Lead compounds that target tubulin are being developed as agents against the human malaria parasite, Plasmodium falciparum. It is important to define the binding sites of these molecules on the ...tubulin dimer: Taxol, Vinca domains or novel binding pockets; however, extraction of native parasite tubulin is difficult.
This report aims to develop assays that allow the rapid assessment of binding sites of compounds on the tubulin dimer.
We have developed a simple growth assay, using a combination of two anti-microtubule drugs that have overlapping binding sites, to study whether the two drugs act in synergistic, antagonistic or neutral manner. Additionally, Molecular docking was used to predict the binding sites of the drugs.
The combination assay shows antagonistic interactions between drugs having overlapping binding sites. In contrast, drugs that do not bind to overlapping sites show no interactions or synergism in this combination assay. Molecular docking predictions show that indeed, drugs with antagonistic interactions in the growth assay do bind to overlapping sites.
These two assays can be a simple preliminary screen for the binding sites of novel anti-tubulin compounds being developed for malaria therapeutics.
The Malaria Evolution in South Asia (MESA) International Center for Excellence in Malaria Research (ICEMR) was established by the US National Institutes of Health (US NIH) as one of 10 malaria ...research centers in endemic countries. In 10 years of hospital-based and field-based work in India, the MESA-ICEMR has documented the changing epidemiology and transmission of malaria in four different parts of India. Malaria Evolution in South Asia-ICEMR activities, in collaboration with Indian partners, are carried out in the broad thematic areas of malaria case surveillance, vector biology and transmission, antimalarial resistance, pathogenesis, and host response. The program integrates insights from surveillance and field studies with novel basic science studies. This is a two-pronged approach determining the biology behind the disease patterns seen in the field, and generating new relevant biological questions about malaria to be tested in the field. Malaria Evolution in South Asia-ICEMR activities inform local and international stakeholders on the current status of malaria transmission in select parts of South Asia including updates on regional vectors of transmission of local parasites. The community surveys and new laboratory tools help monitor ongoing efforts to control and eliminate malaria in key regions of South Asia including the state of evolving antimalarial resistance in different parts of India, new host biomarkers of recent infection, and molecular markers of pathogenesis from uncomplicated and severe malaria.
Enhancer elements regulating the neuronal gene, tyrosine hydroxylase (TH), were identified in TH-expressing peripheral nervous system PATH and central nervous system CATH cell lines. Mutational ...analysis in which rat TH 5′-flanking sequences directed chloramphenicol acetyltransferase (CAT) reporter gene expression demonstrated that mutating the cyclic AMP response element (CRE) at −45 base pair reduced expression by 80-90%. A CRE linked to an enhancerless TH promoter fully supported expression. Cotransfection of a dominant-negative CREB protein reduced expression 50-60%, suggesting that the CRE is bound by CREB or a CREB dimerization partner. Although mutating the AP1/dyad (AD) element at −205 base pair only modestly reduced CAT levels, AD minimal enhancer constructs gave 45-80% of wild type expression when positioned at −91 or −95. However, in its native context at −205, the AD could not support expression. In contrast, a CRE, moved from its normal position at −45 to −206, gave full activity. These results indicate that the CRE is critical for TH transcription in central nervous system CATH and peripheral nervous system PATH cells, whereas the AD is less important and its enhancer activity is context- and/or position-dependent. These results represent the first attempts to map regulatory elements directing TH expression in central nervous system cell lines.
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•The methyltransferase domain of P. falciparum TGS1 (PfTGS1) has an insertion containing a predicted nuclear localization signal (NLS).•This NLS is functional in yeast and is ...recognized by P. falciparum importin-α in vitro.•Assays developed in this report can be used to study NLS from Plasmodium species that cannot be cultured in the laboratory.
Many Plasmodium falciparum proteins do not share homology with, and are generally longer than their respective orthologs. This, to some extent, can be attributed to insertions. Here, we studied a P. falciparum RNA hypermethylase, trimethylguanosine synthase (PfTGS1) that harbors a 76 amino acid insertion in its methyltransferase domain. Bioinformatics analysis revealed that this insertion was present in TGS1 orthologs from other Plasmodium species as well. Interestingly, a classical nuclear localization signal (NLS) was predicted in the insertions of primate parasite TGS1 proteins. To check whether these predicted NLS are functional, we developed an in vivo heterologous system using S. cerevisiae. The predicted NLS when fused to dimeric GFP were able to localize the fusion protein to the nucleus in yeast indicating that it is indeed recognized by the yeast nuclear import machinery. We further showed that the PfTGS1 NLS binds to P. falciparum importin-α in vitro, confirming that the NLS is also recognized by the P. falciparum classical nuclear import machinery. Thus, in this study we report a novel function of the insertion in PfTGS1.
Although common in plants, very few proteins are currently known to be localized to both the plastid and the mitochondrion in
P
lasmodium
falciparum
. One such protein is
P
. falciparum
glutathione ...peroxidase‐like thioredoxin peroxidase (
P
f
TP
x
Gl
) which we show, by immunofluorescence imaging and bioinformatics predictions, is localized to the apicoplast, the mitochondrion and the cytosol. The distribution of
P
f
TP
x
Gl
was random in the population, with the protein localizing to any one organelle in some parasites and to both in others. It has been proposed that targeting to each organelle occurs via independent pathways that do not proceed via the Golgi. However, for
P
f
TP
x
Gl
, both incubation at low temperature (15 °C) and
B
refeldin A treatment reversibly blocked targeting to these organelles, suggesting the involvement of a novel trafficking route, most probably via the endoplasmic reticulum and
G
olgi. This idea is further supported by the lack of cleavage of the putative
N
‐terminal signal sequence of
P
f
TP
x
Gl
, and this
N
‐terminal extension did not compromise
P
f
TP
x
Gl
enzyme activity. In the context of evolution, a common pathway for the dual localization of a single gene product, such as the primitive endoplasmic reticulum–
G
olgi route, may have been retained as opposed to optimization for individual organellar import pathways.
Plasmodium vivax is a protozoan parasite that is one of the causative agents of human malaria. Due to several occult features of its life cycle, P. vivax threatens to be a problem for the recent ...efforts toward elimination of malaria globally. With an emphasis on malaria elimination goals, the authors summarize the major gaps in P. vivax diagnosis and describe how proteomics technologies have begun to contribute toward the discovery of antigens that could be used for various technology platforms and applications. The authors suggest areas where, in the future, proteomics technologies could fill in gaps in P. vivax diagnosis that have proved difficult. The discovery of new parasite antigens, host responses, and immune signatures using proteomics technologies will be a key part of the global malaria elimination efforts.
Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar ...plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure‐guided drug discovery with KNI inhibitors. Compounds used in this study (KNI‐10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high‐resolution crystal structures of PMII–KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI‐10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI‐10333 and KNI‐10343, accommodate different ρ‐substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen‐bonding contacts. KNI‐10743 and KNI‐10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use.
Database
Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.
Four plasmepsins (PMs) in the food vacuole of Plasmodium falciparum degrade human hemoglobin. Being responsible for the survival of the parasite makes them attractive drug targets. Compounds KNI‐10743 and KNI‐10333 show nanomolar inhibition activity against multiple PMs, with significant antiparasitic activity and low cytotoxicity. The cocrystal structures of PMII with potent KNI compounds reveal their inhibition mechanism. Novel KNI derivatives could be designed for future antimalarial drug development.
Plasmodium vivax is the most geographically widespread species responsible for malaria in humans. Our study focused on identifying highly expressed parasite proteins using a shotgun proteomics ...approach. Parasites (P. vivax) are isolated from seven patient samples using saponin lysis. Protein extracts from these parasites are processed and subjected to LC-MS/MS analysis. An overall proteome coverage of 605 P. vivax proteins along with 1670 human host proteins are obtained upon combining the data from LC-MS/MS runs. While a major proportion of the P. vivax proteins are either hypothetical or involved in basic cellular activities, few proteins such as tryptophan-rich antigen (Pv-fam-a; PVX_090265), Pv-fam-d protein (PVX_101520), Plasmodium exported protein (PVX_003545), Pvstp1 (PVX_094303) and hypothetical protein (PVX_083555) are detected in more than 80% of the clinical isolates and found to be unique to P. vivax without orthologs in P. falciparum. Our proteomics study on individual parasite isolates reveals highly expressed P. vivax proteins, few of which may be good candidates for vivax malaria diagnosis due to their abundance and absence in P. falciparum. This study represents the first step towards the identification of biomarkers for P. vivax malaria. In future, their clinical diagnostic values must be explored and validated on large patient cohorts.
Haptoglobin (Hp), an acute phase inflammatory protein is associated with malaria pathogenesis in several proteomics and genomics studies. The Hp gene has two co-dominant alleles: Hp1 and Hp2 that ...produce three genotypes: Hp1/Hp1, Hp1/Hp2 and Hp2/Hp2.
In this study, validation of the proteomics data with Multiple Reaction Monitoring Mass Spectroscopy (MRM-MS) is performed and the association of the Hp gene variants with severe, non-severe malaria and community (healthy) controls using genotyping PCRs and DNA sequencing is analysed.
Highly significant values of Hp is observed in the MRM assay that show a correlation with severity of malaria and is clearly distinguished from another febrile disease, dengue. Moreover, the Hp2/Hp2 genotype is seen in high percentages in non-severe malaria patients (74%) and community controls (72%) whereas patients diagnosed with severe malaria show only (31%) of this genotype. Sequencing of the Hp promoter region reveals three SNPs along with 10 unique haplotypes, out of which five are associated with non-severe and three with severe malaria populations (χ
= 130; df = 18; p < 0.0001).
This proteo-genomic study focuses on the correlation of the Hp protein and gene with malaria, thus highlighting the pivotal role of this acute phase immune gene in malaria pathogenesis.
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