Lysosomal Degradation on Vesicular Membrane Surfaces

Wilkening, Gundo; Linke, Thomas; Sandhoff, Konrad

doi:10.1074/jbc.273.46.30271

Cited by 141 publications

(63 citation statements)

References 29 publications

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“…Thus, sapC is effectively increasing the amount of substrate that is available for hydrolysis in this assay. There is a 10-fold increase in the initial rate of hydrolysis at 2.5 M sapC, which is similar to the Ϸ7-fold increase in GCase activity when incubated with the same concentration of sapC and liposomes containing radiolabeled GlcCer (12). No substrate hydrolysis was observed in the absence of GCase, regardless of the saposin concentration.…”

supporting

confidence: 54%

“…However, the same preparations were shown to contain considerable amounts of lipids (35).] Acidic pH and the presence of the negatively charged lipids 1,2-diacyl-sn-3-phosphoinositol (PI) or bis(monoacylglycero)phosphate (BMP), which are abundant in intralysosomal compartment (36), are required for the activation of GCase activity by sapC (12,27). In our DFUG assay, the activation of GCase by sapC was maximal in the pH range 4.8-5.0 and required PI or BMP (SI Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Different saposins and enzymes are hypothesized to fall into a particular category of enzyme activation. For example, saposin B is thought to be a detergent-like lipid solubilizer (9,10), whereas sapC may act as a liftase at the bilayer surface (2,11,12). Strong binding of sapC to lipid bilayers was shown by using coprecipitation assays (13) and NMR spectroscopic titrations (14).…”

mentioning

confidence: 99%

“…We used a membrane-bound fluorogenic substrate analog to image and assay GCase activity in bilayers. The compound 6,8-dif luoro-4-heptadecylumbelliferyl ␤-Dglucopyranoside (DFUG) is similar to soluble umbelliferylbased glucosides used as GCase substrates in previous studies (12,27,28) but contains a C 17 acyl chain that anchors the molecule to the membrane. DFUG is an excellent analogue of the natural GlcCer substrate for measuring membrane-bound GCase activity, because hydrolysis of the glucoside headgroup yields the f luorescent compound 6,8-dif luoro-7-hydroxy-4-heptadecylcoumarin (DFHU), which also remains in the lipid bilayer.…”

mentioning

confidence: 99%

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Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

Alattia

Shaw²,

Yip³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Acid ␤-glucosidase (GCase) is a soluble lysosomal enzyme responsible for the hydrolysis of glucose from glucosylceramide and requires activation by the small nonenzymatic protein saposin C (sapC) to gain access to the membrane-embedded glycosphingolipid substrate. We have used in situ atomic force microscopy (AFM) with simultaneous confocal and epifluorescence microscopies to investigate the interactions of GCase and sapC with lipid bilayers. GCase binds to sites on membranes transformed by sapC, and enzyme activity occurs at loci containing both GCase and sapC. Using FRET, we establish the presence of GCase/sapC and GCase/ product contacts in the bilayer. These data support a mechanism in which sapC locally alters regions of bilayer for subsequent attack by the enzyme in stably bound protein complexes.atomic force microscopy ͉ confocal microscopy ͉ FRET ͉ interfacial catalysis ͉ lipid storage disease

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supporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

Alattia

Shaw²,

Yip³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Although the detailed mechanisms of SAPLIP-mediated enzyme activation are unknown, it is generally assumed that these proteins remodel the membrane by interacting with lipids; they are also able to interact directly with lipid degrading enzymes, thus providing a platform for lipid hydrolases. [23] SAPLIPs are crucial molecules for the survival of many organisms. For instance, SAPLIP-deficient Caenorhabditis elegans is overcome by bacteria in culture, [24] because the worm is unable to induce bacterial lysis.…”

mentioning

confidence: 99%

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

et al. 2014

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1Mason et al. Structures of parasite proteinsNew interventions against infectious diseases require a detailed knowledge and understanding of pathogen-host interactions and pathogeneses at the molecular level. The combination of the considerable advances in systems biology research with methods to explore the structural biology of molecules is poised to provide new insights into these areas. Importantly, exploring threedimensional structures of proteins is central to understanding disease processes, and establishing structure-function relationships assists in identification and assessment of new drug and vaccine targets. Frequently, the molecular arsenal deployed by invading pathogens, and in particular parasites, reveals a common theme whereby families of proteins with conserved three-dimensional folds play crucial roles in infectious processes, but individual members of such families show high levels of specialisation which is often achieved through grafting particular structural features onto the shared overall fold. Accordingly, the applicability of predictive methodologies based on the primary structure of proteins or genome annotations is limited, particularly when thorough knowledge of molecular level mechanisms is required. Such instances exemplify the need for experimental threedimensional structures provided by protein crystallography, which remain an essential component of this area of research. In the present article, we review two examples of key protein families recently investigated in our laboratories, which could represent intervention targets in the metabolome or secretome of parasites. IntroductionInfectious diseases caused by eukaryotic pathogens represent a major disease burden to humans world-wide. In the absence of effective preventative approaches and new intervention strategies, this burden is likely to increase further, [1,2] compounded by food and water shortages, economic crises, wars and climate change, particularly in disadvantaged countries. In addition, there is evidence of increasing problems with resistance in pathogens against a broad range of chemotherapeutic agents, compromising the treatment of infectious diseases. [3] Moreover, in spite of major efforts, there are very few examples of success in developing new drugs and vaccines against eukaryotic pathogens, [4,5] indicating that alternative methods are needed at a time of major advances in molecular and computer technologies. In our opinion, the development of new treatments requires a detailed understanding of pathogens, host interactions and the molecular processes of disease. For instance, knowledge of the three-dimensional structures of proteins with pivotal roles in disease, and the probing of their underlying biology are crucial for understanding the pathogenesis. Through establishing the relevant structure-function relationships, one can elucidate how individual proteins function, so that new ways of disrupting relevant pathways can be found, in order to facilitate the identification of new drug and vaccine targets. ...

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Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry

Boulla¹,

Randriamampita²,

Raposo³

et al. 2000

Cytometry

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Lysosomal Degradation on Vesicular Membrane Surfaces

Cited by 141 publications

References 29 publications

Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

Molecular imaging of membrane interfaces reveals mode of β-glucosidase activation by saposin C

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry

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