Probing the cathepsin D using a BODIPY FL–pepstatin A: applications in fluorescence polarization and microscopy

Monocyte-derived dendritic cells (DC) possess the unique capacity to capture Ag from live cells through intimate cell contact, a process referred to as nibbling. We sought to define the receptor(s) mediating DC nibbling. Uptake of fluorescently labeled plasma membrane from live cells by DC was inhibited by protease treatment and by a panel of polyanionic ligands, implicating scavenger receptors (SR) in this process. Differential expression of SR on DC and macrophages correlated with the capacity to acquire membrane from live cells. Internalized membrane colocalized with SR ligand and entered the endosomal pathway. DC very efficiently acquired and internalized gp100 tumor Ag expressed at the surface of viable adenocarcinoma cells via recombinant adenoviral infection. Cross-presentation of gp100 by DC to MHC class I-restricted T cells was inhibited by polyanionic SR ligand and an Ab to type A SR (SR-A), whereas Ab to the class B SR CD36, which mediates uptake of apoptotic cells, induced no inhibition. DC capture of fluorescently labeled membrane from live cells was partially blocked by SR-A-specific Ab, suggesting that other SR may also be contributing to nibbling. DC maturation resulted in a switch in expression from type II SR-A (SR-AII) to the SR-AI splice variant. Finally, SR-A was identified on interdigitating DC isolated from monkey lymph nodes. These findings define a novel role for SR-A, and suggest that Ag uptake from live cells by DC may be important in the generation of immunity and in the maintenance of peripheral tolerance in vivo.

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“…4, middle row). In addition, TMRM colocalized with pepstatin A, which binds cathepsin D in late endosomes (27) (Fig. 4, lower row).…”

Section: Subcellular Localization Of Captured Ag In DCmentioning

confidence: 93%

A Role for Class A Scavenger Receptor in Dendritic Cell Nibbling from Live Cells

Harshyne

Zimmer

Watkins

et al. 2003

The Journal of Immunology

165

153

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“…BODIPY FL-pepstatin A is pepstatin A (isovaleryl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid), covalently conjugated with the Boron dipyrromethene difluoride fluorophore (BODIPY). Following treatment, the incorporated fluorophore was examined with a confocal microscope (LSM-510 META, Zeiss), as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

Endocannabinoids Prevent β-Amyloid-mediated Lysosomal Destabilization in Cultured Neurons

Noonan

Tanveer

Klompas

et al. 2010

Journal of Biological Chemistry

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Neuronal cell loss underlies the pathological decline in cognition and memory associated with Alzheimer disease (AD).Recently, targeting the endocannabinoid system in AD has emerged as a promising new approach to treatment. Studies have identified neuroprotective roles for endocannabinoids against key pathological events in the AD brain, including cell death by apoptosis. Elucidation of the apoptotic pathway evoked by ␤-amyloid (A␤) is thus important for the development of therapeutic strategies that can thwart A␤ toxicity and preserve cell viability. We have previously reported that lysosomal membrane permeabilization plays a distinct role in the apoptotic pathway initiated by A␤. In the present study, we provide evidence that the endocannabinoid system can stabilize lysosomes against A␤-induced permeabilization and in turn sustain cell survival. We report that endocannabinoids stabilize lysosomes by preventing the A␤-induced up-regulation of the tumor suppressor protein, p53, and its interaction with the lysosomal membrane. We also provide evidence that intracellular cannabinoid type 1 receptors play a role in stabilizing lysosomes against A␤ toxicity and thus highlight the functionality of these receptors. Given the deleterious effect of lysosomal membrane permeabilization on cell viability, stabilization of lysosomes with endocannabinoids may represent a novel mechanism by which these lipid modulators confer neuroprotection. Alzheimer disease (AD)2 is a debilitating illness of the brain defined by the progressive deterioration of cognition and memory as a result of selective neuronal loss in the hippocampus and surrounding areas of the cerebral cortex (1). There is substantial evidence to suggest that at least a subset of neurons in the AD brain die by apoptosis (2). The principal neuropathological hallmark of the disease, ␤-amyloid peptide (A␤), has been shown to induce apoptosis in neuronal cells in vivo and in vitro (3, 4) through a variety of enzymatic pathways that include activation of caspase-3 (5), calpain (6, 7), and lysosomal cathepsins (8, 9).Recently, the lysosomal system has been implicated in AD pathogenesis (9, 10). Neurons of AD patients demonstrate alterations in the lysosomal system, including the cellular pathways that converge on it, namely endocytosis and autophagy (10, 11). Such alterations include an increase in the size and number of endosomes (10, 12), autophagosomes (13) and lysosomes (10) and an increase in the gene expression and synthesis of all classes of lysosomal hydrolases, including cathepsins (14). In addition to their role in the digestion of cellular waste, it has become clear that partial and selective lysosomal membrane permeabilization (LMP), followed by the release of lysosomal enzymes into the cytosol, can induce apoptotic cell death (15). Cathepsins D and L are among the lysosomal proteases that have been implicated in apoptosis by virtue of their ability to activate apoptotic effectors, such as mitochondrial uncoupling and caspases (16).Among the agents that are ca...

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“…4D, n=5, P>0.6). We have also labelled lysosomes with fluorescent pepstatin A (Molecular Probes), a fluorescent probe for cathepsin D based on a selective cathepsin D inhibitor, which is a major lysosomal aspartic endopeptidase (Chen et al, 2000). We have shown that GPN, both alone or in the presence of CI-1, destroyed lysosomal integrity (Fig.…”

Section: + Poolmentioning

confidence: 99%

NAADP, cADPR and IP3 all release Ca2+ from the endoplasmic reticulum and an acidic store in the secretory granule area

Gerasimenko

Sherwood

Tepikin

et al. 2006

Journal of Cell Science

153

159

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Probing the cathepsin D using a BODIPY FL–pepstatin A: applications in fluorescence polarization and microscopy

Cited by 97 publications

References 24 publications

A Role for Class A Scavenger Receptor in Dendritic Cell Nibbling from Live Cells

A Role for Class A Scavenger Receptor in Dendritic Cell Nibbling from Live Cells

Endocannabinoids Prevent β-Amyloid-mediated Lysosomal Destabilization in Cultured Neurons

NAADP, cADPR and IP3 all release Ca2+ from the endoplasmic reticulum and an acidic store in the secretory granule area

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