scholarly journals Rab7: A Key to Lysosome Biogenesis

2000 ◽  
Vol 11 (2) ◽  
pp. 467-480 ◽  
Author(s):  
Cecilia Bucci ◽  
Peter Thomsen ◽  
Paolo Nicoziani ◽  
Janice McCarthy ◽  
Bo van Deurs
Keyword(s):  
Fluorescent Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Mutant Proteins ◽  
Lysosome Biogenesis ◽  
Late Endosomes ◽  
Early Endosomes ◽  
The Individual

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)–tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein–1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and thetrans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.

LDL Receptor Mediates Effective Endocytosis of Activated Factor V.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1766-1766 ◽  
Author(s):  
Alexander B. Meijer ◽  
Sigrid D. Roosendaal ◽  
Vincent Limburg ◽  
Carmen van der Zwaan ◽  
Kees W. Rodenburg ◽  
...  
Keyword(s):  
Cho Cells ◽  
Fluorescent Protein ◽  
Low Density Lipoprotein ◽  
Yellow Fluorescent Protein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Functional Expression ◽  
Factor V ◽  
Early Endosomes ◽  
Density Lipoprotein Receptor

Abstract The platelet α-granules contain a unique pool of partially activated factor V, which has been suggested to originate form megakaryocyte endocytosis of factor V (FV) from plasma. The presence of activated FV (FVa) in plasma itself should be tightly controlled as dysfunction therein may predispose to thrombotic disorders. Previously, we have reported that FVa but not FV can bind the low-density lipoprotein receptor related protein (LRP). This multifunctional receptor can bind a multitude of ligands including FV’s homologue factor VIII (FVIII). We now investigated whether FV or FVa can, like FVIII, also bind the more restricted LDL receptor (LDLR). To this end, the endocytosis of FV and FVa by CHO cells expressing LDLR (CHO-LDLR+ cells) was assessed utilizing confocal microscopy. In the experimental setup, FV and FVa were visualized employing immuno-fluorescence staining techniques. The results showed that within 10 minutes after addition of FVa, fluorescent spots appeared inside the CHO-LDLR+ cells. In contrast, no fluorescent spots were observed after 10 minutes of incubation with FV. These observations suggest that FVa but not FV effectively interacts with the LDLR expressing CHO cells. We then assessed whether FVa can compete with FVIII for endocytosis by CHO-LDLR+ cells. In the presence of an equimolar amount of FVa and a FVIII derivative (FVIIIYFP) containing yellow fluorescent protein, both proteins were detected within the same vesicles inside the CHO-LDLR+ cells. Employing co-localization studies, we established that these vesicles represented early endosomes. In the presence of an access of FVa, however, the yellow fluorescence of FVIIIYFP was no longer observed. These results demonstrate that FVa can block the FVIII endocytosis by CHO-LDLR+ cells. The observations together suggest that LDLR can not only bind FVIII but also FVa. In line with this notion, we established that CHO-ldlA cells, which lack functional expression of LDLR, were unable to internalize neither FVa nor FVIIIYFP. In addition, the ligand-binding clusters II and cluster IV of LRP effectively inhibited the endocytic uptake of FVa by CHO-LDLR+ cells. This implies that structural elements of FVa involved in LRP binding may overlap with those required for LDLR dependent internalization. Our results suggest a so far unidentified role for members of the LDL receptor family in the regulation of FVa in plasma.

scholarly journals Rapid Transport of Internalized P-Selectin to Late Endosomes and the Tgn

2000 ◽  
Vol 151 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Kimberly S. Straley ◽  
Samuel A. Green
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Endosomal Sorting ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Granule Membrane

Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20–25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.

scholarly journals The Low-Density Lipoprotein Receptor Is Regulated by Estrogen and Forms a Functional Complex with the Estrogen-Regulated Protein Ezrin in Pituitary GH3 Somatolactotropes

Endocrinology ◽  
2004 ◽  
Vol 145 (7) ◽  
pp. 3075-3083 ◽  
Author(s):  
Perry M. Smith ◽  
Ann Cowan ◽  
Bruce A. White
Keyword(s):  
Fluorescent Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Transient Transfection ◽  
Dominant Negative ◽  
Gh3 Cells ◽  
Low Density ◽  
Ldl Particles ◽  
N Terminus ◽  
Ldl Uptake

Abstract Estrogen regulates the function, growth, and proliferation of lactotropes in the pituitary. We report here that low-density lipoprotein (LDL) receptor (LDLR) gene expression and LDL uptake are strongly up-regulated by estrogen in pituitary somatolactotropic GH3 cells. The uptake of LDL was significantly inhibited by the F-actin-severing drug, swinholide A, indicating that LDL uptake is dependent on the integrity of the cortical actin cytoskeleton in GH3 cells. We examined whether the estrogen-inducible cytoskeletal linker protein, ezrin, interacts with the LDLR. The LDLR coimmunoprecipitated with ezrin, and fluorescently labeled LDL bound to regions of the cell membrane that colocalized with the active, phosphorylated form of ezrin (phosphoezrin). Evidence for a functional interaction between ezrin and the LDLR was obtained by transient transfection experiments using ezrin-green fluorescent protein (GFP) expression constructs. We observed that transient transfection of GH3 cells with an ezrin N terminus-GFP dominant-negative construct prevented the uptake of LDL particles, whereas expression of GFP alone or an ezrin C terminus-GFP construct had no effect on LDL uptake. Transfection with the ezrin N terminus dominant- negative construct had no effect on the endocytosis of transferrin. Thus, estrogen stimulates the expression of ezrin and the LDLR in GH3 cells, which interact physically and functionally to facilitate the endocytosis of LDL. We propose that the up-regulation and interaction of ezrin and the LDLR serves to augment the delivery of cholesterol and other lipids in support of the hypertrophic and proliferative response of cells to estrogen.

scholarly journals Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes.

1997 ◽  
Vol 8 (3) ◽  
pp. 517-532 ◽  
Author(s):  
R P Czekay ◽  
R A Orlando ◽  
L Woodward ◽  
M Lundstrom ◽  
M G Farquhar
Keyword(s):  
Gene Family ◽  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Density Lipoprotein Receptor ◽  
Receptor Gene Family

Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.

scholarly journals Application of a New Processing Method to Post-LDL-apheresis Data

2002 ◽  
Vol 4 (3) ◽  
pp. 191-196 ◽  
Author(s):  
Franco Corsini ◽  
Arrigo F. G. Cicero ◽  
Antonia Giannuzzi ◽  
Antonio Gaddi
Keyword(s):  
Relative Error ◽  
Single Case ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Direct Consequence ◽  
Ldl Apheresis ◽  
Theoretical Function ◽  
New Processing ◽  
The Individual ◽  
Estimate Standard Error

Our aim was to elaborate a method to optimise treatment intervals for the individual low-density lipoprotein (LDL)-apheresis treated patients. After each treatment, plasma LDL concentrations show a time-related increase with a decreasing speed until a maximum level.We searched to interpret the post-LDL-apheresis experimental data trend as the physical process that produces the observed curve, so that the fitting presupposed theoretical function is a direct consequence of the physic process, because to establish the better time. Applying the proposed fitting method to a succession of 15 samples obtained from the mean of six plasmapheresis executed on five different subjects, small estimate standard error (5 mg/dl) and relative error (1.7%) with a dispersion evidently related to the experimental error were observed. Obviously, applying the same method to a single case, the dispersion is more marked (relative error <5%), with a SE of 10-13 mg/dl, even though the aspect of a casual phenomenon is conserved. Our physical interpretation appears to be a practical model to predict the LDL-rebound kinetic of the single patient.

scholarly journals Hepatitis C virus entry into the hepatocyte

2011 ◽  
Vol 6 (6) ◽  
pp. 933-945 ◽  
Author(s):  
Sandrine Belouzard ◽  
Laurence Cocquerel ◽  
Jean Dubuisson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Current Knowledge ◽  
Virus Entry ◽  
Low Density Lipoprotein ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
Surface Proteins ◽  
Enveloped Virus ◽  
Early Endosomes

AbstractHepatitis C virus (HCV) is a small enveloped virus with a positive stranded RNA genome belonging to the Flaviviridae family. The virion has the unique ability of forming a complex with lipoproteins, which is known as the lipoviroparticle. Lipoprotein components as well as the envelope proteins, E1 and E2, play a key role in virus entry into the hepatocyte. HCV entry is a complex multistep process involving sequential interactions with several cell surface proteins. The virus relies on glycosaminoglycans and possibly the low-density lipoprotein receptors to attach to cells. Furthermore, four specific entry factors are involved in the following steps which lead to virus internalization and fusion in early endosomes. These molecules are the scavenger receptor SRB1, tetraspanin CD81 and two tight junction proteins, Claudin-1 and Occludin. Although they are essential to HCV entry, the precise role of these molecules is not completely understood. Finally, hepatocytes are highly polarized cells and which likely affects the entry process. Our current knowledge on HCV entry is summarized in this review.

scholarly journals Scavenger receptors: a key player in cardiovascular diseases

2012 ◽  
Vol 3 (4) ◽  
pp. 371-380 ◽  
Author(s):  
Mohammad Z. Ashraf ◽  
Anita Sahu
Keyword(s):  
Cardiovascular Diseases ◽  
Apoptotic Cell ◽  
Low Density Lipoprotein ◽  
Scavenger Receptor ◽  
Density Lipoprotein ◽  
Vascular Lesions ◽  
Great Promise ◽  
Term Therapy ◽  
Membrane Glycoproteins ◽  
The Individual

AbstractThe scavenger receptor (SR) super family consists of integral membrane glycoproteins that are involved in recognition of polyanionic structures of either endogenous (e.g., oxidized low-density lipoprotein) or exogenous (e.g., bacterial lipopolysaccharides) origin. SRs are structurally diverse and can be classified into seven different classes (A–G) based on the multidomain structure of the individual members. SRs are present on various types of tissues, such as vascular, adipose, and steroidogenic tissues. In addition to modified lipoprotein uptake, these proteins are also known to regulate apoptotic cell clearance, initiate signal transduction, and serve as pattern recognition receptors for pathogens. Different SRs are involved in many physiological and pathological processes; more importantly, the function of SRs is highly implicated in the initiation and progression of atherosclerotic plaque. Targeting the SR gene products that mediate the response to and uptake of modified lipids holds great promise in the prevention of cardiovascular diseases. Inhibition of SR expression using a combined gene therapy and RNA interference strategy also appears to be an option for long-term therapy. The present review focuses on the involvement of SRs in atherosclerosis, thrombosis, and other cardiovascular diseases. Moreover, the role of SRs is not restricted to vascular lesions; it is also implicated in a number of different cellular functions.

scholarly journals Triglyceride-containing lipoprotein sub-fractions and risk of coronary heart disease and stroke: A prospective analysis in 11,560 adults

2020 ◽  
Vol 27 (15) ◽  
pp. 1617-1626 ◽  
Author(s):  
Roshni Joshi ◽  
S Goya Wannamethee ◽  
Jorgen Engmann ◽  
Tom Gaunt ◽  
Deborah A Lawlor ◽  
...  
Keyword(s):  
Coronary Heart Disease ◽  
Heart Disease ◽  
Odds Ratio ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipoprotein Cholesterol ◽  
Increased Risk ◽  
The Individual

Aims Elevated low-density lipoprotein cholesterol (LDL-C) is a risk factor for cardiovascular disease; however, there is uncertainty about the role of total triglycerides and the individual triglyceride-containing lipoprotein sub-fractions. We measured 14 triglyceride-containing lipoprotein sub-fractions using nuclear magnetic resonance and examined associations with coronary heart disease and stroke. Methods Triglyceride-containing sub-fraction measures were available in 11,560 participants from the three UK cohorts free of coronary heart disease and stroke at baseline. Multivariable logistic regression was used to estimate the association of each sub-fraction with coronary heart disease and stroke expressed as the odds ratio per standard deviation increment in the corresponding measure. Results The 14 triglyceride-containing sub-fractions were positively correlated with one another and with total triglycerides, and inversely correlated with high-density lipoprotein cholesterol (HDL-C). Thirteen sub-fractions were positively associated with coronary heart disease (odds ratio in the range 1.12 to 1.22), with the effect estimates for coronary heart disease being comparable in subgroup analysis of participants with and without type 2 diabetes, and were attenuated after adjustment for HDL-C and LDL-C. There was no evidence for a clear association of any triglyceride lipoprotein sub-fraction with stroke. Conclusions Triglyceride sub-fractions are associated with increased risk of coronary heart disease but not stroke, with attenuation of effects on adjustment for HDL-C and LDL-C.

Biological variability of cholesterol, triglyceride, low- and high-density lipoprotein cholesterol, lipoprotein(a), and apolipoproteins A-I and B

1994 ◽  
Vol 40 (4) ◽  
pp. 574-578 ◽  
Author(s):  
S M Marcovina ◽  
V P Gaur ◽  
J J Albers
Keyword(s):  
High Density Lipoprotein ◽  
Ldl Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Hdl Cholesterol ◽  
High Density ◽  
Biological Variability ◽  
Apo B ◽  
Lipoprotein A ◽  
The Individual

Abstract Biological variability is a major contributor to the inaccuracy of cardiovascular risk assessments based on measurement of lipids, lipoproteins, or apolipoproteins. We obtained estimates of biological variation (CVb) for 20 healthy adults and calculated the percentiles of CVb as an expression of the variability of CVb among individuals for cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein (apo) A-I, apo B, and lipoprotein(a) [Lp(a)] by four biweekly measurements of these analytes. The CVb for the group was approximately 6-7% for cholesterol, HDL cholesterol, apo A-I, and apo B; approximately 9% for LDL cholesterol; and 28% for triglyceride. However, for each analyte, there was a considerable variation of CVb among individuals. For all analytes except Lp(a), there was no relation between the individual's CVb and the analyte concentration. Lp(a) was inversely related to CVb, and there was a very wide variation in the CVb for Lp(a) among the participants, ranging from 1% to 51%. The number of independent analyses to perform to accurately assess an individual's risk for coronary artery disease should be determined on the basis of the individual CVb for a given analyte rather than the average CVb.

scholarly journals A Cellular Receptor of Human Rhinovirus Type 2, the Very-Low-Density Lipoprotein Receptor, Binds to Two Neighboring Proteins of the Viral Capsid

2003 ◽  
Vol 77 (15) ◽  
pp. 8504-8511 ◽  
Author(s):  
Emmanuelle Neumann ◽  
Rosita Moser ◽  
Luc Snyers ◽  
Dieter Blaas ◽  
Elizabeth A. Hewat
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Low Density Lipoprotein Receptor ◽  
The North ◽  
Density Lipoprotein Receptor ◽  
The Individual ◽  
Viral Protein 1

ABSTRACT The very-low-density lipoprotein receptor (VLDL-R) is a receptor for the minor-group human rhinoviruses (HRVs). Only two of the eight binding repeats of the VLDL-R bind to HRV2, and their footprints describe an annulus on the dome at each fivefold axis. By studying the complex formed between a selection of soluble fragments of the VLDL-R and HRV2, we demonstrate that it is the second and third repeats that bind. We also show that artificial concatemers of the same repeat can bind to HRV2 with the same footprint as that for the native receptor. In a 16-Å-resolution cryoelectron microscopy map of HRV2 in complex with the VLDL-R, the individual repeats are defined. The third repeat is strongly bound to charged and polar residues of the HI and BC loops of viral protein 1 (VP1), while the second repeat is more weakly bound to the neighboring VP1. The footprint of the strongly bound third repeat extends down the north side of the canyon. Since the receptor molecule can bind to two adjacent copies of VP1, we suggest that the bound receptor “staples” the VP1s together and must be detached before release of the RNA can occur. When the receptor is bound to neighboring sites on HRV2, steric hindrance prevents binding of the second repeat.

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