A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics : the case of NPC1 deficiency

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions

Superparamagnetic nanoparticles based isolation of subcellular compartments as a method to identify spatial alterations in protein and lipid composition

Summary The more than ~25,000 human genes give rise to many more proteins, a.o. by alternative splicing. Genome-wide approaches using database annotations attempt to reveal patterns of protein interactions, however generally ignore the protein locations. Mass spectrometry handles a huge number of proteins ranging widely in abundance, yet its resolution can be enhanced by analyzing purified organellar fractions. This proposal aims to generate proteome inventories of plasma membranes (PM) and endosomes in a disease-related context. Classical purification schemes cannot be used as disease-related mutations often alter the physical parameters used for organellar isolation. This caveat is circumvented by administering surface-coated superparamagnetic iron oxide nanoparticles (SPIONs). We have developed lipid-coated SPIONs which target the cell surface and allow purification of PM. By exploring the PM proteome we will search for stage-specific or unique expression of channels, receptors or adhesion molecules that may become novel targets or early biomarkers in disease. SPION variants will further be optimized to load endosomes. Coupling biomolecules (ligands, toxins, drugs) combined with pulse-chase experiments will allow isolating distinct endosome populations. Many diseases including neurodegenerative and metabolic diseases originate from a block in endosomal trafficking and degradation although the causal genes are not always identified. By comparing the endosomal proteomes in patient cell lines with control, we aim to identify aberrant protein expression patterns from which causal genes and/or novel biomarkers could be identified. Production and QC of SPIONs is done at IMEC and VIB11. Mass spectrometry driven proteomics to catalogue organellar proteomes is performed at VIB9.nrpages: 203status: publishe

Organellar omics : a reviving strategy to untangle the biomolecular complexity of the cell

A eukaryotic cell encompasses many membrane-enclosed organelles, each of these holding several types of biomolecules that exhibit tremendous diversity in terms of their localization and expression. Despite the development of increasingly sensitive analytical tools, the enormous biomolecular complexity that exists within a cell cannot yet be fully resolved as low abundant molecules often remain unrecognized. Moreover, a drawback of whole cell analysis is that it does not provide spatial information and therefore it is not capable of assigning distinct biomolecules to specific compartments or analyzing changes in the composition of these compartments. Reduction of the biomolecular complexity of a sample helps to identify low abundant molecules, but such a reductionist approach requires methods that enable proper isolation and purification of individual cellular organelles. Decades of research have led to the development of a plethora of isolation methods for a broad range of subcellular organelles; yet, in particular, intrinsically dynamic compartments belonging to the endocytic machinery, including the plasma membrane, remain difficult to isolate in a sufficiently pure fraction. In this review, we discuss various methods that are commonly used to isolate subcellular organelles from cells and evaluate their advantages and disadvantages

Restricted location of PSEN2/γ-secretase determines substrate specificity and generates an intracellular Aβ pool

gamma-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing gamma-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this gamma-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular A beta that contains longer A beta; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer A beta further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of gamma-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone A beta 42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis