Estimating the Interaction Strength Between PTS1-Peptides and Their Receptor PEX5 in Living Cells Using Flow-Cytometer-Based FRET (flowFRET) Measurements.

The import of many peroxisomal matrix proteins is initiated by the interaction of type-1 peroxisomal targeting signals (PTS1) residing at the extreme C-terminus of cargo proteins and their receptor protein PEX5. This interaction has been amply investigated by biophysical methods using isolated proteins and peptides or heterologous systems such as two-hybrid assays. However, a recently developed novel application of Fluorescence resonance energy transfer (FRET) allows a quantifying measurement of this interaction in living cells. This method combines the systematic measurement of FRET-efficiency in a high number of cells with a well-suited normalization protocol and a fitting algorithm, which together allow the estimation of numerical values for the apparent interaction strength that correlates with other measures of binding strength but can be obtained under rather physiological conditions.

Antagonistic Functions of Androgen Receptor and NF-κB in Prostate Cancer-Experimental and Computational Analyses.

Prostate cancer is very frequent and is, in many countries, the third-leading cause of cancer related death in men. While early diagnosis and treatment by surgical removal is often curative, metastasizing prostate cancer has a very bad prognosis. Based on the androgen-dependence of prostate epithelial cells, the standard treatment is blockade of the androgen receptor (AR). However, nearly all patients suffer from a tumor relapse as the metastasizing cells become AR-independent. In our study we show a counter-regulatory link between AR and NF-κB both in human cells and in mouse models of prostate cancer, implying that inhibition of AR signaling results in induction of NF-κB-dependent inflammatory pathways, which may even foster the survival of metastasizing cells. This could be shown by reporter gene assays, DNA-binding measurements, and immune-fluorescence microscopy, and furthermore by a whole set of computational methods using a variety of datasets. Interestingly, loss of PTEN, a frequent genetic alteration in prostate cancer, also causes an upregulation of NF-κB and inflammatory activity. Finally, we present a mathematical model of a dynamic network between AR, NF-κB/IκB, PI3K/PTEN, and the oncogene c-Myc, which indicates that AR blockade may upregulate c-Myc together with NF-κB, and that combined anti-AR/anti-NF-κB and anti-PI3K treatment might be beneficial.

Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försters resonance energy transfer-based competition assay.

The import of the majority of soluble peroxisomal proteins is initiated by the interaction between type-1 peroxisomal targeting signals (PTS1) and their receptor PEX5. PTS1 motifs reside at the extreme C-terminus of proteins and consist of a characteristic tripeptide and a modulatory upstream region. Various PTS1-PEX5 interactions have been studied by biophysical methods using isolated proteins or in heterologous systems such as two-hybrid assays, but a recently established approach based on Försters resonance energy transfer (FRET) allows a quantifying investigation in living cells. FRET is the radiation-free energy transfer between two fluorophores in close proximity and can be used to estimate the fraction of acceptor molecules bound to a donor molecule. For PTS1-PEX5 this method relies on the measurement of FRET-efficiency between the PTS1-binding TPR-domain of PEX5 tagged with mCherry and EGFP fused to a PTS1 peptide. However, this method is less suitable for binding partners with low affinity and protein complexes involving large proteins such as the interaction between full-length PTS1-carrying cargo proteins and PEX5. To overcome this limitation, we introduce a life-cell competition assay based on the same FRET approach but including a fusion protein of Cerulean with the protein of interest as a competitor. After implementing the mathematical description of competitive binding experiments into a fitting algorithm, we demonstrate the functionality of this approach using known interaction partners, its ability to circumvent previous limitations of FRET-measurements and its ability to study the interaction between PEX5 and its full-length cargo proteins. We find that some proteins (SCP2 and AGXT) bind PEX5 with higher affinity than their PTS1-peptides alone, but other proteins (ACOX3, DAO, PerCR-SRL) bind with lower but reasonable affinity, whereas GSTK1 binds with very low affinity. This binding strength was not increased upon elongating the PEX5 TPR-domain at its N-terminus, PEX5(N-TPR), although it interacts specifically with the N-terminal domain of PEX14. Finally, we demonstrate that the latter reduces the interaction strength between PEX5(N-TPR) and PTS1 by a dose-dependent but apparently non-competitive mechanism. Altogether, this demonstrates the power of this novel FRET-based competition approach for studying cargo recognition by PEX5 and protein complexes including large proteins in general.

Extracellular Vesicles Linking Inflammation, Cancer and Thrombotic Risks.

Extracellular vesicles (EVs) being defined as lipid-bilayer encircled particles are released by almost all known mammalian cell types and represent a heterogenous set of cell fragments that are found in the blood circulation and all other known body fluids. The current nomenclature distinguishes mainly three forms: microvesicles, which are formed by budding from the plasma membrane; exosomes, which are released, when endosomes with intraluminal vesicles fuse with the plasma membrane; and apoptotic bodies representing fragments of apoptotic cells. Their importance for a great variety of biological processes became increasingly evident in the last decade when it was discovered that they contribute to intercellular communication by transferring nucleotides and proteins to recipient cells. In this review, we delineate several aspects of their isolation, purification, and analysis; and discuss some pitfalls that have to be considered therein. Further on, we describe various cellular sources of EVs and explain with different examples, how they link cancer and inflammatory conditions with thrombotic processes. In particular, we elaborate on the roles of EVs in cancer-associated thrombosis and COVID-19, representing two important paradigms, where local pathological processes have systemic effects in the whole organism at least in part via EVs. Finally, we also discuss possible developments of the field in the future and how EVs might be used as biomarkers for diagnosis, and as vehicles for therapeutics.

The inflammatory kinase IKKα phosphorylates and stabilizes c-Myc and enhances its activity.

BACKGROUND: The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKß, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied. METHODS: Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry. RESULTS: We show that IKKα and IKKß bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3ß, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers. CONCLUSIONS: Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.

A Novel FRET Approach Quantifies the Interaction Strength of Peroxisomal Targeting Signals and Their Receptor in Living Cells.

Measuring Förster-resonance-energy-transfer (FRET) efficiency allows the investigation of protein-protein interactions (PPI), but extracting quantitative measures of affinity necessitates highly advanced technical equipment or isolated proteins. We demonstrate the validity of a recently suggested novel approach to quantitatively analyze FRET-based experiments in living mammalian cells using standard equipment using the interaction between different type-1 peroxisomal targeting signals (PTS1) and their soluble receptor peroxin 5 (PEX5) as a model system. Large data sets were obtained by flow cytometry coupled FRET measurements of cells expressing PTS1-tagged EGFP together with mCherry fused to the PTS1-binding domain of PEX5, and were subjected to a fitting algorithm extracting a quantitative measure of the interaction strength. This measure correlates with results obtained by in vitro techniques and a two-hybrid assay, but is unaffected by the distance between the fluorophores. Moreover, we introduce a live cell competition assay based on this approach, capable of depicting dose- and affinity-dependent modulation of the PPI. Using this system, we demonstrate the relevance of a sequence element next to the core tripeptide in PTS1 motifs for the interaction strength between PTS1 and PEX5, which is supported by a structure-based computational prediction of the binding energy indicating a direct involvement of this sequence in the interaction.

Ikk2-mediated inflammatory activation of arterial endothelial cells promotes the development and progression of atherosclerosis.

BACKGROUND AND AIMS: Inflammatory activation of endothelial cells is considered to be the first step in the development of atherosclerosis. Here, we determined the consequences of chronic endothelial activation via the NF-κB activator Ikk2 (Inhibitor of nuclear factor kappa-B kinase 2, Ikk-beta) on the development and progression of atherosclerosis. METHODS: We established a conditional transgenic mouse model, expressing a tamoxifen-inducible, constitutively active form of Ikk2 exclusively in arterial endothelial cells (caIkk2EC mice) on an ApoE-deficient background. Mice were fed a Western-type diet and endothelial Ikk2 was activated either at early or late stages of atherosclerosis. RESULTS: En face preparations of isolated aortas revealed a significant increase in plaque area in caIkk2EC mice at 12 weeks of Western-type diet as compared to ApoE-deficient littermates. This was accompanied by increased infiltration of macrophages and T cells into the lesion. Several chemokine/cytokine and immune cell pathways were significantly upregulated in the aortic transcriptome of caIkk2EC mice. Of note, in mice with established atherosclerosis, activation of endothelial Ikk2 still further accelerated progression of atherosclerosis. This indicates that inflammatory endothelial activation is crucial during all stages of the disease. CONCLUSIONS: Our results show for the first time that chronic inflammatory activation of arterial endothelial cells accelerates the development and progression of atherosclerosis both at early and late stages of disease development. Thus, pharmacological targeting of endothelial inflammation emerges as a promising treatment approach.

Rare Human Missense Variants can affect the Function of Disease-Relevant Proteins by Loss and Gain of Peroxisomal Targeting Motifs.

Single nucleotide variants (SNVs) resulting in amino acid substitutions (i.e., missense variants) can affect protein localization by changing or creating new targeting signals. Here, we studied the potential of naturally occurring SNVs from the Genome Aggregation Database (gnomAD) to result in the loss of an existing peroxisomal targeting signal 1 (PTS1) or gain of a novel PTS1 leading to mistargeting of cytosolic proteins to peroxisomes. Filtering down from 32,985 SNVs resulting in missense mutations within the C-terminal tripeptide of 23,064 human proteins, based on gene annotation data and computational prediction, we selected six SNVs for experimental testing of loss of function (LoF) of the PTS1 motif and five SNVs in cytosolic proteins for gain in PTS1-mediated peroxisome import (GoF). Experimental verification by immunofluorescence microscopy for subcellular localization and FRET affinity measurements for interaction with the receptor PEX5 demonstrated that five of the six predicted LoF SNVs resulted in loss of the PTS1 motif while three of five predicted GoF SNVs resulted in de novo PTS1 generation. Overall, we showed that a complementary approach incorporating bioinformatics methods and experimental testing was successful in identifying SNVs capable of altering peroxisome protein import, which may have implications in human disease.

Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells.

FRET (Fluorescence Resonance Energy Transfer) measurements are commonly applied to proof protein-protein interactions. However, standard methods of live cell FRET microscopy and signal normalization only allow a principle assessment of mutual binding and are unable to deduce quantitative information of the interaction. We present an evaluation and normalization procedure for 3-filter FRET measurements, which reflects the process of complex formation by plotting FRET-saturation curves. The advantage of this approach relative to traditional signal normalizations is demonstrated by mathematical simulations. Thereby, we also identify the contribution of critical parameters such as the total amount of donor and acceptor molecules and their molar ratio. When combined with a fitting procedure, this normalization facilitates the extraction of key properties of protein complexes such as the interaction stoichiometry or the apparent affinity of the binding partners. Finally, the feasibility of our method is verified by investigating three exemplary protein complexes. Altogether, our approach offers a novel method for a quantitative analysis of protein interactions by 3-filter FRET microscopy, as well as flow cytometry. To facilitate the application of this method, we created macros and routines for the programs ImageJ, R and MS-Excel, which we make publicly available.

Novel approach for accurate tissue-based protein colocalization and proximity microscopy.

Fluorescence colocalization microscopy is frequently used to assess potential links between distinct molecules; however, this method can lead to striking false-positive results and erroneous conclusions. Here we developed a novel approach with more sophisticated mathematical colocalization analyses together with visualization of physical proximity using fluorescence resonance energy transfer (FRET). To verify our results we used the proximity ligation assay (PLA). With these methods we could demonstrate that distinct neurodegeneration-related proteins either not or only rarely interact in human brain tissue.

Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation.

The question whether two proteins interact with each other or whether a protein localizes to a certain region of the cell is often addressed with fluorescence microscopy and analysis of a potential colocalization of fluorescence markers. Since a mere visual estimation does not allow quantification of the degree of colocalization, different statistical methods of pixel-intensity correlation are commonly used to score it. We observed that these correlation coefficients are prone to false positive results and tend to show high values even for molecules that reside in different organelles. Our aim was to improve this type of analysis and we developed a novel method combining object-recognition based colocalization analysis with pixel-intensity correlation to calculate an object-corrected Pearson coefficient. We designed a macro for the Fiji-version of the software ImageJ and tested the performance systematically with various organelle markers revealing an improved robustness of our approach over classical methods. In order to prove that colocalization does not necessarily mean a physical interaction, we performed FRET (fluorescence resonance energy transfer) microscopy. This confirmed that non-interacting molecules can exhibit a nearly complete colocalization, but that they do not show any significant FRET signal in contrast to proteins that are bound to each other.

Sequence-function correlations and dynamics of ERG isoforms. ERG8 is the black sheep of the family.

The transcription factor ERG is known to have divergent roles. On one hand, it acts as differentiation factor of endothelial cells. On the other hand, it has pathological roles in various cancers. Genomic analyses of the ERG gene show that it gives rise to several isoforms. However, functional differences between these isoforms, representing potential reasons for distinct effects in diverse cell types have not been addressed in detail so far. We set out to investigate the major protein isoforms and found that ERG8 contains a unique C-terminus. This isoform, when expressed as GFP-fusion protein, localized mainly to the cytosol, whereas the other major isoforms (ERG1-4) were predominantly nuclear. Using site directed mutagenesis and laser scanning microscopy of live cells, we could identify nuclear localization (NLS) and nuclear export sequences (NES). These analyses indicated that ERG8 lacks a classical NLS and the DNA-binding domain, but holds an additional NES within its distinctive C-terminus. All the tested isoforms were shuttling between nucleus and cytosol and showed a high degree of mobility. ERG's 1 to 4 were transcriptionally active on ERG-promoter elements whereas ERG8 was inactive, which is in line with the absence of a DNA-binding domain. Fluorescence resonance energy transfer (FRET) microscopy revealed that ERG8 can bind to the transcriptionally active ERG's. Knockdown of ERG8 in endothelial cells resulted in upregulation of endogenous ERG-transcriptional activity implying ERG8 as an inhibitor of the active ERG isoforms. Quantitative PCR revealed a different ratio of active ERG's to ERG8 in cancer- versus non-transformed cells.

Fluorescent proteins as genetically encoded FRET biosensors in life sciences.

Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.