ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication

The β-coronavirus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the global Covid-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release and pathogenesis. We developed a non-disruptive tagging strategy for SARS-CoV-2 E and find that at steady-state, it localises to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for Endoplasmic Reticulum (ER)-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP Ribosylation Factor-1/Adaptor Protein-1 (ARFRP1/AP-1) dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and by developing a trans-complementation assay for SARS-CoV-2 structural proteins, we show that lysosomal delivery of E and its viroporin activity are necessary for efficient viral replication and release

Inhibition of peptide aggregation by means of enzymatic phosphorylation

As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP- dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β -sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated

Phosphorylation of toxoplasma gondii secreted proteins during acute and chronic stages of infection

ABSTRACT The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion. IMPORTANCE Toxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated

Crystal structure of the human, FIC-Domain containing protein HYPE and implications for its functions

Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition

Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase

Synthetische Phosphopeptide und Phosphoproteine als Werkzeuge zum Studium der Aggregation von Peptiden und Proteinen

The aggregation of soluble proteins to protease-resistant fibrils is a hallmark of neurodegenerative diseases. This phenomenon has been observed in proteins that under certain conditions undergo a structural transition from an unfolded or partially helical state to a β-sheet structure. Factors such as a solution’s pH, protein concentration, presence of metal ions, and temperature have been identified as triggers of protein aggregation. In addition, post translational modifications, e.g. phosphorylation, have been implicated in aberrant protein fibrillization over the last two decades. However, current studies on phosphorylation patterns in natural aggregating systems suffer from major drawbacks; for instance, rapid dephosphorylation of phosphoproteins in vivo renders the identification of key phosphorylated residues difficult, whereas in vitro enzymatic phosphorylations generate heterogeneous mixtures of proteins phosphorylated at various sites and to different extent. This work presents several examples of a successful application of synthetic phosphopeptides as tools for studying amyloid formation. Using such analytical techniques as circular dichroism spectrscopy, Thioflavine T-induced fluorescence, and Transmission Electron Microscopy various aspects of peptide and protein fibrillization were investigated. For example, the influence of site-specific and multiple phosphorylation on the amyloid formation process was examined utilizing an amyloid forming coiled coil peptide model. Structural analysis of phosphorylated peptides revealed that regardless of the quantity and position of modification, the formation of β sheet structure was completely abolished. Furthermore, all phosphorylated peptides completely lost their amyloid forming potential. Subsequently, with the objective to understand and monitor structure switching as well as fibril formation and morphology under conditions that approximate physiological environment, a dephosphorylating enzyme was applied as a natural aggregation trigger. In addition, synthetic phosphopeptides were applied to investigate metal-induced conformational transitions. Coordination of magnesium and manganese ions to phosphate groups induced vigorous structural changes, resulting in stable α helices and helical fibers, respectively. Finally, in addition to the aforementioned investigations of coiled coil model systems, also phosphorylation in a natural aggregating system was probed. Here, a combination of synthetic and recombinant approaches (expressed protein ligation) was used to generate homogenous preparations of site-specifically phosphorylated microtubule-assisted tau protein – a major component of neurofibrillary tangles, the primary marker of Alzheimer’s disease.Die Aggregation löslicher Proteine zu proteolytisch stabilen Fibrillen ist ein Kennzeichen neurodegenerativer Krankheiten. Dieses Phänomen wurde bei Proteinen beobachtet, die unter bestimmten Bedingungen strukturelle Übergänge von einer ungefalteten oder teilweise helikalen Konformation zu einer β-Faltblattstruktur durchlaufen. Umgebungsfaktoren wie der pH-Wert, die Anwesenheit von Metallionen, die Temperatur sowie die Proteinkonzentration selbst wurden als Auslöser der Proteinaggregation identifiziert. In den letzten zwei Jahrzehnten wurde aber auch die Rolle posttranslationaler Modifikationen wie z.B. der Phosphorylierung in diesem Zusammenhang diskutiert. Die Untersuchung der Rolle der Phosphorylierung in natürlichen Systemen sieht sich jedoch mit einigen schwerwiegenden Problemen konfrontiert. Der schnelle Abbau von Phosphoproteinen in vivo macht es beispielsweise schwierig, phosphorylierte Reste genau zu identifizieren. Auf der anderen Seite führt die enzymatische Phosphorylierung in vitro oftmals zu heterogenen Produkten mit unterschiedlichem Phosphorylierungsgrad. Im Rahmen der vorliegenden Arbeit werden einige erfolgreiche Anwendungen synthetischer Phosphopeptide als Modelle zum Studium der Proteinaggregation vorgestellt. Verschiedene Aspekte der Fibrillenbildung wurden mit Hilfe unterschiedlicher analytischer Methoden wie CD-Spektroskopie, Thioflavin-T induzierter Fluoreszenz sowie Transmissionselektronenspektroskopie untersucht. Hierbei wurde z.B. der Einfluss ortsspezifischer und multipler Phosphorylierung auf das Aggregationsverhalten eines Amyloid bildenden Coiled-Coil-Modellpeptids untersucht. Die strukturellen Analysen haben gezeigt, dass die Umfaltung zum β-Faltblatt unabhängig von der Anzahl und Position modifizierter Reste durch die Phosphorylierung vollkommen unterdrückt wird. Sämtliche phosphorylierte Peptide zeigten darüber hinaus keine Neigung zur Bildung vom Amyloiden. Um die Prozesse der Umfaltung und Fibrillenbildung, sowie deren Morphologien unter physiologischen Bedingungen besser verstehen zu können, wurde im Weiteren ein Enzym, das Phosphatgruppen abspaltet, als natürlicher Auslöser der Aggregation eingesetzt. Weiterhin wurden durch Metallionen induzierte konformationelle Übergänge an synthetischen Phosphopeptiden untersucht. Dabei wurde gezeigt, dass die Koordination von Magnesium- und Manganionen an Phosphate ausgeprägte strukturelle Veränderungen, wie zum Beispiel die Bildung stabiler α-Helizes bzw. helikaler Fibrillen zur Folge haben. Zusätzlich zu diesen Untersuchungen wurde auch die Phosphorylierung eines natürlichen aggregierenden Systems untersucht. Dabei kam eine Kombination aus rekombinanter Proteinexpression und Peptidsynthese zum Einsatz, um homogene Präparate ortsspezifisch phosphorylierten Tau-Proteins, einer wichtigen Komponente von Neurofibrillen und dem primären Kennzeichen der Alzheimerschen Krankheit, darzustellen

PerTurboID, a targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria-causing parasites

Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the Plasmodium falciparum-exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria-causing parasite, PfEMP1. We generated independent TurboID fusions of two proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein–protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche

A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells

Abstract The propagation of Toxoplasma gondii is accomplished by repeated lytic cycles of parasite attachment to a host cell, invasion, replication within a parasitophorous vacuole, and egress from the cell. This lytic cycle is delicately regulated by calcium-dependent reversible phosphorylation of the molecular machinery that drives invasion and egress. While much progress has been made elucidating the protein kinases and substrates central to parasite propagation, little is known about the relevant protein phosphatases. In this study, we focused on the five protein phosphatases that are predicted to be membrane-associated either integrally or peripherally. We have determined that of these only PPM5C, a PP2C family member, localizes to the plasma membrane of Toxoplasma. Disruption of PPM5C results in a slow propagation phenotype in tissue culture. Interestingly, parasites lacking PPM5C divide and undergo egress at a normal rate, but have a deficiency in attaching to host cells. Both membrane localization and phosphatase activity are required for PPM5C’s role in attachment. Phosphoproteomic analysis show relatively few phosphorylation sites being affected by PPM5C deletion in extracellular parasites of which several are found on proteins involved in signaling cascades. This implies that PPM5C is part of a wider regulatory network important for attachment to host cells