Mariola Fotin-Mleczek

A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides

The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8–10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.

Cationic cell-penetrating peptides interfere with TNF signalling by induction of TNF receptor internalization.

Cationic cell-penetrating peptides (CPPs) have been used widely as delivery vectors for the import of molecules that otherwise do not cross the plasma membrane of eukaryotic cells. In this work, we demonstrate that the three cationic CPPs, Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction. This inhibition is based on the downregulation of TNF receptors at the cell surface by induction of internalization. In contrast to TNF-dependent receptor internalization, no receptor activation occurs. The receptor downregulation is not restricted to the CPPs. Remarkably, the HIV-1 Tat protein itself also induces the internalization of TNF receptors. The dynamin dependence of the internalization, as well as the fact that epidermal growth factor receptors are also internalized, suggest a general induction of clathrin-dependent endocytosis as the mechanism of action. The significance of these findings for the use of cationic CPPs in the import of bioactive peptides is demonstrated here using a conjugate consisting of Antp and a Smac protein-derived cargo peptide. The cargo alone, when introduced into cells by electroporation, enhanced TNF-induced apoptosis by inhibiting the anti-apoptotic action of IAPs (inhibitor of apoptosis proteins). For the Antp-Smac conjugate at concentrations below 40 μM the inhibitory effect of the Antp peptide compensated for the pro-apoptotic activity of the cargo, and led to the protection of cells against TNF-mediated apoptosis. These data provide important new information for the use of cationic CPPs for the cellular delivery of bioactive molecules.

Tumor Necrosis Factor Receptor-associated Factor (TRAF) 1 Regulates CD40-induced TRAF2-mediated NF-κB Activation

To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-κB activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-κB activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/CD40 ligand-induced NF-κB activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2)2-TRAF1 versus TRAF2-(TRAF1)2) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling.

A targeted protease substrate for a quantitative determination of protease activities in the endolysosomal pathway.

Inside the cell, proteases act in concert in the degradation of proteins and peptides. In order to understand the significance of an individual proteolytic activity within an ensemble of proteases, protocols and probes are required that enable a quantitative determination of the contribution of a protease to the break‐down of a given substrate. Here we present a fluorescence resonance energy transfer‐based probe and protocols for a quantitative determination of proteolytic activities inside the endolysosomal compartment. A peptide substrate that is readily cleaved by different cathepsins is flanked by fluorescein and tetramethylrhodamine‐labeled lysine residues. Efficient endolysosomal targeting of the substrate is achieved by N‐terminal elongation with the cell‐penetrating peptide nona‐arginine. The proteasome inhibitor lactacystin has a small, but significant effect on the break‐down of the substrate, thus demonstrating that only a minor fraction of the peptide reaches the cytoplasm in its intact form. Nona‐arginine therefore constitutes a highly efficient low‐molecular‐weight moiety for targeting the endolysosomal compartment.

Endogenous Membrane Tumor Necrosis Factor (TNF) Is a Potent Amplifier of TNF Receptor 1-mediated Apoptosis

The heat shock protein 90 (Hsp-90) inhibitor, geldanamycin, and the proteasome inhibitor, MG-132, both inhibited tumor necrosis factor receptor 1 (TNF-R1)- but not TRAIL-induced apoptosis in Kym-1 cells, suggesting that TNF-R1-induced cell death is dependent on NF-κB activation in this model. Triggering of TNF-R1 by agonistic antibodies led to cell-type specific induction of endogenous TNF and apoptosis, the latter of which was abrogated by neutralizing TNF specific antibodies. TNF-R1-stimulated cells expressed TNF mainly in a cell-associated form, suggesting that the endogenously produced TNF act in its membrane-bound form. Geldanamycin failed to inhibit apoptosis induction by a combination of agonistic TNF-R1- and TNF-R2-specific antibodies, indicating that both TNF receptors co-operate in TNF-R1-triggered apoptosis in Kym-1 cells. Thus, TNF-R1 stimulation can elicit a strong and rapid apoptotic response via induction of membrane TNF and subsequent cooperation of TNF-R1 and TNF-R2. Moreover, we give evidence that this mechanism circumvents the need of the prolonged presence of exogenous soluble TNF for TNF-R1-mediated apoptosis induction.

Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines

AbstractmRNA represents a promising new vaccine technology platform with high flexibility in regard to development and production. Here, we demonstrate that vaccines based on sequence optimized, chemically unmodified mRNA formulated in optimized lipid nanoparticles (LNPs) are highly immunogenic and well tolerated in non-human primates (NHPs). Single intramuscular vaccination of NHPs with LNP-formulated mRNAs encoding rabies or influenza antigens induced protective antibody titers, which could be boosted and remained stable during an observation period of up to 1 year. First mechanistic insights into the mode of action of the LNP-formulated mRNA vaccines demonstrated a strong activation of the innate immune response at the injection site and in the draining lymph nodes (dLNs). Activation of the innate immune system was reflected by a transient induction of pro-inflammatory cytokines and chemokines and activation of the majority of immune cells in the dLNs. Notably, our data demonstrate that mRNA vaccines can compete with licensed vaccines based on inactivated virus or are even superior in respect of functional antibody and T cell responses. Importantly, we show that the developed LNP-formulated mRNA vaccines can be used as a vaccination platform allowing multiple, sequential vaccinations against different pathogens. These results provide strong evidence that the mRNA technology is a valid approach for the development of effective prophylactic vaccines to prevent infectious diseases.Vaccine technology: Repurposing the genetic messengerVaccines based on mRNA provoke strong immune responses after a single dose. mRNA is commonly known as the ‘genetic messenger’ cousin of DNA and a crucial mediator of protein production. Now, research led by Mariola Fotin-Mleczek, of Germany’s CureVac AG, demonstrates that mRNA can be developed to produce virus fragments, called antigens, that can prime a vaccinee’s immune system against a pathogen. Testing their vaccine platform, the team created mRNA coding for rabies and influenza antigens, and used intramuscular injection to inoculate non-human primates. A single dose elicited strong immune responses, which the team then successfully maintained through booster vaccinations for an observation period of 1 year. The responses outperformed those of licensed vaccines against rabies and influenza type H3N2. This research shows that mRNA has promise as a versatile, cost-effective, rapidly scalable vaccine technology.