Savvas Christoforidis’s research while affiliated with University of Ioannina and other places


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Publications (57)


The Rab5 effector FERRY links early endosomes with mRNA localization
  • Article
  • Full-text available

June 2023

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59 Reads

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20 Citations

Molecular Cell

Jan S. Schuhmacher

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Savvas Christoforidis

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Marino Zerial

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

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Fig. 2. The structure of the PI3Ka heterodimer and its post-translational modifications. (a) The 3D structure of PI3Ka heterodimer and its domains (b) Functionally important residues of PI3Ka. The 3D structure was produced by authors using PDB ID 7MYN structure as a reference (https://doi.org/10.2210/pdb7MYN/pdb) (c) PIK3R1 splice variants. p50a and p55a differ from p85a at N-terminal residues and contain a unique 35-amino-acid and a 5-amino-acid sequence respectively (d) Regulation mediated through posttranslational modifications. Phosphorylation sites are represented as yellow circles. PTMs with positive effects on PI3K activity are shown with arrows, whereas those with negative effects on PI3Ka activity are shown with bars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The orchestrated signaling by PI3Kα and PTEN at the membrane interface

October 2022

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161 Reads

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6 Citations

Computational and Structural Biotechnology Journal

The oncogene PI3Kα and the tumor suppressor PTEN represent two antagonistic enzymatic activities that regulate the interconversion of the phosphoinositide lipids PI(4,5)P2 and PI(3,4,5)P3 in membranes. As such, they are defining components of phosphoinositide-based cellular signaling and membrane trafficking pathways that regulate cell survival, growth, and proliferation, and are often deregulated in cancer. In this review, we highlight aspects of PI3Kα and PTEN interplay at the intersection of signaling and membrane trafficking. We also discuss the mechanisms of PI3Kα- and PTEN- membrane interaction and catalytic activation, which are fundamental for our understanding of the structural and allosteric implications on signaling at the membrane interface and may aid current efforts in pharmacological targeting of these proteins.


Fabricating a Low-cost and Low-immune Xenogeneic Aortic Valve Bio Prosthesis

October 2021

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31 Reads

Background Cardiac valve replacement is the only available treatment for end-stage valvular dysfunction patients. In this treatment, among the available choices of valves, the bio-prosthetic valves are better than the mechanical ones in terms of hemodynamic and infection-resistant properties. However, they tend to fail with time, posing a catastrophic event. This study focuses on fabricating the heart valve to eliminate the flaws of bio-prosthetic valves. Methods Perfusion-based decellularization method was adapted for decellularisation to the sheep heart. Further, decellularised aortic valves were cross-linked with 0.2% Glutaraldehyde (Group C). ResultsAll valves were tested for biochemical and molecular assays including biomechanical tensile testing. Histology, SEM showed a complete lack of cells with intact matrix for decellularised groups. The fibrin glue coated valves leaflet scaffolds showed remodeling of the cells as per the matrix (plasticity). Characterization studies emphasized the cellular behaviour onto matrigel assay, live-dead assay, and the expression of vWF, glycocalyx lectin. Conclusions This study focuses on fabricating a re-endothelialized xenogeneic aortic valve leaflet using cross-linking reaction to mask antigenicity of the host proteins (low-immune humanized) and avoid post-implantation cross-reaction.


Figure 2: A) Scheme of the workflow of the in vitro GST-FERRY interactor screen. B) SEC profile of GST-
Figure 3: A) Results of an electrophoretic mobility shift assay (EMSA) to test the interaction between the FERRY
Figure 6: A) Combination of immunostaining of Fy-2 and Mitochondria (Tomm70a) and smFISH against polyA
The novel Rab5 effector FERRY links early endosomes with the translation machinery

June 2021

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82 Reads

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3 Citations

Local translation is vital to polarized cells such as neurons and requires a precise and robust distribution of different mRNAs and the translation machinery across the entire cell. The underlying mechanisms are poorly understood and important players are still to be identified. Here, we discovered a novel Rab5 effector complex which leads to mental retardation when genetically disrupted. The Five-subunit Endosomal Rab5 and RNA/ribosome intermediarY, FERRY complex localizes to early endosomes and associates with the translation machinery and a subset of mRNAs including mRNAs for mitochondrial proteins. It directly interacts with mRNA, thereby exhibiting different binding efficacies. Deletion of FERRY subunits reduces the endosomal localization of transcripts, indicating a role in mRNA distribution. Accordingly, FERRY-positive early endosomes harboring mRNA encoding mitochondrial proteins were observed in close proximity to mitochondria in neurons. Therefore, the FERRY complex plays a role for mRNA localization by linking early endosomes with the translation machinery.


Intracellular targets: A multiple cargo transporting molecule

June 2021

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53 Reads

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10 Citations

Journal of Peptide Science

The generation of cell‐penetrating peptides as cargo‐delivery systems has produced an immense number of studies owing to the importance of these systems as tools to deliver molecules into the cells, as well as due to the interest to shed light into a yet unclear mechanism of the entrance of these peptides into the cells. However, many cell‐penetrating peptides might present drawbacks due to causing cellular toxicity, or due to being entrapped in endosomes, or as a result of their degradation before they meet their target. In this work, a cargo transporting molecule, the Cell Penetrating Sequential Oligopeptide Carrier (CPSOC), formed by the repetitive ‐Lys‐Aib‐Cys‐ moiety, was tested for its ability to penetrate the cell membrane and transport the conjugated peptides into the cells. The cysteine residue anchors bioactive molecules through a stable thioether bond. The lysine supplies the positive charge to the construct, whereas the α‐amino isobutyric acid is well known to induce helicoid conformation to the peptide backbone and protects from enzymatic degradation. The present study demonstrates that CPSOC penetrates the membrane transporting the conjugated cargo into the cell. When we tested CPSOC‐conjugated peptides carrying critical domains of Cdc42, a small GTPase implicated in exocytosis, the internalized peptides were found to be functional because they inhibited exocytosis of von Willebrand factor from endothelial Weibel–Palade bodies a trafficking event depending on the Cdc42 protein. The data suggest that the carrier can deliver efficiently functional peptides into the cells, and thus, it can be used as a multiple‐cargo transporting molecule. CPSOC can bind up to four copies of biocargoes via thioether bonds and transport them into the cells.


Figure 2. Chemical inhibitors of dynamin consistently block endocytosis of transferrin and inhibit partially the internalization of VEGFR2. (a) Serum-starved (2 h) HUVECs were treated with vehicle (DMSO) or dynasore (100 µM) or dyngo 4a (30 µM) or dynole 34-2 (20 µM) for 30 min. Subsequently, cells were incubated with FITC transferrin (15 min), fixed and processed for fluorescence microscopy analysis. Prior to fixation, membrane-bound transferrin was removed by acid wash (10 µm scale bars). (b) Analysis of VEGF-induced internalization of surface VEGFR2 through biotinylation of plasma membrane proteins. Serum-deprived HUVECs were treated for 30 min with vehicle or inhibitors of dynamin, i.e., dynasore, dyngo 4a or dynole 34-2, and incubated with VEGF for 15 min. Cells were transferred to 4 • C and surface proteins were labeled with cell-impermeable biotin. Surface biotinylated proteins were pulled down by streptavidin beads and processed for Western blotting analysis. Surface VEGFR2 was revealed by anti-VEGFR2 antibodies. Quantification of surface VEGFR2 is shown on the right of the Western blot (n = 3 independent experiments, mean ± S.E.M., *** p < 0.001, t-test).
Figure 4. Chemical inhibitors of dynamin exert differential effects in VEGF signaling. Serum-starved HUVECs were subjected to 30 min treatment with vehicle or inhibitors of dynamin, followed by stimulation with VEGF (50 ng/mL) for 10 min. Subsequently, cells were lysed and were subjected to immunoblotting analysis using antibodies against the total or the phosphorylated protein forms of VEGFR2, ERK1/2 and Akt. Quantification of the effect of inhibitors on the VEGF-induced phosphorylation of VEGFR2 or ERK1/2 or Akt is shown on the right of the immunoblots (n = 3 independent experiments, mean ± S.E.M., *** p < 0.001, ANOVA). The Western blots corresponding to pERK1/2 and total ERK1/2 of vehicle-and dyngo-treated samples are reproduced from Figure S5a of our previous report [18], which falls under Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/, accessed on 1 April 2021).
Figure 5. Comparative validation of the specificity of two endocytic inhibitors (dynasore and EIPA) in VEGF-induced phosphorylation of ERK1/2. (a) EIPA-mediated inhibition of ERK1/2 phosphorylation is reversible. Serum-deprived HUVECs were treated in different steps, as shown above the lanes of the blots. The first two lanes show control samples of vehicle-treated (lane 1) or VEGF-treated (lane 2) cells. In lane 5, HUVECs were treated first with EIPA for 30 min (step 1, EIPA), washed and incubated in serum-free medium for 10 min to allow recovery from the drug (step 2, wash) and stimulated with VEGF (step 3, VEGF). To test that the reversibility of the effect of the drug still depends on activation by VEGF, an identically treated sample (as above), but without VEGF, was analyzed in parallel (lane 4). The arrow indicates
Chemical Inhibitors of Dynamin Exert Differential Effects in VEGF Signaling

April 2021

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75 Reads

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10 Citations

Cells

VEGFR2 is the main receptor and mediator of the vasculogenic and angiogenic activity of VEGF. Activated VEGFR2 internalizes through clathrin-mediated endocytosis and macropinocytosis. As dynamin is a key regulator of the clathrin pathway, chemical inhibitors of dynamin are commonly used to assess the role of the clathrin route in receptor signaling. However, drugs may also exert off-target effects. Here, we compare the effects of three dynamin inhibitors, dynasore, dyngo 4a and dynole, on VEGFR2 internalization and signaling. Although these drugs consistently inhibit clathrin-mediated endocytosis of both transferrin (a typical cargo of this route) and VEGFR2, surprisingly, they exert contradictory effects in receptor signaling. Thus, while dynasore has no effect on phosphorylation of VEGFR2, the other two drugs are strong inhibitors. Furthermore, although dyngo does not interfere with phosphorylation of Akt, dynasore and dynole have a strong inhibitory effect. These inconsistent effects suggest that the above dynamin blockers, besides inhibiting dynamin-dependent endocytosis of VEGFR2, exert additional inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects. Using a recently developed protocol, we comparatively validate the specificity of two endocytic inhibitors, dynasore and EIPA. Our findings highlight the importance of assessing whether the effect of an endocytic drug on signaling is specifically due to its interference with endocytosis or due to off-targets.



Abstract 3918: Νovel small molecule modulators of the hotspot PIK3CA mutants identified by computational and experimental approaches

July 2019

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25 Reads

Cancer Research

Kinases are intensely pursued as drug targets for cancer treatment. Usually, kinase inhibitors target the active center (ATP binding site). However, the similarity of this site across many kinases often results in non-selective inhibition. Accordingly, an alternative approach is being pursued for the identification of allosteric inhibitors targeting relatively less conserved binding sites, thus higher selectivity can be attained. PI3Kα is the most frequently mutated kinase in human cancers, with 80% of the mutations occurring in two hotspots of the PIK3CA gene, which encodes the catalytic subunit of PI3Kα: 1) the H1047R mutation, which is found at the kinase (membrane binding) domain and 2) the E545K mutation, which is found at the helical domain (amino acid replacement with charge reversal). To assess the activation mechanisms of PIK3CA mutants, extensive microsecond Molecular Dynamics (MD) simulations were performed to examine conformational differences between the wild type (WT) and mutant proteins. The MD results were used to predict alterations in the allosteric networks of the WT upon each of the mutation. Binding site analysis of allosteric action in the kinase domain identified cavities in the vicinity of the H1047R mutation and the membrane-binding region. Virtual screening and subsequent biochemical assays were used to identify several PI3Kα inhibitors acting at a micromolar concentration. The effect of the inhibitors on WT and H1047R mutant PI3Kα-membrane associations was subsequently studied by SPR experiments, which showed that these small molecules alter the protein-membrane affinity, unlike other known PI3Kα inhibitors such as wortmannin. It was also observed that the inhibitory concentration remained unchanged even under conditions of increased ATP concentration, indicating that these inhibitors are non-competitive. The most promising small molecules were further tested in vivo using xenografts and genetically modified mouse models. Micro PET/CT imaging of the tumors showed reduction in volume after treatment. Immunohistochemical analysis of the xenografts showed reduction of proliferation and increase in apoptosis after treatment, in comparison to the controls. Details about the results with the E545K will be presented at the meeting. Note: This abstract was not presented at the meeting. Citation Format: Zoe Cournia, Paraskevi Gkeka, Hari Leontiadou, Ioannis Galdadas, Christina Athanasiou, Antriana Papadimitropoulou, Vasiliki Lazani, Maria Pavlaki, Bogos Agianian, Savvas Christoforidis, Argiris Efstratiadis. Νovel small molecule modulators of the hotspot PIK3CA mutants identified by computational and experimental approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3918.


Abstract 3918: Νovel small molecule modulators of the hotspot PIK3CA mutants identified by computational and experimental approaches

July 2019

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20 Reads

Kinases are intensely pursued as drug targets for cancer treatment. Usually, kinase inhibitors target the active center (ATP binding site). However, the similarity of this site across many kinases often results in non-selective inhibition. Accordingly, an alternative approach is being pursued for the identification of allosteric inhibitors targeting relatively less conserved binding sites, thus higher selectivity can be attained. PI3Kα is the most frequently mutated kinase in human cancers, with 80% of the mutations occurring in two hotspots of the PIK3CA gene, which encodes the catalytic subunit of PI3Kα: 1) the H1047R mutation, which is found at the kinase (membrane binding) domain and 2) the E545K mutation, which is found at the helical domain (amino acid replacement with charge reversal). To assess the activation mechanisms of PIK3CA mutants, extensive microsecond Molecular Dynamics (MD) simulations were performed to examine conformational differences between the wild type (WT) and mutant proteins. The MD results were used to predict alterations in the allosteric networks of the WT upon each of the mutation. Binding site analysis of allosteric action in the kinase domain identified cavities in the vicinity of the H1047R mutation and the membrane-binding region. Virtual screening and subsequent biochemical assays were used to identify several PI3Kα inhibitors acting at a micromolar concentration. The effect of the inhibitors on WT and H1047R mutant PI3Kα-membrane associations was subsequently studied by SPR experiments, which showed that these small molecules alter the protein-membrane affinity, unlike other known PI3Kα inhibitors such as wortmannin. It was also observed that the inhibitory concentration remained unchanged even under conditions of increased ATP concentration, indicating that these inhibitors are non-competitive. The most promising small molecules were further tested in vivo using xenografts and genetically modified mouse models. Micro PET/CT imaging of the tumors showed reduction in volume after treatment. Immunohistochemical analysis of the xenografts showed reduction of proliferation and increase in apoptosis after treatment, in comparison to the controls. Details about the results with the E545K will be presented at the meeting. Note: This abstract was not presented at the meeting. Citation Format: Zoe Cournia, Paraskevi Gkeka, Hari Leontiadou, Ioannis Galdadas, Christina Athanasiou, Antriana Papadimitropoulou, Vasiliki Lazani, Maria Pavlaki, Bogos Agianian, Savvas Christoforidis, Argiris Efstratiadis. Νovel small molecule modulators of the hotspot PIK3CA mutants identified by computational and experimental approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3918.



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Citations (43)


... Conversely, a molecular tether and relative mRNA targets have been described for early endosomes. The FERRY complex is composed of Tbck, Ppp1r21, C12orf4, Cryzl1, and Gatd1 proteins, and it tethers NEM-mRNAs to early endosomes through interaction with Rab5 [119]. This complex binds mRNAs encoding components of the mitochondrial matrix, mitochondrial ribosome, cellular respiration, and tricarboxylic acid cycle in HeLa cells. ...

Reference:

Mitochondrial Dynamics and mRNA Translation: A Local Synaptic Tale
The Rab5 effector FERRY links early endosomes with mRNA localization

Molecular Cell

... The class I phosphoinositide 3-kinase alpha isoform (PI3Kα) is a lipid kinase that mediates activation of the protein kinase B (Akt)/mTOR signaling pathway, playing fundamental roles in cell proliferation, growth, and survival. When PI3Kα binds to activated Receptor Tyrosine Kinases (RTKs) and activated RAS on cell membranes, it phosphorylates the 3′-position hydroxyl group of the inositol ring of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) to produce phosphatidylinositol (3,4,5)trisphosphate (PIP 3 ) [1,2]. PIP 3 acts as a second messenger activating numerous membrane signaling molecules and kinases, and is thus regulating protein synthesis, cell growth, and proliferation [3]. ...

The orchestrated signaling by PI3Kα and PTEN at the membrane interface

Computational and Structural Biotechnology Journal

... Other unknown RBPs or factors (light gray) may also be involved to stabilize the mRNP complex and define the connection to endosomes. (C) Working model of the transport of mRNAs encoding mitochondrial proteins via trafficking endosomes in rat neuron cell (adapted from preprint studies of [66,67]). A FERRY (Five-subunit Endosomal Rab5 and RNA/ribosome intermediary) complex, composed of Fy-1 to 5, is required for transport. ...

The Novel Rab5 Effector FERRY Links Early Endosomes With the Translation Machinery
  • Citing Article
  • January 2021

SSRN Electronic Journal

... In plants, two RRM-type RNAbinding proteins form a complex with cargo mRNAs and the endosomal component N-ethylmaleimide-sensitive factor NSF as well as Rab5a [7,12,49]. Comparably, the FERRY complex (Five-subunit Endosomal Rab5 and RNA/ribosome intermediarY) interacts with the activated GTP-bound form of Rab5 during endosomal mRNA transport in neurons [10,11]. Further examples are the membrane-associated protein Annexin 11 that links large RNA granules to lysosomal vesicles during mRNA transport in neuronal axons and dendrites [9]. ...

The novel Rab5 effector FERRY links early endosomes with the translation machinery

... In addition, Sequential Oligopeptide Carriers (SOCn) with the moiety [Lys(antigen)-Aib-Gly] 4 , (Aib: 2 aminoisobutyric acid), as well as the modified versions [Lys-Aib-Cys] 4 (C-SOC 4 ) and [Lys-Aib-Cys (3,9 Acm)] 4 (CPSOC (3,9 Acm)), have been developed for anchoring multiple copies of peptide epitopes, enhancing their antigenic/immunogenic potency [9][10][11]. These carriers allow the construction of high molecular weight molecules (>3000 Da) capable of inducing a potent immune response. ...

Intracellular targets: A multiple cargo transporting molecule
  • Citing Article
  • June 2021

Journal of Peptide Science

... Thus, the specificity of these inhibitors and their potential unexpected effect on other cellular functions were not enough documented, although some published works indicated off-target effects of endocytosis inhibitors. [45][46][47][48] We previously observed that well-known endocytosis inhibitors (Table S1) had negligible inhibitory effects, lacked specificity on the cellular uptake of endocytosis markers (Tf, CTB, and dextran), or reduced cell viability. 14 For example, sucrose, a clathrin-mediated endocytosis inhibitor, completely abolished the cellular uptake of all endocytosis markers. ...

Chemical Inhibitors of Dynamin Exert Differential Effects in VEGF Signaling

Cells

... Endothelial cells use macropinocytosis for signaling of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) [23][24][25]. Amiloride is a Na + /H + exchange inhibitors that inhibit macropinocytosis [26]. To investigate whether amiloride inhibits PAI1-HA uptake, endothelial cells were pre-treated with amiloride for 30 minutes prior to addition of PAI1-HA enriched media. ...

VEGF induces signalling and angiogenesis by directing VEGFR2 internalisation through macropinocytosis
  • Citing Article
  • January 2016

Journal of Cell Science

... Golgi-bypass mechanism (Sánchez-León et al, 2011 these studies concern trafficking routes operating only under specific conditions or 764 stress, and do not seem to reflect a major trafficking mechanism for the biogenesis of 765 essential cargoes, as transporters or receptors, proposed through our work. Notice also 766 that in A. nidulans transporters and apical cargoes use different endocytic mechanisms 767 for their internalization, turnover or recycling (Martzoukou et al, 2017). 768 ...

The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi

eLife

... Thus, we hypothesized that flow may enhance endocytosis, thereby clustering ALK1 receptors and increasing pSMAD1/5/9 signaling. Considering that dynamin-2 is crucial for ALK1 endocytosis [73], we used dynasore, a potent and specific dynamin1/2 inhibitor, to interfere with endocytosis [74]. We compared the ASI between vehicle-and 100 µM dynasore-treated HUVECs subjected to static conditions or 19 dyn/cm 2 SS, with or without added BMP9. ...

Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner
Scientific Reports

... Considering that dynamin-2 is crucial for ALK1 endocytosis [70], we used dynasore, a potent and specific dynamin1/2 inhibitor, to interfere with endocytosis [71]. We compared the ASI between vehicle and 100 µM dynasore-treated HUVECs subjected to static conditions or 19 dyn/cm 2 SS, with or without added BMP9. ...

Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner
  • Citing Article
  • March 2017

Scientific Reports