A method to assess the lysosomal residence of proteins in cultured cells

Gasingirwa, Marie-Christine; Thirion, Jacqueline; Costa, C. Z. F.; Flamion, Bruno; Lobel, Peter; Jadot, Michel

doi:10.1016/j.ab.2007.11.021

Cited by 7 publications

(4 citation statements)

References 34 publications

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“…The late endosomes/lysosomes were isolated from human fibroblasts using differential and isopycnic sucrose density gradient centrifugation [Gasingirwa et al, 2008]. In brief, cells were rinsed three times with cold PBS, scraped from the plates with cold PBS containing protease inhibitors, and then homogenized with a hypotonic buffer (10 mM Tris pH 8.0, 2 mM EDTA, and protease inhibitors) using a small‐clearance 15‐ml Dounce homogenizer.…”

Section: Methodsmentioning

confidence: 99%

Physiological and coordinate downregulation of the NPC1 and NPC2 genes are associated with the sequestration of LDL‐derived cholesterol within endocytic compartments

Jelínek

Patrick

Kitt

et al. 2009

J of Cellular Biochemistry

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The Niemann-Pick C1 and C2 (NPC1 and NPC2) proteins have a central role in regulating the transport of lipoprotein-derived cholesterol from endocytic compartments to the endoplasmic reticulum for esterification by acyl-CoA:cholesterol acyltransferase (ACAT) and feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway. Since the NPC1 gene/protein has recently been shown to be downregulated by feedback inhibition of the SREBP pathway, the present study was performed to determine whether physiological downregulation of the NPC1 gene/protein alters the transport and metabolism of low-density lipoprotein (LDL)-derived cholesterol in human fibroblasts. To perform this study, three different culture conditions were used that included fibroblasts grown in lipoprotein-deficient serum (LPDS), LPDS supplemented with LDL, and LPDS supplemented with LDL, followed by equilibration in the absence of LDL to allow the transport of LDL-derived cholesterol from endocytic compartments and equilibration of cellular sterol pools. The results from this study indicated that in addition to the NPC1 gene/protein, the NPC2 gene/protein was also downregulated by LDL-derived cholesterol-dependent feedback inhibition and that downregulation of both the NPC1 and NPC2 genes/proteins was associated with the sequestration of LDL-derived cholesterol within endocytic compartments, including late endosomes/lysosomes after equilibration. Therefore, it is proposed that physiological and coordinate downregulation of the NPC1 and NPC2 genes/proteins promotes the sequestration of LDL-derived cholesterol within endocytic compartments and serves a role in maintaining intracellular cholesterol homeostasis.

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Section: Methodsmentioning

confidence: 99%

Physiological and coordinate downregulation of the NPC1 and NPC2 genes are associated with the sequestration of LDL‐derived cholesterol within endocytic compartments

Jelínek

Patrick

Kitt

et al. 2009

J of Cellular Biochemistry

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“…274 Unfortunately, the discovery and identification of lysosomal proteins has been difficult due to the presence of other contaminating organelles (such as mitochondria) in the lysosomal fraction when using conventional s ubcellular fractionation techniques. Using lysosomal density shifting techniques (treatment with either Triton WR-1339 275 or progesterone 276 ), investigators have been able to assign proteins to the lysosome in Wistar rat livers and human hepatocellular carcinoma (HepG2) cells, respectively, that were n ot previously assigned to that organelle. In addition, they were able to identify proteins associated with other organelles in the same fraction that were previously assigned to the lysosome using a quadratic discriminant analysis.…”

Section: Organelle Compositionmentioning

confidence: 99%

Bioanalysis of Eukaryotic Organelles

et al. 2013

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“…Loss of NPC2, a lysosomal cholesterol binding protein, results in an accumulation of cholesterol and other lipids within the lysosome with a concomitant decrease in density [27]. In addition, treatment of cultured cells with progesterone also results in a shift in the buoyant density of lysosomes [28], presumably by blocking cholesterol egress [29]. For each of these methods, the density of other organelles remains largely unchanged, thus the shift in distribution represents a specific test for lysosomal localization.…”

Section: Organellar Proteomics Subcellular Fractionation and Applmentioning

confidence: 99%

Proteomics of the lysosome

Lübke

Lobel²,

Sleat³

2009

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

238

202

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Defects in lysosomal function have been associated with numerous monogenic human diseases typically classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal proteins are also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this is the identification of its constituent proteins by proteomic analyses. To date, the mammalian lysosome has been shown to contain ~ 60 soluble luminal proteins and ~25 transmembrane proteins. However, recent proteomic studies based upon affinity purification of soluble components or subcellular fractionation to obtain both soluble and membrane components suggest that there may be many more of both classes of protein resident within this organelle than previously appreciated. Discovery of such proteins has important implications for understanding the function and the dynamics of the lysosome but can also lead the way towards the discovery of the genetic basis for human diseases of hitherto unknown etiology. Here, we describe current approaches to lysosomal proteomics and data interpretation and review the new lysosomal proteins that have recently emerged from such studies.

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A method to assess the lysosomal residence of proteins in cultured cells

Cited by 7 publications

References 34 publications

Physiological and coordinate downregulation of the NPC1 and NPC2 genes are associated with the sequestration of LDL‐derived cholesterol within endocytic compartments

Physiological and coordinate downregulation of the NPC1 and NPC2 genes are associated with the sequestration of LDL‐derived cholesterol within endocytic compartments

Bioanalysis of Eukaryotic Organelles

Proteomics of the lysosome

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