129 research outputs found
Functional and structural charaterization of the unique bifunctional enzyme complex involved in regulation of polyamine metabolism in Plasmodium falciparum
Malaria remains one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapies. The need for novel therapeutic targets and drugs is therefore of the utmost importance and detailed knowledge of the biochemistry of the parasite is imperative. This study was directed at the biochemical characterisation of the polyamine metabolic pathway of P. falciparum in order to elucidate differences between the parasite and its human host that can be exploited in the design of novel antimalarials. The thesis focussed on the two rate-limiting enzymes in polyamine biosynthesis, S¬adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), which occur as a unique bifunctional complex in P. falciparum. The genomic structure of the bifunctional gene indicated a single, monocistronic transcript with large untranslated regions that were predicted to be involved in unique translational regulatory mechanisms. This gives rise to a bifunctional protein containing both decarboxylase activities on a single polypeptide forming a heterotetrameric complex. Activity of the decarboxylases decreases dramatically if these proteins are expressed in their monofunctional forms as homodimeric ODC and heterotetrameric AdoMetDC. The deduced amino acid sequence indicated that all the essential residues for catalysis are conserved and highlighted the presence of three parasite-specific insertions. The parasite-specific inserts were shown to be essential for the catalytic activity of the respective domains and also to influence the activity of the neighbouring domain, indicating that intramolecular communication exists in the heterotetrameric complex. The most structured and smallest insert was also shown to mediate protein-protein interactions between the two domains and to stabilise the complex. Further structure- functional characterisations of specifically the ODC domain were deduced from a comparative homology model. The model predicted an overall structure corresponding to those of other homologous proteins. The validity of the model is supported by mutagenesis results. However, certain parasite-specific properties were identified in the active site pocket and dimerisation interface. The former was exploited in the rational design of novel putative ODC inhibitors directed only against the P. falciparumprotein by in silico screening of chemical structure libraries. This study therefore describes the identification of certain parasite-specific properties in a unique bifunctional protein involved in regulation of polyamine metabolism of P. falciparum. Such discoveries are invaluable in strategies aimed at elucidating biochemical and metabolic differences between the parasite and its human host that could be exploited in the design of alternative, parasite-specific chemotherapies. Moreover, the thesis also contributed new knowledge on certain less well-understood biological phenomena characteristic of P. falciparum, the nature and origin of bifunctional proteins and the functional properties of parasite-specific inserts found in some proteins of the parasite.Thesis (PhD (Biochemistry))--University of Pretoria, 2002.Biochemistryunrestricte
Deletion mutagenesis of large areas in Plasmodium falciparum genes: a comparative study
BACKGROUND: The increasing emergence of Plasmodium falciparum parasites resistant to most of the cost-effective drugs has necessitated the identification of novel leads and drug targets. Parasitespecific inserts in enzymes that are essential for the differentiation and proliferation of malarial parasites have received considerable interest since it distinguishes these proteins from their human counterparts. The functions of these inserts, which include mediations of protein activities or protein-protein interactions, are being investigated by several strategies including deletion mutagenesis. A comparative study of five widely used PCR-based mutagenesis methods identified a
modified inverse PCR method as particularly suitable for the deletion of large areas (>100 bp) in malaria parasite genes.
METHODS: The restriction enzyme-mediated inverse PCR method described here incorporates unique restriction enzyme sites at the 5'-ends of inverse tail-to-tail primers. The entire genecontaining vector is amplified except the desired region to be deleted and cloned using the unique restriction sites to increase ligation efficiency. This method was compared in its efficiency to delete a ~400 bp parasite-specific insert in malarial S-adenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC) to existing PCR-based site-directed deletion mutagenesis methods including the QuickChangeâ„¢ site-directed mutagenesis, ExSiteâ„¢, overlapping primer and inverse PCR. In addition, the modified method was applied in the deletion of a >600 bp parasitespecific insert in another malarial gene, pyridoxal kinase (PfPdxK).
RESULTS: The modified and optimized restriction enzyme-mediated inverse PCR method resulted in 80% compared to 40% deletion mutagenesis efficiency of the overlapping primer method in the
deletion of a large area (411 bp) from a large malaria gene (PfAdoMetDC/ODC, gene size 4257 bp).
In contrast, deletion mutagenesis methods such as the well-known QuickChangeâ„¢ site-directed
mutagenesis, ExSiteâ„¢ and inverse PCR methods produced insignificant results. A 100%
mutagenesis efficiency was obtained with the restriction enzyme-mediated inverse PCR method to
delete 618 bp from a smaller gene (PfPdxK). CONCLUSION: An efficient method was developed for the deletion of large areas (>100 bp) in
significantly sized genes such as those of the A+T-rich P. falciparum genome
Transmission-blocking drugs for malaria elimination
Preventing human-to-mosquito transmission of malaria parasites provides possible solutions to interrupt the malaria parasite life cycle for malaria elimination. The development of validated routine assays enabled the discovery of such transmission-blocking compounds. Currently, one development priority remains on combinations of dual-active compounds with equipotent activity against both the disease-causing asexual and transmissible, sexual erythrocytic stages. Additionally, transmission-blocking compounds that target gametocyte-specific biology could be used in combination with compounds against asexual parasites. In either case, preventing transmission will reduce the risk of reinfection and, if different processes are targeted, also curb the spread of drug resistance. Here, we provide an updated roadmap to the discovery and development of new antimalarials with transmission-blocking activity to guide drug discovery for malaria elimination.The South African Department of Science and Innovation (DSI); the National Research Foundation (SA NRF) South African Research Chair Initiative; drug discovery funding from the Italy/South Africa Joint Research Program (ISARP) from the SA DSI and NRF and the Italian Ministries of Health and of Foreign Affairs and International Cooperation.http://www.journals.elsevier.com/trends-in-parasitologyhj2022BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC
Histone modification landscapes as a roadmap for malaria parasite development
Plasmodium falciparum remains the deadliest parasite species in the world, responsible for
229 million cases of human malaria in 2019. The ability of the P. falciparum parasite to
progress through multiple life cycle stages and thrive in diverse host and vector species
hinges on sophisticated mechanisms of epigenetic regulation of gene expression.
Emerging evidence indicates such epigenetic control exists in concentric layers,
revolving around core histone post-translational modification (PTM) landscapes. Here,
we provide a necessary update of recent epigenome research in malaria parasites,
focusing specifically on the ability of dynamic histone PTM landscapes to orchestrate
the divergent development and differentiation pathways in P. falciparum parasites. In
addition to individual histone PTMs, we discuss recent findings that imply functional
importance for combinatorial PTMs in P. falciparum parasites, representing an operational
histone code. Finally, this review highlights the remaining gaps and provides strategies to
address these to obtain a more thorough understanding of the histone modification
landscapes that are at the center of epigenetic regulation in human malaria parasites.The South African Research Chairs Initiative of the Department of Science and Innovation, administered through the South African National Research Foundation.https://www.frontiersin.org/journals/cell-and-developmental-biologydm2022BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC
Resisting resistance : is there a solution for malaria?
INTRODUCTION : Currently, widely used antimalarial drugs have a limited clinical lifespan due to parasite resistance development. With resistance continuously rising, antimalarial drug discovery requires strategies to decrease the time of delivering a new antimalarial drug while simultaneously increasing the drug's therapeutic lifespan. Lessons learnt from various chemotherapeutic resistance studies in the fields of antibiotic and cancer research offer potentially useful strategies that can be applied to antimalarial drug discovery.
AREAS COVERED : In this review the authors discuss current strategies to circumvent resistance in malaria and alternatives that could be employed. EXPERT OPINION : Scientists have been 'beating back' the malaria parasite with novel drugs for the past 49 years but the constant rise in antimalarial drug resistance is forcing the drug discovery community to explore alternative strategies. Avant-garde anti-resistance strategies from alternative fields may assist our endeavors to manage, control and prevent antimalarial drug resistance to progress beyond beating the resistant parasite back, to stopping it dead in its tracks.http://www.tandfonline.com/loi/iedc202017-02-28hb2016Biochemistr
Streamlined and robust stage-specific profiling of gametocytocidal compounds against Plasmodium falciparum
Malaria elimination is dependent on the ability to target both the pathogenic and transmissible stages of the human malaria parasite, Plasmodium falciparum. These forms of the parasite are differentiated by unique developmental stages, each with their own biological mechanisms and processes. These individual stages therefore also respond differently to inhibitory compounds, and this complicates the discovery of multistage active antimalarial agents. The search for compounds with transmission-blocking activity has focused on screening for activity on mature gametocytes, with only limited descriptions available for the activity of such compounds on immature stage gametocytes. This therefore poses a gap in the profiling of antimalarial agents for pan-reactive, multistage activity to antimalarial leads. Here, we optimized an effective and robust strategy for the simple and cost-effective description of the stage-specific action of gametocytocidal antimalarial compounds.The South African Medical Research Council Strategic Health Innovation Partnership and the Department of Science and Innovation South African Research Chairs Initiative, administered through the South African National Research Foundation; a BMGF Grand Challenges Africa grant; the Medicines for Malaria Venture as Global Test center for stage-specific gametocytocidal assays.http://www.frontiersin.org/Cellular_and_Infection_Microbiologyhj2022BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC
Adapt or die : targeting unique transmission-stage biology for malaria elimination
Plasmodium parasites have a complex life cycle that includes development in the human
host as well as the Anopheles vector. Successful transmission of the parasite between its
host and vector therefore requires the parasite to balance its investments in asexual
replication and sexual reproduction, varying the frequency of sexual commitment to
persist within the human host and generate future opportunities for transmission. The
transmission window is extended further by the ability of stage V gametocytes to circulate
in peripheral blood for weeks, whereas immature stage I to IV gametocytes sequester in
the bone marrow and spleen until final maturation. Due to the low gametocyte numbers in
blood circulation and with the ease of targeting such life cycle bottlenecks, transmission
represents an efficient target for therapeutic intervention. The biological process of
Plasmodium transmission is a multistage, multifaceted process and the past decade
has seen a much deeper understanding of the molecular mechanisms and regulators
involved. Clearly, specific and divergent processes are used during transmission
compared to asexual proliferation, which both poses challenges but also opportunities
for discovery of transmission-blocking antimalarials. This review therefore presents an
update of our molecular understanding of gametocyte and gamete biology as well as the
status of transmission-blocking activities of current antimalarials and lead development
compounds. By defining the biological components associated with transmission,
considerations for the development of new transmission-blocking drugs to target such
untapped but unique biology is suggested as an important, main driver for transmissionblocking drug discovery.https://www.frontiersin.org/journals/cellular-and-infection-microbiologydm2022BiochemistryGeneticsMicrobiology and Plant PathologySchool of Health Systems and Public Health (SHSPH)UP Centre for Sustainable Malaria Control (UP CSMC
Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum
Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating
cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual
blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions,
but does not cure infections in vivo. This may be due to replenishment of the parasite’s intracellular polyamine
pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine
uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines,
putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their
host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine
were taken up into isolated parasites via a temperature-dependent process that showed cross-competition
between different polyamines. There was also some inhibition of polyamine uptake by basic amino
acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine
taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated
parasites was independent of extracellular Na+ but increased with increasing external pH. Uptake also
showed a marked dependence on the parasite’s membrane potential, decreasing with membrane depolarization
and increasing with membrane hyperpolarization. The data are consistent with polyamines
being taken up into the parasite via an electrogenic uptake process, energised by the parasite’s inwardly
negative membrane potential.J.N. was supported by the Carl and Emily Fuchs
foundation (South Africa), the Ernst and Ethel Eriksen Trust (South
Africa) and research performed in Australia was funded by AusAID,
the University of Pretoria (South Africa), Postgraduate Mentorship
Programme and a University of Pretoria Study Abroad Bursary. This
work was supported by the South African Medical Research
Council (L.M.B.), the South African National Research Foundation
KISC programme (L.M.B., Grant No. 67444) and the Australian
National Health and Medical Research Council (K.K., Grant No.
525428).http://www.elsevier.com/locate/ijpar
A 2-methoxyestradiol bis-sulphamoylated derivative induces apoptosis in breast cell lines
INTRODUCTION : Research involving antimitotic compounds identified 2-methoxyestradiol (2ME2), as a promising anticancer
endogenous metabolite. Owing to its low bioavailability, several in silico-designed 2ME2 analogues were synthesized.
Structure-activity relationship studies indicated that an already existing 17-β-estradiol analogue, namely
(8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl
bis(sulphamate) (EMBS) to exert potential in vitro anticancer activity.
METHODS : This study investigated the in vitro apoptotic influence of EMBS in an estrogen receptor-positive breast
adenocarcinoma epithelial cell line (MCF-7); an estrogen receptor-negative breast epithelial cell line (MDA-MB-231) and a
non-tumorigenic breast cell line (MCF-12A). Cell cycle progression, a phosphatidylserine flip, caspase 6-, 7- and 8 enzyme
activity levels, Bcl-2 phosphorylation status at serine 70 and Bcl-2- and p53 protein levels were investigated to identify a
possible action mechanism for apoptotic induction.
RESULTS : The xCELLigence real-time label-independent approach revealed that EMBS exerted antiproliferative activity in all
three cell lines after 24 h of exposure. A G2M block was observed and apoptosis induction was verified by means of flow
cytometry using propidium iodide and Annexin V-FITC respectively. EMBS-treated cells demonstrated a reduced
mitochondrial membrane potential. EMBS exposure resulted in a statistically significant increase in p53 protein
expression, decreased Bcl-2 protein expression and a decrease in pBcl-2(s70) phosphorylation status in all three
cell lines. Results support the notion that EMBS induces apoptosis in all three cell lines.
CONCLUSION : This study includes investigation into the apoptotic hallmarks exerted by EMBS after exposure of
three cell lines namely MCF-7-, MDA-MDA-231- and MCF-12A cells. Increased caspase 6-, caspase 7- and caspase
8 activities, upregulation of p53 protein expression and a decrease in phosphorylation status of Bcl-2 at serine 70
in tumorigenic and non-tumorigenic lines were demonstrated.The Cancer Association of South
Africa, the Struwig Germeshuysen Trust, RESCOM (Research Council of the
University of Pretoria), the South African National Research Foundation and
Medical Research Council.http://www.cellandbioscience.comam201
17-beta-estradiol analog inhibits cell proliferation by induction of apoptosis in breast cell lines
Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2-Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several
analogs were synthesized. This study investigated the antiproliferative influence of an 2-methoxyestradiol analog, (8R, 13S, 14S, 17S)-2-Ethyl-13-methyl-7, 8, 9, 11, 12,13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrane-3, 17-diyl bis(sulfamate) (EMBS) on cell proliferation,
morphology and apoptosis induction in a estrogen receptor-positive breast adenocarcinoma cells line (MCF-7), estrogen receptor-negative highly metastatic breast cell line (MDAMB- 231) and a non-tumorigenic breast epithelial cell line (MCF-12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell
lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 lM after 24 h of exposure. Light microscopy and polarization-optical transmitted light differential interference contrast demonstrated compromised cell density, cells
blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and
cell debris in EMBS-exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non-tumorigenic breast cell lines. Findings warrant in-depth analysis
of specific targets in vitro and subsequent in vivo investigation for anticancer therapy.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-0029hb2014ay201
- …