Search

Cereal and legume seeds are the primary source of food protein for humans and farm animals. Both major classes of seed proteins, globulins and prolamins, are multimeric proteins that start their life in the ER, where they are co-translationally folded and assembled. Biotic and abiotic stresses alter endoplasmic reticulum (ER) proteostasis and may therefore disturb proper seed protein accumulation, inducing the unfolded protein response (UPR) an as adaptive reaction to restore proper cellular homeostasis. Climate change, which obviously affects plant growth and crop production, may therefore also influence the nutritional quality and technological properties of seed proteins. After folding and assembly in the ER, legume globulins are then deposited in storage vacuoles (PSV) via the secretory pathway, whether cereal prolamins accumulate directly in the ER lumen, forming protein bodies. Thus, during seed development, the functions of the ER are challenged by the massive synthesis of storage proteins, but it is unknown if this physiological process engages stress response mechanisms. In order to assess the physiological extent of the ER stress response during the accumulation of storage proteins, we compared by RT-PCR the expression of UPR/autophagy genes in wild type A. thaliana plants and in transgenic Arabidopsis constitutively expressing 27kD gamma-zein, 16kD gamma-zein, 27kD 1C (a mutated version of 27kd gamma-zein that is not retained in the ER lumen) or phaseolin (PHSL, the 7S vacuolar globulin of P. vulgaris). The analysis shows that gamma-zein polypeptides, but not PHSL or 27kD 1C, trigger the UPR. This indicates that the engagement of UPR is physiological during prolamin deposition in protein bodies, but is not necessary to sustain the synthesis of an oligomeric storage protein that traffic out of the ER. Supported by the FILAGRO Project of CNR-Regione Lombardia

700

Tonoplast, the membrane delimiting plant vacuoles, regulates ion, water and nutrient movement between the cytosol and the vacuolar lumen through the activity of its membrane proteins. Correct traffic of proteins from the endoplasmic reticulum (ER) to the tonoplast requires (i) approval by the ER quality control, (ii) motifs for exit from the ER and (iii) motifs that promote sorting to the tonoplast. Recent evidence suggests that this traffic follows different pathways that are protein-specific and could also reflect vacuole specialization for lytic or storage function. The routes can be distinguished based on their sensitivity to drugs such as brefeldin A and C834 as well as using mutant plants that are defective in adaptor proteins of vesicle coats, or dominant-negative mutants of Rab GTPases.

Maize seed storage proteins (zeins) accumulate as insoluble heteropolymers (protein bodies) in the endoplasmic reticulum (ER). Gamma-zein is a major component, assembles into protein bodies also when synthesized in the absence of its partners and is composed of a C-terminal domain with four intrachain disulfide bonds, preceded by an N-terminal part that contains seven additional Cys residues and a Pro-rich repeated region. By transient expression in tobacco protoplasts, we have analyzed the relationships between assembly, solubility and intracellular traffic of gamma-zein in which either the first two, six or all Cys residues of the N-terminal part have been mutated to Ser. Our results indicate that gamma-zein can assemble as soluble dimers, most probably through hydrophobic interactions between the repeated domains. In the absence of further assembly, these dimers are largely secreted and in part sorted to the vacuole The formation of interchain disulfide bonds, to an extent that depends on the availability of Cys residues in the N-terminal half, allows further assembly into insoluble polymers and inhibits traffic from the ER. Supported by the 2006 Accordo Quadro CNR-Regione Lombardia.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is therefore a potential issue in the fight against T1DM but current production platforms are expensive. GAD65 has previously been expressed in transgenic tobacco plants. Here, we show that a catalytically-inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein, which is more than10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be due to a higher rate of protein synthesis, and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.

The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non -cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 37,000 people in Italy and has caused widespread socioeconomic disruption. Urgent measures are needed to contain and control the virus, particularly diagnostic kits for detection and surveillance, therapeutics to reduce mortality among the severely affected, and vaccines to protect the remaining population. Here we discuss the potential role of plant molecular farming in the rapid and scalable supply of protein antigens as reagents and vaccine candidates, antibodies for virus detection and passive immunotherapy, other therapeutic proteins, and virus-like particles as novel vaccine platforms. We calculate the amount of infrastructure and production capacity needed to deal with predictable subsequent waves of COVID-19 in Italy by pooling expertise in plant molecular farming, epidemiology and the Italian health system. We calculate the investment required in molecular farming infrastructure that would enable us to capitalize on this technology, and provide a roadmap for the development of diagnostic reagents and biopharmaceuticals using molecular farming in plants to complement production methods based on the cultivation of microbes and mammalian Accepted manuscript:https://www.frontiersin.org/articles/10.3389/fpls.2020.609910/full

What happens when we use the word context, in research and learning? Are we aware of its different connotations? Or do we simply use it to evoke anything “surrounding” learning? When we begin to analyse the contexts of learning, we stumble over many antinomies and conceptual polarities. Different perspectives define what a context is, and what it does to us. There are at least four of them: the materialist/structural, the constructivist/symbolic, the reflexive/recursive, and the enactive/co-evolutionary. The chapter plays with all these lenses, using etymology, imagination, and narratives to suggest a generative theory of context.

In our society the school is one of the most exclusive places to educate the new generations, so we put trust and many expectations on it. As institution, it has a particular organization of spaces, times, rules, roles and its practices are based on rigorous hierarchies, discipline and authority, training children and young people to become suitable for the society in which they live. School reflects the dominant educational culture and spreads ideas that even if unconsciously internalized, have an indepth action on students. Its spaces are often detached and devoid of beauty and sensibility; inside school everything is under control and quantified, making learning and knowledge influenced by the market laws of our society. It rather focuses the attention on profit and results than on every student's personal path and makes culture something to get close to in a bored attitude and without any interest and passion. That's why in these years Paolo Mottana, a professor of the University of Milan-Bicocca, elaborated a theoretical reflection called "joyful and diffused education" to think about education again and to create educational experiences out of the school as we know it; in this new way of thinking about education the entire society and not only the school is the best context in which children and young people can learn and live rich, deep and exciting experiences. The town becomes an important experience place, in which young people can be real actors and not only spectators of their learning activities and of the city life. But town has to change its spaces, times and rhythms to welcome young generations, to pay them attention, to take care of them and to set their vitality, creativity and competences off. Starting from the joyful and diffused education reflection, Paolo Mottana and his research group elaborated the "Educational District", an experimental project in collaboration with some lower secondary schools of a district in Milan. It wants to foster a more inductive way of learning, which starts from the students' curiosity and the experiences lived out of the school, building real and diversified ways of discovering, far from the simulations and simplified experiences lived in the traditional school system. It also wants to satisfy young people's need for independence and involvement, promoting their attitudes, making them co-planners and letting them play a main role in their schooling, which needs to be more individual and personalized. The school needs to become a more open and flexible place, a "hole", a base, a meeting point to go and come back to talk, discuss and rework the experiences lived in the town. In this way school could get closer to young people's life and create strong links with the territory, being not the only educational institution and recognizing the importance of sharing this responsibility, this task with the territory and the entire society, trying to make a real cultural revolution

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.

For many years our knowledge on hepatitis C virus (HCV) replication has been based on in vitro experiments or transfection studies. Recently, the first reliable system for studying viral replication in tissue culture cells was developed. Taking advantage of this system, we examined in detail the localization of viral nonstructural (NS) proteins in cells containing functional replication complexes. By fractionation experiments and immunomicroscopy, we observed that all NS proteins were associated with the endoplasmic reticulum (ER) membranes, confirming the hypothesis that the ER is the site of membrane-associated HCV RNA replication. Interestingly, NS3 and NS4A were preferentially localized in endoplasmic reticulum cisternae surrounding mitochondria, suggesting additional subcellular compartment-related functions for these viral proteins. Furthermore, the immunoelectron microscopy revealed the loss of the organization and other morphological alterations of the ER (convoluted cisternae and paracrystalline structures), resembling alterations observed in liver biopsies of HCV-infected individuals and in flavivirus-infected cells.

Endoplasmic reticulum (ER) proteins maintain their residency by static retention, dynamic retrieval, or a combination of the two. Tail-anchored proteins that contain a cytosolic domain associated with the lipid bilayer via a hydrophobic stretch close to the COOH terminus are sorted within the secretory pathway by largely unknown mechanisms. Here, we have investigated the mode of insertion in the bilayer and the intracellular trafficking of cytochrome b(5) (b[5]), taken as a model for ER-resident tail-anchored proteins. We first demonstrated that b(5) can acquire a transmembrane topology posttranslationally, and then used two tagged versions of b(5), N-glyc and O-glyc b(5), containing potential N- and O-glycosylation sites, respectively, at the COOH-terminal lumenal extremity, to discriminate between retention and retrieval mechanisms. Whereas the N-linked oligosaccharide provided no evidence for retrieval from a downstream compartment, a more stringent assay based on carbohydrate acquisition by O-glyc b(5) showed that b(5) gains access to enzymes catalyzing the first steps of O-glycosylation. These results suggest that b(5) slowly recycles between the ER and the cis-Golgi complex and that dynamic retrieval as well as retention are involved in sorting of tail-anchored proteins.