The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling.

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine-rich repeat receptor-like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per µg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1.

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

An Exploratory Review of the Potential of Lytic Proteins and Bacteriophages for the Treatment of Tuberculosis.

Tuberculosis (TB) is a highly prevalent infectious disease that causes more than 1.5 million deaths a year. More than 25% of TB deaths occur in Africa, and TB is South Africa's leading cause of death, with about 89,000 people dying of it yearly. The emergence of multidrug-resistant TB (MDR-TB) poses a significant threat to health security and could reverse the positive gains already made in the fight against TB. Antibiotic treatments are available, but side effects and the alarming increase in the prevalence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) will compromise the control of the spread and treatment of the disease. A promising option is to employ specialized enzymes encoded by bacteriophages, which destroy bacterial cell membranes and walls to treat tuberculosis. Phage therapy against bacteria is a known treatment that is now reemerging with lytic proteins. These proteins provide an alternative means to treat infectious diseases where conventional antibiotic regimens do not meet the requirements. This review explores and discusses the potential of lytic protein therapy as an antimicrobial strategy against M. tuberculosis and multidrug-resistant tuberculosis.

South Africa's indigenous microbial diversity for industrial applications: A review of the current status and opportunities.

The unique metagenomic, metaviromic libraries and indigenous micro diversity within Southern Africa have the potential for global beneficiation in academia and industry. Microorganisms that flourish at high temperatures, adverse pH conditions, and high salinity are likely to have enzyme systems that function efficiently under those conditions. These attributes afford researchers and industries alternative approaches that could replace existing chemical processes. Thus, a better understanding of African microbial/genetic diversity is crucial for the development of "greener" industries. A concerted drive to exploit the potential locked in biological resources has been previously seen with companies such as Diversa Incorporated and Verenium (Badische Anilin-und SodaFabrik-BASF) both building business models that pioneered the production of high-performance specialty enzymes for a variety of different industrial applications. The market potential and accompanying industry offerings have not been fully exploited in South Africa, nor in Africa at large. Utilization of the continent's indigenous microbial repositories could create long-lasting, sustainable growth in various production sectors, providing economic growth in resource-poor regions. By bolstering local manufacture of high-value bio-based products, scientific and engineering discoveries have the potential to generate new industries which in turn would provide employment avenues for many skilled and unskilled laborers. The positive implications of this could play a role in altering the face of business markets on the continent from costly import-driven markets to income-generating export markets. This review focuses on identifying microbially diverse areas located in South Africa while providing a profile for all associated microbial/genetically derived libraries in this country. A comprehensive list of all the relevant researchers and potential key players is presented, mapping out existing research networks for the facilitation of collaboration. The overall aim of this review is to facilitate a coordinated journey of exploration, one which will hopefully realize the value that South Africa's microbial diversity has to offer.

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants.

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate K(m) of 7.5 µm and V(max) of 1800 nmol min(-1) mg(-1) of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg(2+) or Mn(2+). Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains.

Recombinant Expression, Purification and PEGylation of DNA Ligases.

BACKGROUND: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source. OBJECTIVE: The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases. METHODS: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions. RESULTS: In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates. CONCLUSION: Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.

Protective immunity of plant-produced African horse sickness virus serotype 5 chimaeric virus-like particles (VLPs) and viral protein 2 (VP2) vaccines in IFNAR-/- mice.

Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.

A plant-biotechnology approach for producing highly potent anti-HIV antibodies for antiretroviral therapy consideration.

Despite a reduction in global HIV prevalence the development of a pipeline of new therapeutics or pre-exposure prophylaxis to control the HIV/AIDS epidemic are of high priority. Antibody-based therapies offer several advantages and have been shown to prevent HIV-infection. Plant-based production is efficient for several biologics, including antibodies. We provide a short review on the work by Singh et al., 2020 who demonstrated the transient production of potent CAP256-VRC26 broadly neutralizing antibodies. These antibodies have engineered posttranslational modifications, namely N-glycosylation in the fragment crystallizable region and O-sulfation of tyrosine residues in the complementary-determining region H3 loop. The glycoengineered Nicotiana benthamiana mutant (ΔXTFT) was used, with glycosylating structures lacking ß1,2-xylose and/or α1,3-fucose residues, which is critical for enhanced effector activity. The CAP256-VRC26 antibody lineage targets the first and second variable region of the HIV-1 gp120 envelope glycoprotein. The high potency of this lineage is mediated by a protruding O-sulfated tyrosine in the CDR H3 loop. Nicotiana benthamiana lacks human tyrosyl protein sulfotransferase 1, the enzyme responsible for tyrosine O-sulfation. The transient coexpression of the CAP256-VRC26 antibodies with tyrosyl protein sulfotransferase 1 in planta had restored the efficacy of these antibodies through the incorporation of the O-sulfation modification. This approach demonstrates the strategic incorporation of posttranslational modifications in production systems, which may have not been previously considered. These plant-produced CAP256-VRC26 antibodies have therapeutic as well as topical and systemic pre-exposure prophylaxis potential in enabling the empowerment of young girls and women given that gender inequalities remain a major driver of the epidemic.

Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications.

Broadly neutralising antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1), such as CAP256-VRC26 are being developed for HIV prevention and treatment. These Abs carry a unique but crucial post-translational modification (PTM), namely O-sulfated tyrosine in the heavy chain complementarity determining region (CDR) H3 loop. Several studies have demonstrated that plants are suitable hosts for the generation of highly active anti-HIV-1 antibodies with the potential to engineer PTMs. Here we report the expression and characterisation of CAP256-VRC26 bNAbs with posttranslational modifications (PTM). Two variants, CAP256-VRC26 (08 and 09) were expressed in glycoengineered Nicotiana benthamiana plants. By in planta co-expression of tyrosyl protein sulfotransferase 1, we installed O-sulfated tyrosine in CDR H3 of both bNAbs. These exhibited similar structural folding to the mammalian cell produced bNAbs, but non-sulfated versions showed loss of neutralisation breadth and potency. In contrast, tyrosine sulfated versions displayed equivalent neutralising activity to mammalian produced antibodies retaining exceptional potency against some subtype C viruses. Together, the data demonstrate the enormous potential of plant-based systems for multiple posttranslational engineering and production of fully active bNAbs for application in passive immunisation or as an alternative for current HIV/AIDS antiretroviral therapy regimens.

Intramolecular crosstalk between catalytic activities of receptor kinases.

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Recombinant expression and functional testing of candidate adenylate cyclase domains.

Adenylate cyclases (ACs) are enzymes capable of converting adenosine-5'-triphosphate to cyclic 3', 5'--adenosine monophosphate (cAMP). In animals and lower eukaryotes, ACs and their product cAMP have firmly been established as important signalling molecules with important roles in several cellular signal transduction pathways. However, in higher plants, the only annotated and experimentally confirmed AC is a Zea mays pollen protein capable of generating cAMP. Recently a number of candidate AC-encoding genes in Arabidopsis thaliana have been proposed based on functionally assigned amino acids in the catalytic center of annotated and/or experimentally tested nucleotide cyclases in lower and higher eukaryotes. Here we detail the cloning and recombinant expression of functional candidate AC domains using, as an example, the A. thaliana pentatricopeptide repeat-containing protein (AtPPR-AC; At1g62590). Through a complementation test, in vivo adenylate cyclase activity of candidate recombinant molecules can be prescreened and promising candidates can subsequently be further evaluated in an in vitro AC immunoassay.

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks.

Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and / or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

The Arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes.

BACKGROUND: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3',5'-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. PRINCIPAL FINDINGS: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10(431-700) can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. CONCLUSIONS: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP.

Plant nucleotide cyclases: an increasingly complex and growing family.

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, cGMP, has long been known to be critical to many different processes in higher plants while guanylyl cyclases (GCs), enzymes that catalyse the formation of cGMP from GTP have largely remained elusive. This is somewhat surprising considering that the unicellular green alga Chlamydomonas reinhardtii contains >90 annotated GCs. We have recently shown (PLoS ONE 2(5): e449) that a recombinant cytoplasmic domain of the Arabidopsis brassinosteroid receptor AtBRI has GC activity in vitro. This finding may suggest that other leucine-rich receptor kinases such as the phystosulfokine receptor may also confer GC activity as it has a high degree of similarity in the domain that has been delineated as essential for catalysis. In addition, the discovery of increasing complexities in the molecular architecture of higher plant nucleotide cyclases (NCs) is entirely compatible with findings in Chlamydomonas where such domains appear in >20 different combinations suggesting a role in highly diverse and complex signaling events.

The Arabidopsis thaliana brassinosteroid receptor (AtBRI1) contains a domain that functions as a guanylyl cyclase in vitro.

BACKGROUND: Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) from guanosine 5'-triphosphate (GTP). Cyclic GMP has been implicated in an increasing number of plant processes, including responses to abiotic stresses such as dehydration and salt, as well as hormones. PRINCIPLE FINDINGS: Here we used a rational search strategy based on conserved and functionally assigned residues in the catalytic centre of annotated GCs to identify candidate GCs in Arabidopsis thaliana and show that one of the candidates is the brassinosteroid receptor AtBR1, a leucine rich repeat receptor like kinase. We have cloned and expressed a 114 amino acid recombinant protein (AtBR1-GC) that harbours the putative catalytic domain, and demonstrate that this molecule can convert GTP to cGMP in vitro. CONCLUSIONS: Our results suggest that AtBR1 may belong to a novel class of GCs that contains both a cytosolic kinase and GC domain, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors, NPR1 and NPR2. The findings also suggest that cGMP may have a role as a second messenger in brassinosteroid signalling. In addition, it is conceivable that other proteins containing the extended GC search motif may also have catalytic activity, thus implying that a significant number of GCs, both in plants and animals, remain to be discovered, and this is in keeping with the fact that the single cellular green alga Chlamydomonas reinhardtii contains over 90 annotated putative CGs.