The polyphenol EGCG directly targets intracellular amyloid-ß aggregates and promotes their lysosomal degradation.

The accumulation of amyloidogenic protein aggregates in neurons is a pathogenic hallmark of a large number of neurodegenerative diseases including Alzheimer's disease (AD). Small molecules targeting such structures and promoting their degradation may have therapeutic potential for the treatment of AD. Here, we searched for natural chemical compounds that decrease the abundance of stable, proteotoxic ß-sheet-rich amyloid-ß (Aß) aggregates in cells. We found that the polyphenol (-)-epigallocatechin gallate (EGCG) functions as a potent chemical aggregate degrader in SH-EP cells. We further demonstrate that a novel, fluorescently labeled EGCG derivative (EGC-dihydroxybenzoate (DHB)-Rhodamine) also shows cellular activity. It directly targets intracellular Aß42 aggregates and competes with EGCG for Aß42 aggregate binding in vitro. Mechanistic investigations indicated a lysosomal accumulation of Aß42 aggregates in SH-EP cells and showed that lysosomal cathepsin activity is critical for efficient EGCG-mediated aggregate clearance. In fact, EGCG treatment leads to an increased abundance of active cathepsin B isoforms and increased enzymatic activity in our SH-EP cell model. Our findings suggest that intracellular Aß42 aggregates are cleared through the endo-lysosomal system. We show that EGCG directly targets intracellular Aß42 aggregates and facilitates their lysosomal degradation. Small molecules, which bind to protein aggregates and increase their lysosomal degradation could have therapeutic potential for the treatment of amyloid diseases.

Sclerotiorin Stabilizes the Assembly of Nonfibrillar Abeta42 Oligomers with Low Toxicity, Seeding Activity, and Beta-sheet Content.

The self-assembly of the 42-residue amyloid-ß peptide, Aß42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aß42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aß42 oligomers. These structures exhibit low ß-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, ß-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aß42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aß42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aß42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, ß-sheet-rich Aß42 fibrils in AD, while structures with low ß-sheet content may be less relevant.

The Anti-amyloid Compound DO1 Decreases Plaque Pathology and Neuroinflammation-Related Expression Changes in 5xFAD Transgenic Mice.

Self-propagating amyloid-ß (Aß) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aß42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aß42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aß42 peptides and decreases the seeding activity of Aß aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aß plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aß structures with small molecules as a valid therapeutic strategy.

mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease.

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells.

Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC). The double-readout procedure detects interactions with higher sensitivity than traditional single-readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease-causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult-onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease-causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease-associated mutations impair protein activity in biological systems.

A Filter Retardation Assay Facilitates the Detection and Quantification of Heat-Stable, Amyloidogenic Mutant Huntingtin Aggregates in Complex Biosamples.

N-terminal mutant huntingtin (mHTT) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously form stable, amyloidogenic protein aggregates with a fibrillar morphology. Such structures are detectable in brains of Huntington's disease (HD) patients and various model organisms, suggesting that they play a critical role in pathogenesis. Heat-stable, fibrillar mHTT aggregates can be detected and quantified in cells and tissues using a denaturing filter retardation assay (FRA). Here, we describe step-by-step protocols and experimental procedures for the investigation of mHTT aggregates in complex biosamples using FRAs. The methods are illustrated with examples from studies in cellular, transgenic fly, and mouse models of HD, but can be adapted for any disease-relevant protein with amyloidogenic polyQ tracts.

Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis.

Alzheimer's disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-ß (Aß) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aß peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aß in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching.

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q48 The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Caenorhabditis elegans Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q97 aggregation and represents a target of future therapeutic avenues for HD.

Different Enzymatic Processing of γ-Phosphoramidate and γ-Phosphoester-Modified ATP Analogues.

Monitoring the activity of ATP-consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate-modified ATP analogues are accepted by the α-ß-phosphoanhydride-cleaving ubiquitin-activating enzyme 1 (UBA1) and the ß-γ-phosphoanhydride-cleaving focal adhesion kinase (FAK), we tested phosphoramidate- and phosphoester-modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate-modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester-modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.

Current Approaches Toward Quantitative Mapping of the Interactome.

Protein-protein interactions (PPIs) play a key role in many, if not all, cellular processes. Disease is often caused by perturbation of PPIs, as recently indicated by studies of missense mutations. To understand the associations of proteins and to unravel the global picture of PPIs in the cell, different experimental detection techniques for PPIs have been established. Genetic and biochemical methods such as the yeast two-hybrid system or affinity purification-based approaches are well suited to high-throughput, proteome-wide screening and are mainly used to obtain qualitative results. However, they have been criticized for not reflecting the cellular situation or the dynamic nature of PPIs. In this review, we provide an overview of various genetic methods that go beyond qualitative detection and allow quantitative measuring of PPIs in mammalian cells, such as dual luminescence-based co-immunoprecipitation, Förster resonance energy transfer or luminescence-based mammalian interactome mapping with bait control. We discuss the strengths and weaknesses of different techniques and their potential applications in biomedical research.

The Tyrosine Kinase Pyk2 Contributes to Complement-Mediated Phagocytosis in Murine Macrophages.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin αMß2, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized Escherichia coli and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the pyk2 gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis.

DULIP: A Dual Luminescence-Based Co-Immunoprecipitation Assay for Interactome Mapping in Mammalian Cells.

Mapping of protein-protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening and validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns.

Integrin-mediated internalization of Staphylococcus aureus does not require vinculin.

BACKGROUND: Disease manifestations of Staphylococcus aureus are connected to the fibronectin (Fn)-binding capacity of these Gram-positive pathogens. Fn deposition on the surface of S. aureus allows engagement of α5ß1 integrins and triggers uptake by host cells. For several integrin- and actin-associated cytoplasmic proteins, including FAK, Src, N-WASP, tensin and cortactin, a functional role during bacterial invasion has been demonstrated. As reorganization of the actin cytoskeleton is critical for bacterial entry, we investigated whether vinculin, an essential protein linking integrins with the actin cytoskeleton, may contribute to the integrin-mediated internalization of S. aureus. RESULTS: Complementation of vinculin in vinculin -/- cells, vinculin overexpression, as well as shRNA-mediated vinculin knock-down in different eukaryotic cell types demonstrate, that vinculin does not have a functional role during the integrin-mediated uptake of S. aureus. CONCLUSIONS: Our results suggest that vinculin is insignificant for the integrin-mediated uptake of S. aureus despite the critical role of vinculin as a linker between integrins and F-actin.

Grb14 is a negative regulator of CEACAM3-mediated phagocytosis of pathogenic bacteria.

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a phagocytic receptor on human granulocytes, which mediates the opsonin-independent recognition and internalization of a restricted set of Gram-negative bacteria such as Neisseria gonorrhoeae. In an unbiased screen using a SH2 domain microarray we identified the SH2 domain of growth factor receptor-bound protein 14 (Grb14) as a novel binding partner of CEACAM3. Biochemical assays and microscopic studies demonstrated that the Grb14 SH2 domain promoted the rapid recruitment of this adaptor protein to the immunoreceptor-based activation motif (ITAM)-like sequence within the cytoplasmic domain of CEACAM3. Furthermore, FRET-FLIM analyses confirmed the direct association of Grb14 and CEACAM3 in intact cells at the sites of bacteria-host cell contact. Knockdown of endogenous Grb14 by RNA interference as well as Grb14 overexpression indicate an inhibitory role for this adapter protein in CEACAM3-mediated phagocytosis. Therefore, Grb14 is the first negative regulator of CEACAM3-initiated bacterial phagocytosis and might help to focus granulocyte responses to the subcellular sites of pathogen-host cell contact.

HemITAM signaling by CEACAM3, a human granulocyte receptor recognizing bacterial pathogens.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to the immunoglobulin superfamily and contribute to cell-cell adhesion and signal modulation in various tissues. In humans, several CEACAMs are targeted by pathogenic bacteria. One peculiar member of this family, CEACAM3, is exclusively expressed by human granulocytes and functions as an opsonin-independent phagocytic receptor for CEACAM-binding bacteria. Here, we will discuss CEACAM3-dependent processes by summarizing recent insight into the phosphotyrosine-based signaling complex formed upon CEACAM3 engagement. Compared to different well-studied phagocytic receptors, such as Fcγ receptors and Dectin-1, CEACAM3 appears as an example of a hemITAM-containing innate immune receptor, which promotes rapid internalization and intracellular destruction of a diverse group of CEACAM-binding bacteria. The particular efficiency of CEACAM3 arises from the direct coupling of upstream activators and downstream effectors of the small GTPase Rac by the cytoplasmic domain of CEACAM3, which co-ordinates actin cytoskeleton re-arrangements and bactericidal effector mechanisms of granulocytes.

Phosphatidylinositol 3'-kinase activity is critical for initiating the oxidative burst and bacterial destruction during CEACAM3-mediated phagocytosis.

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAM-binding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylation-dependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens.

Caveolin limits membrane microdomain mobility and integrin-mediated uptake of fibronectin-binding pathogens.

Staphylococcus aureus, which is a leading cause of hospital-acquired infections, binds via fibronectin to integrin α5ß1, a process that can promote host colonization in vivo. Integrin engagement induces actin cytoskeleton rearrangements that result in the uptake of S. aureus by non-professional phagocytic cells. Interestingly, we found that fibronectin-binding S. aureus trigger the redistribution of membrane microdomain components. In particular, ganglioside GM1 and GPI-linked proteins were recruited upon integrin ß1 engagement, and disruption of membrane microdomains blocked bacterial internalization. Several membrane-microdomain-associated proteins, such as flotillin-1 and flotillin-2, as well as caveolin, were recruited to sites of bacterial attachment. Whereas dominant-negative versions of flotillin-2 did not affect bacterial attachment or internalization, cells deficient for caveolin-1 (Cav1(-/-)) showed increased uptake of S. aureus and other Fn-binding pathogens. Recruitment of membrane microdomains to cell-associated bacteria was unaltered in Cav1(-/-) cells. However, fluorescence recovery after photobleaching (FRAP) revealed an enhanced mobility of membrane-microdomain-associated proteins in the absence of caveolin-1. Enhanced membrane microdomain mobility and increased uptake of S. aureus was repressed by expression of wild-type caveolin-1, but not caveolin-1 G83S, which harbors a point mutation in the caveolin scaffolding domain. Similarly, chemical or physical stimulation of membrane fluidity led to increased uptake of S. aureus. These results highlight a crucial role for caveolin-1 in negative regulation of membrane microdomain mobility, thereby affecting endocytosis of bacteria-engaged integrins. This process might not only limit host cell invasion by integrin-binding bacterial pathogens, but might also be physiologically relevant for integrin-mediated cell adhesion.

Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling.

BACKGROUND: Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET) to visualize pathogen-initiated signaling events in human cells. RESULTS: Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3). In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2) domain of Hck (Hck-SH2) was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet)-Hck-SH2) and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet) and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. CONCLUSION: These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.