Spyros Petrakis

Aggregation of polyQ-extended proteins is promoted by interaction with their natural coiled-coil partners

Polyglutamine (polyQ) diseases are genetically inherited neurodegenerative disorders. They are caused by mutations that result in polyQ expansions of particular proteins. Mutant proteins form intranuclear aggregates, induce cytotoxicity and cause neuronal cell death. Protein interaction data suggest that polyQ regions modulate interactions between coiled-coil (CC) domains. In the case of the polyQ disease spinocerebellar ataxia type-1 (SCA1), interacting proteins with CC domains further enhance aggregation and toxicity of mutant ataxin-1 (ATXN1). Here, we suggest that CC partners interacting with the polyQ region of a mutant protein, increase its aggregation while partners that interact with a different region reduce the formation of aggregates. Computational analysis of genetic screens revealed that CC-rich proteins are highly enriched among genes that enhance pathogenicity of polyQ proteins, supporting our hypothesis. We therefore suggest that blocking interactions between mutant polyQ proteins and their CC partners might constitute a promising preventive strategy against neurodegeneration.

Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.

Evaluation of the possible transmission of BSE and scrapie to gilthead sea bream (Sparus aurata).

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). While the precise mechanism of the PrPC to PrPSc conversion is not understood, it is clear that host PrPC expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrPSc, or of a prion disease developing in farmed fish is alarming and requires further evaluation.

Cellular prion protein co‐localizes with nAChR β4 subunit in brain and gastrointestinal tract

PrPC, the cellular isoform of prion protein, is widely expressed in most tissues, including brain, muscle and gastrointestinal tract. Despite its involvement in several bioprocesses, PrP has still no apparent physiological role. During propagation of transmissible spongiform encephalopathies (TSE), prion protein is converted to the pathological isoform, PrPSc, in a process believed to be mediated by unknown host factors. The identification of proteins associated with PrP may provide information about both the biology of prions and the pathogenesis of TSE. Thus far, PrPC has been shown to interact with synaptic proteins, components of the cytoskeleton and intracellular proteins involved in signalling pathways. Here, we describe the association of PrP with the β4 subunit of nicotinic acetylcholine receptor (nAChR), as indicated by co‐immunoprecipitation assays and double‐label immunofluorescence. The interaction between prion protein and native β4 subunit was further studied by affinity chromatography, using immobilized and refolded recombinant PrP as a bait and brain homogenates from normal individuals. Additionally, the participation of β4 subunit in the pathogenesis of TSE was studied by in vivo assays. β4–/– and wild‐type mice were challenged with the RML (Rocky Mountain Laboratories) infectious agent. Transgenic animals displayed altered incubation times but the deletion of β4 subunit did not result in a significant change of the incubation period of the disease. Our results suggest that PrPC is a member of a multiprotein membrane complex participating in the formation and function of α3β4 nAChR.

Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells.

The Gateway technology cloning system and transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools allows rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway technology-compatible transposon plasmid that combines the advantages of Gateway recombination cloning with the Sleeping Beauty (SB) transposon-mediated transgene integrations. In our system the transposition is catalyzed by the novel hyperactive SB100x transposase, and provides highly efficient and precise transgene integrations into the host genome. A Gateway-compatible transposon plasmid was generated in which the potential target gene can be fused with a yellow fluorescent protein (YFP) tag at the N-terminal. The vector utilizes the CAGGS promoter to control fusion protein expression. The transposon expression vector encoding the YFP-interferon-β protein (IFNB1) fusion protein together with the hyperactive SB100x transposase was used to generate stable cell lines in human embryonic kidney (HEK293) and rat adipose-derived stromal cells (ASC). ASCs and HEK293 cells stably expressed and secreted the human IFNB1 for up to 4 weeks after transfection. The generated Gateway-compatible transposon plasmid can be utilized for numerous experimental approaches, such as gene therapy or high-throughput screening methods in primary cells, representing a valuable molecular tool for laboratory applications.

Tissue plasminogen activator in brain tissues infected with transmissible spongiform encephalopathies

Prion propagation involves conversion of host PrP(C) to a disease-related isoform, PrP(Sc), which accumulates during disease and is the principal component of the transmissible agent. Proteolysis seems to play an important role in PrP metabolism. Plasminogen, a serine protease precursor, has been shown to interact with PrP(Sc). Plasminogen can be proteolytically activated by tissue plasminogen activator (tPA). Recent reports imply a crosstalk between tPA-mediated plasmin activation and PrP. In our study, both tPA activity and tPA gene expression were found elevated in TSE-infected brains as compared to their normal counterparts. Furthermore, it was proved that PrP(Sc), in contrast to PrP(C), could not be degraded by plasmin. In addition, it was observed that TSE symptoms and subsequent death of plasminogen-deficient and tPA-deficient scrapie challenged mice preceded that of wild-type controls. Our data imply that enhanced tPA activity observed in prion infected brains may reflect a neuro-protective response.

Antigenic profile of human recombinant PrP: generation and characterization of a versatile polyclonal antiserum

We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antiserum was evident from the range of animal species and immunochemical techniques where it could be applied successfully. Antigen absorption studies revealed differences in the location and number of epitopes remaining after incubation with soluble or aggregated antigen.Our knowledge concerning immunoprophylaxis against prion diseases and the important role played by conformational changes of PrP is increasing rapidly. The findings reported here should add to this body of knowledge.

Proteasome activation enhances stemness and lifespan of human mesenchymal stem cells

ABSTRACT The age‐associated decline of adult stem cell function contributes to the physiological failure of homeostasis during aging. The proteasome plays a key role in the maintenance of proteostasis and its failure is associated with various biological phenomena including senescence and aging. Although stem cell biology has attracted intense attention, the role of proteasome in stemness and its age‐dependent deterioration remains largely unclear. By employing both Whartons‐Jelly‐ and Adipose‐derived human adult mesenchymal stem cells (hMSCs), we reveal a significant age‐related decline in proteasome content and peptidase activities, accompanied by alterations of proteasomal complexes. Additionally, we show that senescence and the concomitant failure of proteostasis negatively affects stemness. Remarkably, the loss of proliferative capacity and stemness of hMSCs can be counteracted through proteasome activation. At the mechanistic level, we demonstrate for the first time that Oct4 binds at the promoter region of &bgr;2 and &bgr;5 proteasome subunits and thus possibly regulates their expression. A firm understanding of the mechanisms regulating proteostasis in stem cells will pave the way to innovative stem cell‐based interventions to improve healthspan and lifespan. Graphical abstract Figure. No Caption Available. HighlightsProteasome dysfunction is linked with the senescence‐related decline of MSC function.Proteasome activation through &bgr;5 overexpression promotes MSCs stemness.The expression of proteasome subunits is affected by pluripotency factors.Oct4 possibly regulates the expression of &bgr;2 and &bgr;5 proteasome subunits by binding to their promoter region.

Intradermal injection of GFP-producing adipose stromal cells promotes survival of random-pattern skin flaps in rats

BackgroundTissue necrosis is a common complication in operations that use skin flaps for reconstructive surgery. Here we demonstrate the beneficial effect of autologous genetically modified adipose-derived stromal cells (ASCs) in the survival of random-pattern skin flaps.MethodsASCs were isolated from the inguinal fat pad of Wistar rats and genetically modified in order to permanently produce green fluorescent protein (GFP) using the Sleeping Beauty transposon technology. Autologous GFP-producing cells were then injected intradermally into random-pattern skin flaps planned on the dorsal area of rats.ResultsInjection of ASCs resulted in significant improvement of skin flap survival. Histological analysis showed that the connective tissue was almost intact in skin flaps treated with ASCs in contrast to disorganized tissues from mock-treated skin flaps. GFP ASCs were detected in the endothelium of blood vessels co-expressing the endothelial marker von Willebrand factor, thus suggesting that they promote blood vessel regeneration.ConclusionsThese findings indicate that transplantation of autologous GFP ASCs improve survival of skin flaps. This methodology suggests that the use of genetically modified ASCs producing, e.g., angiogenic factors may facilitate survival and integration of flaps in plastic surgery.

Human mesenchymal stem cells with enhanced telomerase activity acquire resistance against oxidative stress-induced genomic damage

BACKGROUND Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Replicative senescence has been linked to macromolecular damage, and especially oxidative stress-induced DNA damage. Recent studies on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has a telomere-length independent function that promotes survival. METHODS Here, we studied the DNA damage accumulation and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that overexpress the catalytic subunit of telomerase (hTERT MSCs). RESULTS We showed that hTERT MSCs at high passages have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation of such defects. Finally, hTERT MSCs were found to possess higher activities of the basic antioxidant enzymes, superoxide dismutase and catalase, than control MSCs. DISCUSSION On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cells basic antioxidant machinery.