Refinement of an ovine-based immunoglobulin therapy against SARS-CoV-2, with comparison of whole IgG versus F(ab')2 fragments.

The development of new therapies against SARS-CoV-2 is required to extend the toolkit of intervention strategies to combat the global pandemic. In this study, hyperimmune plasma from sheep immunised with whole spike SARS-CoV-2 recombinant protein has been used to generate candidate products. In addition to purified IgG, we have refined candidate therapies by removing non-specific IgG via affinity binding along with fragmentation to eliminate the Fc region to create F(ab')2 fragments. These preparations were evaluated for in vitro activity and demonstrated to be strongly neutralising against a range of SARS-CoV-2 strains, including Omicron B2.2. In addition, their protection against disease manifestations and viral loads were assessed using a hamster SARS-CoV-2 infection model. Results demonstrated protective effects of both IgG and F(ab')2, with the latter requiring sequential dosing to maintain in vivo activity due to rapid clearance from the circulation.

Development of a cost-effective ovine antibody-based therapy against SARS-CoV-2 infection and contribution of antibodies specific to the spike subunit proteins.

Antibodies against SARS-CoV-2 are important to generate protective immunity, with convalescent plasma one of the first therapies approved. An alternative source of polyclonal antibodies suitable for upscaling would be more amendable to regulatory approval and widespread use. In this study, sheep were immunised with SARS-CoV-2 whole spike protein or one of the subunit proteins: S1 and S2. Once substantial antibody titres were generated, plasma was collected and samples pooled for each antigen. Non-specific antibodies were removed via affinity-purification to yield candidate products for testing in a hamster model of SARS-CoV-2 infection. Affinity-purified polyclonal antibodies to whole spike, S1 and S2 proteins were evaluated for in vitro for neutralising activity against SARS-CoV-2 Wuhan-like virus (Australia/VIC01/2020) and a recent variant of concern, B.1.1.529 BA.1 (Omicron), antibody-binding, complement fixation and phagocytosis assays were also performed. All antibody preparations demonstrated an effect against SARS-CoV-2 disease in the hamster model of challenge, with those raised against the S2 subunit providing the most promise. A rapid, cost-effective therapy for COVID-19 was developed which provides a source of highly active immunoglobulin specific to SARS-CoV-2 with multi-functional activity.

RNA-Seq and metabolic flux analysis of Tetraselmis sp. M8 during nitrogen starvation reveals a two-stage lipid accumulation mechanism.

To map out key lipid-related pathways that lead to rapid triacylglyceride accumulation in oleaginous microalgae, RNA-Seq was performed with Tetraselmis sp. M8 at 24h after exhaustion of exogenous nitrogen to reveal molecular changes during early stationary phase. Further gene expression profiling by quantitative real-time PCR at 16-72h revealed a distinct shift in expression of the fatty acid/triacylglyceride biosynthesis and ß-oxidation pathways, when cells transitioned from log-phase into early-stationary and stationary phase. Metabolic reconstruction modeling combined with real-time PCR and RNA-Seq gene expression data indicates that the increased lipid accumulation is a result of a decrease in lipid catabolism during the early-stationary phase combined with increased metabolic fluxes in lipid biosynthesis during the stationary phase. During these two stages, Tetraselmis shifts from reduced lipid consumption to active lipid production. This process appears to be independent from DGAT expression, a key gene for lipid accumulation in microalgae.

Isolation and evaluation of oil-producing microalgae from subtropical coastal and brackish waters.

Microalgae have been widely reported as a promising source of biofuels, mainly based on their high areal productivity of biomass and lipids as triacylglycerides and the possibility for cultivation on non-arable land. The isolation and selection of suitable strains that are robust and display high growth and lipid accumulation rates is an important prerequisite for their successful cultivation as a bioenergy source, a process that can be compared to the initial selection and domestication of agricultural crops. We developed standard protocols for the isolation and cultivation for a range of marine and brackish microalgae. By comparing growth rates and lipid productivity, we assessed the potential of subtropical coastal and brackish microalgae for the production of biodiesel and other oil-based bioproducts. This study identified Nannochloropsis sp., Dunaniella salina and new isolates of Chlorella sp. and Tetraselmis sp. as suitable candidates for a multiple-product algae crop. We conclude that subtropical coastal microalgae display a variety of fatty acid profiles that offer a wide scope for several oil-based bioproducts, including biodiesel and omega-3 fatty acids. A biorefinery approach for microalgae would make economical production more feasible but challenges remain for efficient harvesting and extraction processes for some species.

The family of Deg/HtrA proteases in plants.

BACKGROUND: The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms from bacteria to human and vascular plants. In recent years, multiple deg/htrA protease genes were identified in various plant genomes. During genome annotations most proteases were named according to the order of discovery, hence the same names were sometimes given to different types of Deg/HtrA enzymes in different plant species. This can easily lead to false inference of individual protease functions based solely on a shared name. Therefore, the existing names and classification of these proteolytic enzymes does not meet our current needs and a phylogeny-based standardized nomenclature is required. RESULTS: Using phylogenetic and domain arrangement analysis, we improved the nomenclature of the Deg/HtrA protease family, standardized protease names based on their well-established nomenclature in Arabidopsis thaliana, and clarified the evolutionary relationship between orthologous enzymes from various photosynthetic organisms across several divergent systematic groups, including dicots, a monocot, a moss and a green alga. Furthermore, we identified a "core set" of eight proteases shared by all organisms examined here that might provide all the proteolytic potential of Deg/HtrA proteases necessary for a hypothetical plant cell. CONCLUSIONS: In our proposed nomenclature, the evolutionarily closest orthologs have the same protease name, simplifying scientific communication when comparing different plant species and allowing for more reliable inference of protease functions. Further, we proposed that the high number of Deg/HtrA proteases in plants is mainly due to gene duplications unique to the respective organism.

Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal ß-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance.

Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism.

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

A new principle of oligomerization of plant DEG7 protease based on interactions of degenerated protease domains.

Deg/HtrA proteases are a large group of ATP-independent serine endoproteases found in almost every organism. Their usual domain arrangement comprises a trypsin-type protease domain and one or more PDZ domains. All Deg/HtrA proteases form homo-oligomers with trimers as the basic unit, where the active protease domain mediates the interaction between individual monomers. Among the members of the Deg/HtrA protease family, the plant protease DEG7 is unique since it contains two protease domains (one active and one degenerated) and four PDZ domains. In the present study, we investigated the oligomerization behaviour of this unusual protease using yeast two-hybrid analysis in vivo and with recombinant protein in vitro. We show that DEG7 forms trimeric complexes, but in contrast with other known Deg/HtrA proteases, it shows a new principle of oligomerization, where trimerization is based on the interactions between degenerated protease domains. We propose that, during evolution, a duplicated active protease domain degenerated and specialized in protein-protein interaction and complex formation.

Deg/HtrA proteases as components of a network for photosystem II quality control in chloroplasts and cyanobacteria.

Organisms that perform oxygenic photosynthesis are subjected to photoinhibition of their photosynthetic function when exposed to excessive illumination. The main target of photoinhibition is the D1 protein in the reaction center of the photosystem II complex. Rapid degradation of photodamaged D1 protein and its replacement by a de novo synthesized functional copy represent an important repair mechanism crucial for cell survival under light stress conditions. This review summarizes the literature on the ATP-independent Deg/HtrA family of serine endopeptidases in cyanobacteria and chloroplasts of higher plants, and discusses their role in D1 protein degradation. We propose that Deg/HtrA proteases are part of a larger network of enzymes that ensure protein quality control, including photosystem II, in plants and cyanobacteria.

The DEG15 serine protease cleaves peroxisomal targeting signal 2-containing proteins in Arabidopsis.

Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. Recently, candidates for the responsible processing protease were identified from plants (DEG15) and mammals (TYSND1). We demonstrate that plants lacking DEG15 show an expressed phenotype potentially linked to reduced beta-oxidation, indicating the impact of protein processing on peroxisomal functions in higher eukaryotes. Mutational analysis of Arabidopsis (Arabidopsis thaliana) DEG15 revealed that conserved histidine, aspartic acid, and serine residues are essential for the proteolytic activity of this enzyme in vitro. This indicates that DEG15 and related enzymes are trypsin-like serine endopeptidases. Deletion of a plant-specific stretch present in the protease domain diminished, but did not abolish, the proteolytic activity of DEG15 against the PTS2-containing glyoxysomal malate dehydrogenase. Fluorescence microscopy showed that a DEG15-green fluorescent protein fusion construct is targeted to peroxisomes in planta. In vivo studies with isolated homozygous deg15 knockout mutants and complemented mutant lines suggest that this enzyme mediates general processing of PTS2-containing proteins.

Photodamaged D1 protein is degraded in Arabidopsis mutants lacking the Deg2 protease.

In plants exposed to high irradiances of visible light, the D1 protein in the reaction center of photosystem II is oxidatively damaged and rapidly degraded. Earlier work in our laboratory showed that the serine protease Deg2 performs the primary cleavage of photodamaged D1 protein in vitro. Here, we demonstrate that the rate of D1 protein degradation under light stress conditions in Arabidopsis mutants lacking the Deg2 protease is similar to those in wild-type plants. Therefore, we propose that several redundant D1 protein degradation pathways might exist in vivo.

The linear pentadecapeptide gramicidin is assembled by four multimodular nonribosomal peptide synthetases that comprise 16 modules with 56 catalytic domains.

Linear gramicidin is a membrane channel forming pentadecapeptide that is produced via the nonribosomal pathway. It consists of 15 hydrophobic amino acids with alternating l- and d-configuration forming a beta-helix-like structure. It has an N-formylated valine and a C-terminal ethanolamine. Here we report cloning and sequencing of the entire biosynthetic gene cluster as well as initial biochemical analysis of a new reductase domain. The biosynthetic gene cluster was identified on two nonoverlapping fosmids and a 13-kilobase pair (kbp) interbridge fragment covering a region of 74 kbp. Four very large open reading frames, lgrA, lgrB, lgrC, and lgrD with 6.8, 15.5, 23.3, and 15.3 kbp, were identified and shown to encode nonribosomal peptide synthetases with two, four, six, and four modules, respectively. Within the 16 modules identified, seven epimerization domains in alternating positions were detected as well as a putative formylation domain fused to the first module LgrA and a putative reductase domain attached to the C-terminal module of LgrD. Analysis of the substrate specificity by phylogenetic studies using the residues of the substrate-binding pockets of all 16 adenylation domains revealed a good agreement of the substrate amino acids predicted with the sequence of linear gramicidin. Additional biochemical analysis of the three adenylation domains of modules 1, 2, and 3 confirmed the colinearity of this nonribosomal peptide synthetase assembly line. Module 16 was predicted to activate glycine, which would then, being the C-terminal residue of the peptide chain, be reduced by the adjacent reductase domain to give ethanolamine, thereby releasing the final product N-formyl-pentadecapeptide-ethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro.