A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids

The amyloid precursor protein (APP) binds the PIKfyve complex and modulates its function

Phosphoinositides are important components of eukaryotic membranes that are required for multiple forms of membrane dynamics. Phosphoinositides are involved in defining membrane identity, mediate cell signalling and control membrane trafficking events. Due to their pivotal role in membrane dynamics, phosphoinositide de-regulation contributes to various human diseases. In this review, we will focus on the newly emerging regulation of the PIKfyve complex, a phosphoinositide kinase that converts the endosomal phosphatidylinositol-3-phosphate [PI(3)P] to phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2)], a low abundance phosphoinositide of outstanding importance for neuronal integrity and function. Loss of PIKfyve function is well known to result in neurodegeneration in both mousemodels and human patients. Our recent work has surprisingly identified the amyloid precursor protein (APP), the central molecule in Alzheimer s disease aetiology, as a novel interaction partner of a subunit of the PIKfyve complex, Vac14. Furthermore, it has been shown that APP modulates PIKfyve function and PI(3,5)P2 dynamics, suggesting that the APP gene family functions as regulator of PI(3,5)P2 metabolism. The recent advances discussed in this review suggest a novel, unexpected, â-amyloid-independent mechanism for neurodegeneration in Alzheimer s disease

The amyloid precursor protein controls PIKfyve function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease

APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve complex

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer’s disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein–protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer’s disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer’s disease

Overexpression of APL-1 reduces vacuolar pathology caused by hypomorphic PIKfyve complex mutants.

<p>(A, B) Overexpression of APL-1::GFP (established in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref032" target="_blank">32</a>]) significantly reduced the number of vacuoles in PPK-3 partial loss-of-function mutant animals (Wilcoxon rank test, p<0.01, n≥25 per strain). Note that in the case of the stronger hypomorphic ppk-3(n2668) mutant APL-1::GFP expression, while strongly ameliorating the phenotype, failed to fully rescue the vacuolar phenotype.</p

Vac14 associates with the intracellular domain of APP (AICD) biochemically and in HeLa cells.

<p>(A) Proteo-liposome recruitment analysed by mass spectrometry allowed the identification of the intracellular interactome of APP. A Vulcan plot showing that PIKfyve, Vac14 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.g004" target="_blank">Fig 4</a> of the mammalian PIKfyve complex were significantly enriched in AICD proteo-liposomes (right quadrant) compared to controls (left). The dashed line indicates the 0.05 significance threshold. (B) Western blotting confirmed the enrichment of Vac14 by AICD presenting proteo-liposomes while the intracellular domain of the none-related receptor Sortilin and two additional controls (a non-related control peptide designated as 'control 1' or coupled cysteine 'control 2'–both described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref027" target="_blank">27</a>]) failed to bind Vac14, showing that Vac14 specifically associates with the intracellular domain of APP. (C) APP expressed as a C-terminal CFP fusion displayed strong colocalisation with Vac14-mCit in HeLa cells. Limited colocalisation of APP and Vac14 could be observed on early endosomes (labelled with EEA1) and late endosomes/lysosomes labelled with LampI, as indicated by arrows on the insets. Bar, 20 μm. (D) Line scans (indicated by the red line in (C) demonstrated strong colocalisation between APP and Vac14. (E) In live-cell imaging APP fused to mCherry displayed co-movement with Vac14-mCit positive vesicles and tubular carriers (arrows) that track through the cytoplasm. (F, G) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.s007" target="_blank">S1 Video</a>). AICD fused to mCherry also colocalised with Vac14-mCit in live cell imaging (arrows) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.s008" target="_blank">S2 Video</a>). (G) Four individual vesicles positive for both AICD and Vac14 were tracked and the first image of the sequence overlayed with the traces to illustrate comigration of AICD and Vac14. Note that in live cell imaging due to the delay caused by the change of filters on fast moving vesicles the staining in the red and green channels are slightly displaced. Bar, 5μm.</p

APL-1 interacts genetically with the PIKfyve complex genes vacl-14 and ppk-3.

<p>(A) Mutations in apl-1, ppk-3 and vacl-14 led to the formation of vacuoles in the C. elegans intestine and hypoderm (indicated by arrows) apparent in the anterior tips of the worms as observed by Differential Interference Contrast (DIC) microscopy. Combination of apl-1, ppk-3 and vacl-14 mutations in double mutant worms strongly enhanced this phenotype (* indicate very large vacuoles), particularly evident in the apl-1(yn5) ppk-3(n2668) double mutant. (B) Box plots demonstrated that the number of vacuoles in apl-1/ppk-3 and apl-1/vacl-14 double mutants is significantly increased compared to the single mutants (Wilcoxon rank test, p<0.01, n≥20 per strain), demonstrating that APL-1 functionally interacts with the PPK-3 complex. This showed that the C-terminal domain of APL-1 is necessary for suppressing vacuole formation induced by loss of PPK-3 and VACL-14 activity. Bar, 50 μm.</p

APL-1 overexpression, while able to rescue partial loss of PPK-3 function, failed to rescue the ppk-3(mc46) null allele.

<p>(A) Expression of APL-1::GFP failed to rescue lethality of ppk-3 null animals and failed to rescue vacuolation in apl-1::GFP; ppk-3(mc46) animals. Bar, 50μm. (B) Quantification of the relative vacuolated area in ppk-3(mc46) and apl-1::GFP; ppk-3(mc46) animals showed that APL-1 overexpression failed to rescue complete loss of PPK-3 function (n≥38, p = 0.73 (two-tailed t-test), suggesting that APL-1 functions upstream of PPK-3.</p