E and M SARS-CoV-2 membrane protein expression and enrichment with plant lipid droplets.

Plants are gaining traction as a cost-effective and scalable platform for producing recombinant proteins. However, expressing integral membrane proteins in plants is challenging due to their hydrophobic nature. In our study, we used transient and stable expression systems in Nicotiana benthamiana and Camelina sativa respectively to express SARS-CoV-2 E and M integral proteins, and target them to lipid droplets (LDs). LDs offer an ideal environment for folding hydrophobic proteins and aid in their purification through flotation. We tested various protein fusions with different linkers and tags and used three dimensional structure predictions to assess their effects. E and M mostly localized in the ER in N. benthamiana leaves but E could be targeted to LDs in oil accumulating tobacco when fused with oleosin, a LD integral protein. In Camelina sativa seeds, E and M were however found associated with purified LDs. By enhancing the accumulation of E and M within LDs through oleosin, we enriched these proteins in the purified floating fraction. This strategy provides an alternative approach for efficiently producing and purifying hydrophobic pharmaceuticals and vaccines using plant systems.

PUX10 Is a CDC48A Adaptor Protein That Regulates the Extraction of Ubiquitinated Oleosins from Seed Lipid Droplets in Arabidopsis.

Postgerminative mobilization of neutral lipids stored in seed lipid droplets (LDs) is preceded by the degradation of oleosins, the major structural LD proteins that stabilize LDs in dry seeds. We previously showed that Arabidopsis thaliana oleosins are marked for degradation by ubiquitination and are extracted from LDs before proteolysis. However, the mechanisms underlying the dislocation of these LD-anchored proteins from the LD monolayer are yet unknown. Here, we report that PUX10, a member of the plant UBX-domain containing (PUX) protein family, is an integral LD protein that associates with a subpopulation of LDs during seed germination. In pux10 mutant seedlings, PUX10 deficiency impaired the degradation of ubiquitinated oleosins and prevented the extraction of ubiquitinated oleosins from LDs. We also showed that PUX10 interacts with ubiquitin and CDC48A, the AAA ATPase Cell Division Cycle 48, through its UBA and UBX domains, respectively. Collectively, these results strongly suggest that PUX10 is an adaptor recruiting CDC48A to ubiquitinated oleosins, thus facilitating the dislocation of oleosins from LDs by the segregase activity of CDC48A. We propose that PUX10 and CDC48A are core components of a LD-associated degradation machinery, which we named the LD-associated degradation system. Importantly, PUX10 is also the first determinant of a LD subpopulation described in plants, suggesting functional differentiation of LDs in Arabidopsis seedlings.

Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis.

In oleaginous seeds, lipids--stored in organelles called oil bodies (OBs)--are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1-OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1-OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub(2)) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub(2)-oleosin aggregates. These results indicate that K48Ub(2)-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization.

Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds.

Oil bodies (OBs) are seed-specific lipid storage organelles that allow the accumulation of neutral lipids that sustain plantlet development after the onset of germination. OBs are covered with specific proteins embedded in a single layer of phospholipids. Using fluorescent dyes and confocal microscopy, we monitored the dynamics of OBs in living Arabidopsis (Arabidopsis thaliana) embryos at different stages of development. Analyses were carried out with different genotypes: the wild type and three mutants affected in the accumulation of various oleosins (OLE1, OLE2, and OLE4), three major OB proteins. Image acquisition was followed by a detailed statistical analysis of OB size and distribution during seed development in the four dimensions (x, y, z, and t). Our results indicate that OB size increases sharply during seed maturation, in part by OB fusion, and then decreases until the end of the maturation process. In single, double, and triple mutant backgrounds, the size and spatial distribution of OBs are modified, affecting in turn the total lipid content, which suggests that the oleosins studied have specific functions in the dynamics of lipid accumulation.

The context of HLA-DR/CD18 complex in the plasma membrane governs HLA-DR-derived signals in activated monocytes.

HLA-DR-derived signals in activated monocytes mediate both pro-inflammatory cytokine production and caspase-independent death, and have been postulated to play a role in inflammation and in its resolution, respectively. Herein, using the monocytic/macrophagic human cell line THP-1 primed with IFNgamma (IFNgamma-primed THP-1), we investigated how HLA-DR may integrate both signals. Our inhibition studies demonstrated that if cell death is dependent on PKCbeta activation, the induction of TNFalpha gene expression relies on PTK activation, in particular the Src family of kinases, but both cell responses implicate the beta2-integrin CD18. Accordingly, sequential immunoprecipitation experiments demonstrated that following engagement of HLA-DR on IFNgamma-primed THP-1 cells, the HLA-DR/CD18 complex physically associates with PKCbeta and with PTK. Pharmacological disruption of lipid rafts microdomains abolished the assembly of HLA-DR/CD18/PTK signaling complex, HLA-DR-mediated tyrosine activation, and the PTK-dependent TNFalpha expression in IFNgamma-primed THP-1 cells. In contrast, HLA-DR/CD18/PKCbeta complex was still formed and able to mediate cell death after cholesterol depletion of these cells. These results indicate that while the integrity of lipid rafts is necessary for the transduction of cytokine gene expression through the HLA-DR/CD18 complex, it is not necessary for the induction of the HLA-DR/CD18-dependent cell death. Thus, our study provides experimental evidence indicating the compartmentalization of HLA-DR/CD18 complex within or outside lipid rafts as a mechanism through which HLA-DR can integrate both PTK and PKCbeta signals leading to activation and death, respectively, of activated monocytes. This might provide new insights into how MHC class II signaling may regulate inflammatory response.

Class II transactivator (CIITA) isoform expression and activity in melanoma.

In contrast with melanocytes, melanomas display constitutive expression of HLA-DR (HLA-DR+). This abnormal expression has been associated with tumour progression and metastatic dissemination. We have previously reported that this deregulation of HLA-D genes is due to the abnormal constitutive expression of the lymphocyte-specific isoform of class II transactivator (B-CIITA), in addition to its fibroblast form (F-CIITA), which is usually expressed in major histocompatibility complex (MHC) class II-negative interferon-gamma-induced cell types, such as melanocytes. In this study, we investigated the abnormal expression of B-CIITA in a panel of melanoma cell lines displaying differential HLA-DR expression profiles, and analysed whether such a molecular event can participate in tumour progression. Our results showed that the abnormal expression of B-CIITA did not have any particular effect, in comparison with F-CIITA, on the classical activity of CIITA HLA-D gene regulation. As CIITA has also been shown to regulate genes other than HLA-D, we evaluated the modulation of those encoding cyclin D1, YARS (tyrosyl-tRNA synthetase) and TRIP1 (transforming growth factor (TGF)-beta receptor-interacting protein), proteins involved in cell cycle/apoptosis balance, angiogenesis and resistance to TGF-beta, respectively. In contrast with other cell types, neither B-CIITA nor F-CIITA was able to modulate these genes in melanoma cell lines. Thus, the activity of CIITA, whether lymphocyte-specific or fibroblast-specific, is restricted to HLA-D gene expression in these tumours. Accordingly, our data suggest that CIITA is not involved per se in tumour progression; rather, it is the MHC class II molecules themselves, through tumour antigen presentation and the induction of tumour antigen-specific CD4 lymphocyte anergy, that may participate in immune escape and melanoma progression.