesicular trafficking regulation (RAS-related protein Rab-8B, RAS-related protein Rab-8A, RAS-related protein Rab-10, ADPribosylation factor 1) and d)-defense activities (Myeloperoxidase, Cathepsin G, Lysozyme C, Plasma serine protease inhibitor, Alpha-2-macroglobulin). It is interesting to note that all groups are somehow related to monocyte-macrophage transition since differentiation is triggered by mitochondrial functions, induces a dramatic rearrangement of cytoskeleton due to increasing motility and enhances vesicular trafficking for more intense intra- and intercellular interactions, these including also defense strategies.
Indeed, mitochondrial functions produce Reactive Oxygen Species (ROS), which are recognized triggers of monocyte activation [22]. Normally, the diffusion of ROS and damaged mitochondrial contents into the cytosol is prevented by autophagy. During autophagy cytosolic constituents are enclosed within a double-layered lipid vesicle addressed to fuse with lysosomes for degradation and recycling of the internal contents. Impaired autophagy may interfere with mitochondrial turnover [23].
In brief, the extensive down regulation of mitochondrial enzymes and vesicular trafficking agents along with cytoskeletal and defense proteins in monocyte proteome (Figure 3) suggest a reduced activation ability of monocytes from TD homozygous patient.
As expected, a lower number of proteins were identified in the secretome (128) and of these only 16 resulted differentially released within 24 h (Table 3). The majority (8 out of 16) are actin binding or related factors, underlining a reassessment of monocyte cytoskeleton.
Conclusion
In conclusion, it was possible to obtain a preliminary plasma fingerprint and a monocyte molecular map of Tangier disease. This study could be preparatory for deeper and more specific analyses that could help to understand general mechanisms of lipid transport and metabolism and their linkage to inflammatory processes.
Limitation of this work is the difficulty to establish if the results obtained with a TD patient can be extrapolated to other TD patients, due to the infrequence of this genetic disorder. Nevertheless, this case can be exploited as model for the study of general, common mechanisms in cell biology.
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