<p dir="ltr">Membrane-dependent enzymes play crucial roles in cellular signaling by transducing extracellular signals into intracellular responses. Phospholipase Cepsilon (PLCe) and diacylglycerol kinase alpha (DGKa) are membrane-associated enzymes regulated by G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), controlling signaling pathways essential for numerous cellular processes. PLCe catalyzes the hydrolysis of phosphatidylinositol phosphates into inositol phosphates (IPX) and diacylglycerol (DAG), triggering calcium release from intracellular stores and activating protein kinase C (PKC)-dependent pathways. While PLCe is crucial for normal cardiovascular function, hyperactivation or sustained activation can lead to hypertrophy. Due to structural heterogeneity, previous studies focused on isolated regulatory domains or the catalytic core. In this work, I present the first cryo-EM reconstruction of the largest PLCe fragment to date in complex with an antigen-binding fragment (Fab). This structure reveals the domain architecture of the N-terminal regions of the lipase and defines an extended membrane-binding surface critical for maximal basal and G protein-dependent activity. These findings lay the groundwork for high-resolution structures of the full-length enzyme and its complexes with the small GTPase Rap1A. Additionally, I explored the role of mAKAP in the Rap1A–PLCe pathway, alongside the guanine nucleotide exchange factor (GEF) function of PLCe toward Rap1A. In parallel, cryo-EM studies of DGKa bound to a covalent inhibitor were initiated. DGKa reduces DAG, thereby limiting PKC activity, and its inhibition is emerging as a promising cancer immunotherapy target. We have established a protocol for structural studies of full-length DGKa, which will elucidate its structures in basal and inhibited states.</p>

Viruses are entities which are made of a few genes and are reliant on obligate parasitism to propagate. Due to the obligate connection to their hosts, virus evolution is constrained to the type of host. Viruses however do transmit to evolutionary distinct hosts; in these cases, the phylogenetic relationship of the hosts usually are close. In some instances, RNA-viruses have made host jumps between evolutionary distant hosts, such as the host jump from invertebrates to vertebrates, and fungi to arthropod. Partitiviruses are double stranded RNA viruses which mainly infect fungi and plants. The defining characteristic of these double stranded RNA viruses are the double layered capsids which are formed by a single open reading frame (ORF). The capsid proteins form icosahedral virus particles which are in the magnitude of 30-40 nm. Metagenomic studies have discovered partitiviruses originating from an insect in the Odanata family, a finding which contradicts the fungal host specificity of partitiviruses. The finding of the Hubei.PLV 11 thus implies the existence of a partitiviruses containing structural elements in their capsids which could be involved in the infection of arthropods. Thus, this virus could be used as a model for a structural comparison with its fungi infecting relatives with hopes to identify common viral structural factors necessary for the infection of arthropods. For this purpose, the Hubei.PLV ORF was cloned and then transfected into insect Spodoptera frugiperda (Sf-9) cells using a baculovirus expression system, “bac-to-bac” expression system. The FLAG-tagged capsid proteins were expressed by the Sf-9 cells to be approximately 60 kDa. After ultra-centrifugation in a sucrose gradient, some spontaneous assembly into the expected ~40 nm icosahedral virus-like particles were observed using low resolution scanning electron microscopy. The observed particles were also confirmed by a dynamic light scattering experiment (DLS) and a higher resolution cryo-EM microscope. Thus, the bac-to-bac expression system can be used to produce VLPs from this genus of viruses, and this metagenomically derived virus genome. However, for future success in defining a high-resolution model of this virus, it is recommended that the Sf-9 culture volume is sufficiently high for enough particle production which is necessary for a high-resolution map. The other virus, the Southern pygmy squid Flavilike virus (SpSFV) has been suggested to be the oldest relative of the land based flaviviruses. The SpSFV was found to be the most divergent of the flaviviruses, and to infect invertebrates. Solving for the structure of the SpSFV and comparing it to vertebrate infecting flaviviruses could therefore lead to the identification of factors necessary for the adaptation to vertebrates and thus the humoral immunity by flaviviruses. The soluble E-protein was expressed using the bac-to-bac expression system. The protein was indicated to be multiglycosylated and approximately 50 kDa which is in line with other strains in the genus. Affinity chromatography did not elute this protein, likely due to the His-tag not being spatially available. Cation exchange could elute some protein, but not much from the small ~30 mL culture. To conclude, VLP assembly was confirmed by the Hubei.PLV, thus, solving for the structure is a distinct possibility when a larger Sf-9 culture is used to produce the VLPs. For the SpSFV soluble E-protein, the protein is secreted into the supernatant of the Sf-9 cultures, making purification a possibility. For this, a large Sf-9 culture can be used to produce this protein and then purify it with a cat-ion exchange chromatography.