A Novel Strategy to Identify Drugs that Interfere with Endosomal Lipids

Lipids are major components of the cell and, like proteins, exhibit much diversity and are highly regulated. And yet, our knowledge of lipids remains limited primarily because their study is difficult. We will use novel Systems Biology approaches, and in particular high content screening techniques, to investigate the mechanisms that regulate the cellular lipid content and function. Our project is to carry out a small compound screen using lipid imaging techniques to identify conditions that interfere with cellular levels and distribution of cholesterol, lysobisphosphatic acid and phosphoinositol-3-phosphate. This forward chemical genetic screen approach should reveal new molecular tools to investigate the molecular mechanism involved in the regulation of these lipids. The aim is to apply chemical proteomic techniques to identify the molecular target(s) of compounds able to affect the intracellular cholesterol regulation and to assess if these are novel druggable targets. This will be the ideal complementary study to the RNAi screen, currently run in our group, as the effect of the inhibition caused by a small molecule can be rapidly reversed when this is removed. Such a small molecule can be administered to a cell or an animal for a very short time to study the function of the target protein and to look at biological mechanisms in a short time-frame. This project is highly interdisciplinary, and will benefit from the help of the screening core facility, currently developed with the support of the NCCR

Sonic Hedgehog Induces the Segregation of Patched and Smoothened in Endosomes

AbstractBackground: Sonic hedgehog (Shh) signal transduction involves the ligand binding Patched1 (Ptc1) protein and a signaling component, Smoothened (Smo). A select group of compounds inhibits both Shh signaling, regulated by Ptc1, and late endosomal lipid sorting, regulated by the Ptc-related Niemann-Pick C1 (NPC1) protein. This suggests that Ptc1 regulates Smo activity through a common late endosomal sorting pathway also utilized by NPC1. During signaling, Ptc accumulates in endosomal compartments, but it is unclear if Smo follows Ptc into the endocytic pathway.Results: We characterized the dynamic subcellular distributions of Ptc1, Smo, and activated Smo mutants individually and in combination. Ptc1 and Smo colocalize extensively in the absence of ligand and are internalized together after ligand binding, but Smo becomes segregated from Ptc1/Shh complexes destined for lysosomal degradation. In contrast, activated Smo mutants do not colocalize with nor are cotransported with Ptc1. Agents that block late endosomal transport and protein sorting inhibit the ligand-induced segregation of Ptc1 and Smo. We show that, like NPC1-regulated lipid sorting, Shh signal transduction is blocked by antibodies that specifically disrupt the internal membranes of late endosomes, which provide a platform for protein and lipid sorting.Conclusions: These data support a model in which Ptc1 inhibits Smo only when in the same compartment. Ligand-induced segregation allows Smo to signal independently of Ptc1 after becoming sorted from Ptc1/Shh complexes in the late endocytic pathway

Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation

The phospholipid-binding annexin A2 (AnxA2) is known to play a role in the regulation of membrane and actin dynamics, in particular in the endocytic pathway. The protein is present on early endosomes, where it regulates membrane traffic, including the biogenesis of multivesicular transport intermediates destined for late endosomes. AnxA2 membrane association depends on the protein N terminus and membrane cholesterol but does not involve the AnxA2 ligand p11/S100A10. However, the precise mechanisms that control AnxA2 membrane association and function are not clear. In the present study, we have investigated the role of AnxA2 N-terminal phosphorylation in controlling association to endosomal membranes and functions. We found that endosomal AnxA2 was partially tyrosine-phosphorylated and that mutation of Tyr-23 to Ala (AnxA2Y23A), but not of Ser-25 to Ala, impaired AnxA2 endosome association. We then found that the AnxA2Y23A mutant was unable to bind endosomes in vivo, whereas a phospho-mimicking AnxA2 mutant (Y23D) showed efficient endosome binding capacity. Similarly, we found that AnxA2Y23D interacted more efficiently with liposomes in vitro when compared with AnxA2Y23A. To investigate the role of Tyr-23 in vivo, AnxA2 was knocked down with small interfering RNAs, and then cells were recomplemented with RNA interference-resistant forms of the protein. Using this strategy, we could show that AnxA2Y23D, but not AnxA2Y23A, could restore early-to-late endosome transport after AnxA2 depletion. We conclude that phosphorylation of Tyr-23 is essential for proper endosomal association and function of AnxA2, perhaps because it stabilizes membrane-associated protein via a conformational change

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3′(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation

Secondary structure and 1H, 13C, 15N resonance assignments of the endosomal sorting protein sorting nexin 3

Sorting nexin 3 (SNX3) belongs to a sub-family of sorting nexins that primarily contain a single Phox homology domain capable of binding phosphoinositides and membranes. We report the complete (1)H, (13)C and (15)N resonance assignments of the full-length human SNX3 protein and identification of its secondary structure elements, revealing a canonical fold and unstructured termini

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility - two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to-exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex

Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation

Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking

Background Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Methodology/Principal Findings Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. Conclusions/Significance These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation