ResearchSpace :: Browsing by Author "Kwezi, Lusisizwe"

Browsing by Author "Kwezi, Lusisizwe"

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The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling
(Wiley, 2017-06) Wheeler, J; Wong, A; Marondedze, C; Groen, AJ; Kwezi, Lusisizwe
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.
Development and evaluation of Poly(Lactic-Co-Glycolic Acid) encapsulated betulinic acid nanocarrier for improved anti-tumor efficacy
(2024-12) Selepe, Cyril T; Dhlamini, Khanyisile S; Tshweu, Lesego L; Kwezi, Lusisizwe; Ramalapa, Bathabile E; Ray, Suprakas S
Betulinic acid (BA) is a promising natural anti-tumor agent renowned for its activity against various tumor cell types. Despite its favorable profile of low cytotoxicity to normal cells, BA’s inherent hydrophobic nature and relatively short systematic half-life impose hurdles for clinical application. This study introduces a strategy to surmount these obstacles by developing a drug delivery system employing poly(lactic-co-glycolic acid) (PLGA)-encapsulated BA nanoparticles (PLGA-BA NPs). Rigorous characterization techniques such as dynamic light scattering (DLS), x-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses are employed to confirm the integrity of the drug within the nanocarriers. The PLGA-BA NPs demonstrated a mean particle size of 196 ± 6.80 nm. XRD analysis demonstrated the amorphous state of the PLGA-BA formulation, a characteristic vital for sustained drug release and enhanced bioavailability. The PLGA-BA NPs exhibited spherical morphology with encapsulation and loading efficiency of 83 ± 9.24% and 7.0 ± 0.4%, respectively, highlighting efficient encapsulation of the drug within the PLGA NPs. In vitro, cytotoxicity assessments demonstrated enhanced anti-proliferative efficacy against breast and lung tumor cells when utilizing PLGA-BA NPs in comparison to free BA. This research underlines the potential of employing the developed PLGA-based nanocarrier to optimize the therapeutic efficacy of BA.
Enantioselective biocatalytic hydrolysis of ß-aminonitriles to ß-amino-amides using Rhodococcus rhodochrous ATCC BAA-870
(Elsevier, 2012-04) Chhiba, Varsha P; Bode, M; Mathiba, K; Kwezi, Lusisizwe; Brady, D
A range of ß-aminonitriles (3-amino-3-phenylpropanenitrile and derivatives) were synthesised by reaction of various benzonitriles with acetonitrile and subsequent reduction of the resulting acrylonitrile products. These compounds were hydrolysed to the corresponding amides using the nitrile biocatalytic activity of Rhodococccus rhodochrousATCCBAA-870. Results showed that the nitrile hydratase enzyme was enantioselective for these compounds, in particular 3-amino-3-p-tolylpropanenitrile and 3-amino-3-(4- methoxyphenyl)propanenitrile and the corresponding amides (up to 85% in one case). The reactions were performed at pH 9.0 after initial attempts at pH 7.0 were unsuccessful, most likely as a result of protonation of the 3-amino group at the lower pH.
Intramolecular crosstalk between catalytic activities of receptor kinases
(Taylor & Francis, 2018-02) Kwezi, Lusisizwe; Wheeler, JI; Marondedze, C; Gehring, C; Irving, HR
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.
Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1
(Portland Press, 2016-10) Muleya, V; Marondedze, C; Wheeler, JI; Thomas, L; Mok, YF; Griffin, MD; Manallack, DT; Kwezi, Lusisizwe; Lilley, KS; Gehring, C; Irving, HR
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.
Plant molecular pharming to overcome the global impact of neglected tropical diseases
(Springer, 2024-04) Moralo, Maabo; Singh, Advaita A; Pillay, Priyen; Kwezi, Lusisizwe; Tsekoa, Tsepo L
Neglected tropical diseases (NTD) include a broad group of diseases that affect close to two billion people mainly from low and lower-middle income countries. NTD’s account for 12% of the global disease burden and approximately 200,000 deaths annually. The updated 2017 World Health Organisation’s NTD list recognises 20 major NTDs, 19 of these are infectious diseases caused by helminths, protozoa, bacteria or viruses. Snakebite envenoming is the only non-infectious disease on the list, and it has been included due to its high disease burden and impact, especially on young people and children. Global interventions for achieving 90% reduction in NTD treatment by 2030 NTD include prevention, control, elimination and eradication strategies. However, there are still gaps in the research and development of therapeutics and vaccines and limited access to therapeutics, vaccines and diagnostics. This chapter broadly discusses NTDs, progress made and the insufficiencies in the landscape of therapeutics and vaccines and the critical role of plant molecular farming in response to the global disease burden of NTDs. Plant molecular farming presents an opportunity to develop new cost-effective NTD-targeting therapeutics and vaccines and cost-effective NTD diagnostic tools for increased equitable access by vulnerable populations in resource-limited settings.
Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications
(2020-04) Singh, Advaita Acarya; Pooe, O; Kwezi, Lusisizwe; Lotter-Stark, T; Stoychev, Stoyan H; Alexandra, Kabamba B; Gerber, Isak B; Bhiman, JN; Vorster, J; Pauly, M; Zeitlin, L; Whaley, K; Mach, L; Steinkellner, H; Morris, L; Tsekoa, Tsepo L; Chikwamba, Rachel K
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.
A plant-biotechnology approach for producing highly potent anti-HIV antibodies for antiretroviral therapy consideration
(2021-12) Singh, Advaita A; Pillay, Priyen; Kwezi, Lusisizwe; Tsekoa, Tsepo L
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 ( XTFT) was used, with glycosylating structures lacking ß1,2-xylose and/or a1,3-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.
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
(2022-08) O'Kennedy, Maretha M; Coetzee, P; Koekemoer, O; Du Plessis, L; Lourens, CW; Kwezi, Lusisizwe; Du Preez, Ilse; Mamputha, Sipho; Rutkowska, Daria A; Lemmer, Yolandy
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.
Recombinant expression, purification and PEGylation of DNA Ligases
(2022-04) Zuma, LK; Gasa, NL; Mazibuko, X; Simelane, BC; Pillay, Priyen; Kwezi, Lusisizwe; Tsekoa, Tsepo L; Pooe, OJ
Background: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are routinely 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. Method: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins were conjugated 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 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-fold for 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.

Browsing by Author "Kwezi, Lusisizwe"

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