Intracellular delivery of Parkin-RING0-based fragments corrects Parkin-induced mitochondrial dysfunction through interaction with SLP-2.

BACKGROUND: Loss-of-function mutations in the PRKN gene, encoding Parkin, are the most common cause of autosomal recessive Parkinson's disease (PD). We have previously identified mitoch ondrial Stomatin-like protein 2 (SLP-2), which functions in the assembly of respiratory chain proteins, as a Parkin-binding protein. Selective knockdown of either Parkin or SLP-2 led to reduced mitochondrial and neuronal function in neuronal cells and Drosophila, where a double knockdown led to a further worsening of Parkin-deficiency phenotypes. Here, we investigated the minimal Parkin region involved in the Parkin-SLP-2 interaction and explored the ability of Parkin-fragments and peptides from this minimal region to restore mitochondrial function. METHODS: In fibroblasts, human induced pluripotent stem cell (hiPSC)-derived neurons, and neuroblastoma cells the interaction between Parkin and SLP-2 was investigated, and the Parkin domain responsible for the binding to SLP-2 was mapped. High resolution respirometry, immunofluorescence analysis and live imaging were used to analyze mitochondrial function. RESULTS: Using a proximity ligation assay, we quantitatively assessed the Parkin-SLP-2 interaction in skin fibroblasts and hiPSC-derived neurons. When PD-associated PRKN mutations were present, we detected a significantly reduced interaction between the two proteins. We found a preferential binding of SLP-2 to the N-terminal part of Parkin, with a highest affinity for the RING0 domain. Computational modeling based on the crystal structure of Parkin protein predicted several potential binding sites for SLP-2 within the Parkin RING0 domain. Amongst these, three binding sites were observed to overlap with natural PD-causing missense mutations, which we demonstrated interfere substantially with the binding of Parkin to SLP-2. Finally, delivery of the isolated Parkin RING0 domain and a Parkin mini-peptide, conjugated to cell-permeant and mitochondrial transporters, rescued compromised mitochondrial function in Parkin-deficient neuroblastoma cells and hiPSC-derived neurons with endogenous, disease causing PRKN mutations. CONCLUSIONS: These findings place further emphasis on the importance of the protein-protein interaction between Parkin and SLP-2 for the maintenance of optimal mitochondrial function. The possibility of restoring an abolished binding to SLP-2 by delivering the Parkin RING0 domain or the Parkin mini-peptide involved in this specific protein-protein interaction into cells might represent a novel organelle-specific therapeutic approach for correcting mitochondrial dysfunction in Parkin-linked PD.

Analysis of the evolution of COVID-19 disease understanding through temporal knowledge graphs.

The COVID-19 pandemic highlighted two critical barriers hindering rapid response to novel pathogens. These include inefficient use of existing biological knowledge about treatments, compounds, gene interactions, proteins, etc. to fight new diseases, and the lack of assimilation and analysis of the fast-growing knowledge about new diseases to quickly develop new treatments, vaccines, and compounds. Overcoming these critical challenges has the potential to revolutionize global preparedness for future pandemics. Accordingly, this article introduces a novel knowledge graph application that functions as both a repository of life science knowledge and an analytics platform capable of extracting time-sensitive insights to uncover evolving disease dynamics and, importantly, researchers' evolving understanding. Specifically, we demonstrate how to extract time-bounded key concepts, also leveraging existing ontologies, from evolving scholarly articles to create a single temporal connected source of truth specifically related to COVID-19. By doing so, current knowledge can be promptly accessed by both humans and machines, from which further understanding of disease outbreaks can be derived. We present key findings from the temporal analysis, applied to a subset of the resulting knowledge graph known as the temporal keywords knowledge graph, and delve into the detailed capabilities provided by this innovative approach.

Rapid, novel screening of toxicants in poison baits, and autopsy specimens by ambient mass spectrometry.

Animal poisoning and dissemination of baits in the environment have public health and ethological implications, which can be followed by criminal sanctions for those responsible. The reference methods for the analysis of suspect baits and autopsy specimens are founded on chromatographic-based techniques. They are extremely robust and sensitive, but also very expensive and laborious. For this reason, we developed an ambient mass spectrometry (AMS) method able to screen for 40 toxicants including carbamates, organophosphate and chlorinated pesticides, coumarins, metaldehyde, and strychnine. Spiked samples were firstly purified and extracted by dispersive solid phase extraction (QuEChERS) and then analyzed by direct analysis in real time high-resolution mass spectrometry (DART-HRMS). To verify the performance of this new approach, 115 authentic baits (n = 59) and necropsy specimens (gastrointestinal content and liver, n = 56) were assessed by the official reference methods and combined QuEChERS-DART-HRMS. The agreement between the results allowed evaluation of the performances of the new screening method for a variety of analytes and calculation of the resultant statistical indicators (the new method had overall accuracy 89.57%, sensitivity of 88.24%, and a specificity of 91.49%). Taking into account only the baits, 96.61% of overall accuracy was achieved with 57/59 samples correctly identified (statistical sensitivity 97.50%, statistical specificity 94.74%). Successful identification of the bitter compound, denatonium benzoate, in all the samples that contained rodenticides (28/28) was also achieved. We believe initial screening of suspect poison baits could guide the choice of reference confirmatory methods, reduce the load in official laboratories, and help the early stages of investigations into cases of animal poisoning.

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation.

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson's disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

Ambient mass spectrometry for rapid authentication of milk from Alpine or lowland forage.

Metabolomics approaches, such as direct analysis in real time-high resolution mass spectrometry (DART-HRMS), allow characterising many polar and non-polar compounds useful as authentication biomarkers of dairy chains. By using both a partial least squares discriminant analysis (PLS-DA) and a linear discriminant analysis (LDA), this study aimed to assess the capability of DART-HRMS, coupled with a low-level data fusion, discriminate among milk samples from lowland (silages vs. hay) and Alpine (grazing; APS) systems and identify the most informative biomarkers associated with the main dietary forage. As confirmed also by the LDA performed against the test set, DART-HRMS analysis provided an accurate discrimination of Alpine samples; meanwhile, there was a limited capacity to correctly recognise silage- vs. hay-milks. Supervised multivariate statistics followed by metabolomics hierarchical cluster analysis allowed extrapolating the most significant metabolites. Lowland milk was characterised by a pool of energetic compounds, ketoacid derivates, amines and organic acids. Seven informative DART-HRMS molecular features, mainly monoacylglycerols, could strongly explain the metabolomic variation of Alpine grazing milk and contributed to its classification. The misclassification between the two lowland groups confirmed that the intensive dairy systems would be characterised by a small variation in milk composition.

The mitochondrial chaperone TRAP1 regulates F-ATP synthase channel formation.

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

Assessing direct analysis in real-time mass spectrometry for the identification and serotyping of Legionella pneumophila.

AIMS: The efficacy of ambient mass spectrometry to identify and serotype Legionella pneumophila was assessed. To this aim, isolated waterborne colonies were submitted to a rapid extraction method and analysed by direct analysis in real-time mass spectrometry (DART-HRMS). METHODS AND RESULTS: The DART-HRMS profiles, coupled with partial least squares discriminant analysis (PLS-DA), were first evaluated for their ability to differentiate Legionella spp. from other bacteria. The resultant classification model achieved an accuracy of 98.1% on validation. Capitalising on these encouraging results, DART-HRMS profiling was explored as an alternative approach for the identification of L. pneumophila sg. 1, L. pneumophila sg. 2-15 and L. non-pneumophila; therefore, a different PLS-DA classifier was built. When tested on a validation set, this second classifier reached an overall accuracy of 95.93%. It identified the harmful L. pneumophila sg. 1 with an impressive specificity (100%) and slightly lower sensitivity (91.7%), and similar performances were reached in the classification of L. pneumophila sg. 2-15 and L. non-pneumophila. CONCLUSIONS: The results of this study show the DART-HMRS method has good accuracy, and it is an effective method for Legionella serogroup profiling. SIGNIFICANCE AND IMPACT OF THE STUDY: These preliminary findings could open a new avenue for the rapid identification and quick epidemiologic tracing of L. pneumophila, with a consequent improvement to risk assessment.

The nuclear import of the transcription factor MyoD is reduced in mesenchymal stem cells grown in a 3D micro-engineered niche.

Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, we show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein are reduced. Our results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, our results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.

Rational Design of Allosteric and Selective Inhibitors of the Molecular Chaperone TRAP1.

TRAP1 is the mitochondrial paralog of the heat shock protein 90 (HSP90) chaperone family. Its activity as an energy metabolism regulator has important implications in cancer, neurodegeneration, and ischemia. Selective inhibitors of TRAP1 could inform on its mechanisms of action and set the stage for targeted drug development, but their identification was hampered by the similarity among active sites in HSP90 homologs. We use a dynamics-based approach to identify a TRAP1 allosteric pocket distal to its active site that can host drug-like molecules, and we select small molecules with optimal stereochemical features to target the pocket. These leads inhibit TRAP1, but not HSP90, ATPase activity and revert TRAP1-dependent downregulation of succinate dehydrogenase activity in cancer cells and in zebrafish larvae. TRAP1 inhibitors are not toxic per se, but they abolish tumorigenic growth of neoplastic cells. Our results indicate that exploiting conformational dynamics can expand the chemical space of chaperone antagonists to TRAP1-specific inhibitors with wide therapeutic opportunities.

Advantages and limitations of a supernegative GFP in facilitating MyoD intracellular tracking.

Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (-30)GFP, to a myogenic transcription factor, MyoD. The (-30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (-30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (-30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

An antipsychotic drug exerts anti-prion effects by altering the localization of the cellular prion protein.

Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrPC), an endogenous membrane glycoprotein of uncertain function, into PrPSc, a pathological isoform that replicates by imposing its abnormal folding onto PrPC molecules. A great deal of evidence supports the notion that PrPC plays at least two roles in prion diseases, by acting as a substrate for PrPSc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrPC may transduce neurotoxic signals elicited by other disease-associated protein aggregates. Thus, PrPC may represent a convenient pharmacological target for prion diseases, and possibly other neurodegenerative conditions. Here, we sought to characterize the activity of chlorpromazine (CPZ), an antipsychotic previously shown to inhibit prion replication by directly binding to PrPC. By employing biochemical and biophysical techniques, we provide direct experimental evidence indicating that CPZ does not bind PrPC at biologically relevant concentrations. Instead, the compound exerts anti-prion effects by inducing the relocalization of PrPC from the plasma membrane. Consistent with these findings, CPZ also inhibits the cytotoxic effects delivered by a PrP mutant. Interestingly, we found that the different pharmacological effects of CPZ could be mimicked by two inhibitors of the GTPase activity of dynamins, a class of proteins involved in the scission of newly formed membrane vesicles, and recently reported as potential pharmacological targets of CPZ. Collectively, our results redefine the mechanism by which CPZ exerts anti-prion effects, and support a primary role for dynamins in the membrane recycling of PrPC, as well as in the propagation of infectious prions.

Tat-MyoD fused proteins, together with C2c12 conditioned medium, are able to induce equine adult mesenchimal stem cells towards the myogenic fate.

The Tat protein is able to translocate through the plasma membrane and when it is fused with other peptides may acts as a protein transduction system. This ability appears particularly interesting to induce tissue-specific differentiation when the Tat protein is associated to transcription factors. In the present work, the potential of the complex Tat-MyoD in inducing equine peripheral blood mesenchymal stem cells (PB-MSCs) towards the myogenic fate, was evaluated. Results showed that the internalization process of Tat-MyoD happens only in serum free conditions and that the nuclear localization of the fused complex is observed after 15 hours of incubation. However, the supplement of Tat-MyoD only was not sufficient to induce myogenesis and, therefore, in order to achieve the myogenic differentiation of PB-MSCs, conditioned medium from C2C12 cells was added without direct contact. Real Time PCR and immunofluorescence methods evaluated the establishment of a myogenic program. Our results suggest that TAT- transduction of Tat-MyoD, when supported by conditioned medium, represents a useful methodology to induce myogenic differentiation.

In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration.

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury.

A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein.

Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrP(Sc) is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrP(C) may provide an opportunity to overcome these problems. PrP(C) ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrP(C), and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrP(C)-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrP(C)-dependent synaptotoxicity of amyloid-ß (Aß) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrP(C) may produce a dual effect of blocking prion replication and inhibiting PrP(C)-mediated toxicity.

The Parkinson's disease-related protein DJ-1 protects dopaminergic neurons in vivo and cultured cells from alpha-synuclein and 6-hydroxydopamine toxicity.

BACKGROUND: Dopaminergic degeneration is a major finding in brains of patients with Parkinson's disease (PD), together with Lewy bodies, intraneuronal inclusions mainly composed of the fibrillogenic protein α-synuclein (α-syn). The familial-PD-related protein DJ-1 was reported to reduce dopaminergic degeneration triggered by α-syn or by the dopaminergic-selective neurotoxin 6-hydroxydopamine (6-OHDA). OBJECTIVE: The aim was to further investigate the role of DJ-1 in dopaminergic degeneration and to see whether a cell-permeable recombinant form of DJ-1 (TAT-DJ-1) could restore dopamine depletion in vivo, thus representing an innovative therapeutic approach. METHODS: We developed in vitro (PC12/TetOn cells and mouse primary mesencephalic neurons) and in vivo models [including DJ-1 knockout (-/-) mice] to investigate DJ-1 in dopaminergic degeneration. RESULTS: We found that in PC12/TetOn cells overexpressing α-syn with the familial-PD linked mutation A30P, DJ-1 silencing increased α-syn (A30P) toxicity. Primary mesencephalic neurons from DJ-1 (-/-) mice were more vulnerable to a cell-permeable form of α-syn (TAT-α-syn) and to 6-OHDA. Intrastriatally administered TAT-DJ-1 reduced 6-OHDA toxicity in vivo in C57BL/6 mice. Finally, when we injected TAT-α-syn (A30P) in the striatum of DJ-1 (-/-) animals, dopamine was depleted more than in the control strain. CONCLUSION: DJ-1 appears to have a protective role against dopaminergic degeneration triggered by α-syn or 6-OHDA, reinforcing the possible therapeutic importance of this protein in PD.

A novel prion protein-tyrosine hydroxylase interaction.

The prion protein (PrP) is currently one of the most studied molecules in the neurosciences. It is the main cause of a group of neurological diseases collectively called transmissible spongiform encephalopathies that severely affect both humans and a variety of mammals. Much effort has been directed to understanding the molecular basis of PrP activity, both in physiological and pathological terms. In this context, identification of neuronally-relevant interactors of PrP may play a crucial role. We recently discovered a specific, high-affinity (nanomolar KD) interaction with tyrosine hydroxylase (TH), a enzyme catalyzing the rate-limiting step in the synthesis of the neurotransmitter dopamine. Using molecular biological, biochemical and biophysical techniques we identified the C-terminal structured domain of PrP and the Nterminal regulatory domain of TH as interacting domains between these two proteins. This interaction does not affect TH activity in vitro, although co-expression experiments in HeLa and Chinese hamster ovary cells revealed that PrP is able to internalize TH. Moreover, TH modulated the level of expression of PrP and its localization at the plasma membrane. This novel interaction between two proteins of central importance in nervous system function may shed new light on our understanding of PrP in neurological diseases.

The small heat shock protein HspB8: role in nervous system physiology and pathology.

The accumulation and aggregation of misfolded proteins can be highly cytotoxic and may underlie several human degenerative diseases characterized by neuronal inclusions such as Alzheimer's, Parkinson's, prion-like and polyglutamine repeat diseases. In this context small heat shock proteins, molecular chaperones known to be induced by cell stress, play a fundamental role by facilitating folding of nascent polypeptides, preventing aggregation of misfolded proteins and enhancing their degradation. A recently identified member of the small heat shock protein family, HspB8, is of particular interest in the field of neurological diseases since mutations in its sequence correlate with development of distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 expression has been detected in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington disease and spinocerebellar ataxia type 3. In the latter, HspB8 appears to be involved in protecting the cell from accumulation of insoluble aggregates either by preventing aggregation or by promoting degradation of improperly folded proteins. These data propose that HspB8 may be a major player in the neuroprotective response and a promising target for the development of therapeutic strategies.

Role of lipid rafts and GM1 in the segregation and processing of prion protein.

The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.

The intracellular delivery of TAT-aequorin reveals calcium-mediated sensing of environmental and symbiotic signals by the arbuscular mycorrhizal fungus Gigaspora margarita.

Arbuscular mycorrhiza (AM) is an ecologically relevant symbiosis between most land plants and Glomeromycota fungi. The peculiar traits of AM fungi have so far limited traditional approaches such as genetic transformation. The aim of this work was to investigate whether the protein transduction domain of the HIV-1 transactivator of transcription (TAT) protein, previously shown to act as a potent nanocarrier for macromolecule delivery in both animal and plant cells, may translocate protein cargoes into AM fungi. We evaluated the internalization into germinated spores of Gigaspora margarita of two recombinant TAT fusion proteins consisting of either a fluorescent (GFP) or a luminescent (aequorin) reporter linked to the TAT peptide. Both TAT-fused proteins were found to enter AM fungal mycelia after a short incubation period (5-10 min). Ca2+ measurements in G. margarita mycelia pre-incubated with TAT-aequorin demonstrated the occurrence of changes in the intracellular free Ca2+ concentration in response to relevant stimuli, such as touch, cold, salinity, and strigolactones, symbiosis-related plant signals. These data indicate that the cell-penetrating properties of the TAT peptide can be used as an effective strategy for intracellularly delivering proteins of interest and shed new light on Ca2+ homeostasis and signalling in AM fungi.

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7.

BACKGROUND: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. METHODS: Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. RESULTS: Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>10(3)M(-1)s(-1)), but not by Trx. By surface plasmon resonance analysis, a KD=5.2µM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. CONCLUSIONS: GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. GENERAL SIGNIFICANCE: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.