Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4

Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.

Download Full-text

Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites

Biochemical Journal ◽

10.1042/bj20020753 ◽

2002 ◽

Vol 368 (1) ◽

pp. 273-281 ◽

Cited By ~ 30

Author(s):

Ajay SINGH ◽

Bhaskar R. SHENAI ◽

Youngchool CHOE ◽

Jiri GUT ◽

Puran S. SIJWALI ◽

...

Keyword(s):

Amino Acid ◽

Drug Discovery ◽

Drug Targets ◽

Critical Role ◽

Cysteine Proteases ◽

Malaria Parasites ◽

Ph Optimum ◽

Peptide Substrates ◽

Rodent Malaria

Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity (kcat/Km increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.

Download Full-text

Non-human primate and human malaria: past, present and future

Journal of Travel Medicine ◽

10.1093/jtm/taab036 ◽

2021 ◽

Author(s):

Spinello Antinori ◽

Cecilia Bonazzetti ◽

Andrea Giacomelli ◽

Mario Corbellino ◽

Massimo Galli ◽

...

Keyword(s):

Plasmodium Knowlesi ◽

Plasmodium Species ◽

Molecular Techniques ◽

Malaria Parasites ◽

Human Malaria ◽

Complex Interactions ◽

Zoonotic Malaria ◽

The Impact ◽

Human Plasmodium ◽

Human Primate

Abstract Background Studies of the malaria parasites infecting various non-human primates (NHPs) have increased our understanding of the origin, biology and pathogenesis of human Plasmodium parasites. This review considers the major discoveries concerning NHP malaria parasites, highlights their relationships with human malaria and considers the impact that this may have on attempts to eradicate the disease. Results The first description of NHP malaria parasites dates back to the early 20th century. Subsequently, experimental and fortuitous findings indicating that some NHP malaria parasites can be transmitted to humans have raised concerns about the possible impact of a zoonotic malaria reservoir on efforts to control human malaria. Advances in molecular techniques over the last 15 years have contributed greatly to our knowledge of the existence and geographical distribution of numerous Plasmodium species infecting NHPs, and extended our understanding of their close phylogenetic relationships with human malaria parasites. The clinical application of such techniques has also made it possible to document ongoing spillovers of NHP malaria parasites (Plasmodium knowlesi, P. cynomolgi, P. simium, P. brasilianum) in humans living in or near the forests of Asia and South America, thus confirming that zoonotic malaria can undermine efforts to eradicate human malaria. Conclusions Increasing molecular research supports the prophetic intuition of the pioneers of modern malariology who saw zoonotic malaria as a potential obstacle to the full success of malaria eradication programmes. It is, therefore, important to continue surveillance and research based on one-health approaches in order to improve our understanding of the complex interactions between NHPs, mosquito vectors and humans during a period of ongoing changes in the climate and the use of land, monitor the evolution of zoonotic malaria, identify the populations most at risk and implement appropriate preventive strategies.

Download Full-text

Assessing Diversity, Plasmodium Infection and Blood Meal Sources in Mosquitoes (Diptera: Culicidae) from a Brazilian Zoological Park with Avian Malaria Transmission

Insects ◽

10.3390/insects12030215 ◽

2021 ◽

Vol 12 (3) ◽

pp. 215

Author(s):

Lilian de Oliveira Guimarães ◽

Roseli França Simões ◽

Carolina Romeiro Fernandes Chagas ◽

Regiane Maria Tironi de Menezes ◽

Fabiana Santos Silva ◽

...

Keyword(s):

Blood Meal ◽

Avian Malaria ◽

Mosquito Species ◽

Plasmodium Species ◽

Sao Paulo ◽

São Paulo ◽

Malaria Parasites ◽

Ecological Relationships ◽

Lack Of Information ◽

Avian Malaria Parasites

Avian malaria parasites are widespread parasites transmitted by Culicidae insects belonging to different genera. Even though several studies have been conducted recently, there is still a lack of information about potential vectors of Plasmodium parasites, especially in Neotropical regions. Former studies with free-living and captive animals in São Paulo Zoo showed the presence of several Plasmodium and Haemoproteus species. In 2015, a pilot study was conducted at the zoo to collect mosquitoes in order to find out (i) which species of Culicidae are present in the study area, (ii) what are their blood meal sources, and (iii) to which Plasmodium species might they be potential vectors. Mosquitoes were morphologically and molecularly identified. Blood meal source and haemosporidian DNA were identified using molecular protocols. A total of 25 Culicidae species were identified, and 6 of them were positive for Plasmodium/Haemoproteus DNA. Ten mosquito species had their source of blood meal identified, which were mainly birds, including some species that were positive for haemosporidian parasites in the former study mentioned. This study allowed us to expand the list of potential vectors of avian malaria parasites and to improve our knowledge of the evolutionary and ecological relationships between the highly diverse communities of birds, parasites, and vectors present at São Paulo Zoo.

Download Full-text

How prevalent is Plasmodium malariae in Rondônia, Western Brazilian Amazon?

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822000000500011 ◽

2000 ◽

Vol 33 (5) ◽

pp. 489-492 ◽

Cited By ~ 32

Author(s):

Marisa Torres Vidal Cavasini ◽

Weber Luidi Ribeiro ◽

Fumihiko Kawamoto ◽

Marcelo Urbano Ferreira

Keyword(s):

Polymerase Chain Reaction ◽

Plasmodium Species ◽

Brazilian Amazon ◽

Plasmodium Malariae ◽

Mixed Species ◽

Chain Reaction ◽

Nested Polymerase Chain Reaction ◽

Polymerase Chain ◽

Potential Impact ◽

And Control

We have compared results of Plasmodium species identification obtained with conventional on-site microscopy of Giemsa-stained thick smears (GTS) and a semi-nested polymerase chain reaction (PCR) in 96 malaria patients from Rondônia, Western Brazilian Amazon. Mixed-species infections were detected by PCR in 30% patients, but no such case had been found on GTS. Moreover, P. malariae infections were detected in 9 of 96 patients (10%) by PCR, but were not identified by local microscopists. The potential impact of species misidentification on malaria treatment and control is discussed.

Download Full-text

Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation

Cell Death Discovery ◽

10.1038/s41420-021-00799-0 ◽

2022 ◽

Vol 8 (1) ◽

Author(s):

Hridindu Roychowdury ◽

Philip A. Romero

Keyword(s):

Large Scale ◽

Cysteine Proteases ◽

Amino Acid Substitutions ◽

Substrate Preferences ◽

Functional Differences ◽

Caspase Family ◽

Executioner Caspases ◽

The Impact ◽

Human Caspase

AbstractThe human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspase-specific therapeutics.

Download Full-text

Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action

10.1101/2020.08.13.249425 ◽

2020 ◽

Author(s):

Nelson V. Simwela ◽

Katie R. Hughes ◽

Michael T. Rennie ◽

Michael P. Barrett ◽

Andrew P. Waters

Keyword(s):

Anticancer Agents ◽

Drug Targets ◽

Reverse Genetics ◽

Enzyme Inhibitors ◽

Artemisinin Resistance ◽

Drug Repurposing ◽

Antimalarial Drugs ◽

Malaria Parasites

AbstractCurrent malaria control efforts rely significantly on artemisinin combinational therapies which have played massive roles in alleviating the global burden of the disease. Emergence of resistance to artemisinins is therefore, not just alarming but requires immediate intervention points such as development of new antimalarial drugs or improvement of the current drugs through adjuvant or combination therapies. Artemisinin resistance is primarily conferred by Kelch13 propeller mutations which are phenotypically characterised by generalised growth quiescence, altered haemoglobin trafficking and downstream enhanced activity of the parasite stress pathways through the ubiquitin proteasome system (UPS). Previous work on artemisinin resistance selection in a rodent model of malaria, which we and others have recently validated using reverse genetics, has also shown that mutations in deubiquitinating enzymes, DUBs (upstream UPS component) modulates susceptibility of malaria parasites to both artemisinin and chloroquine. The UPS or upstream protein trafficking pathways have, therefore, been proposed to be not just potential drug targets, but also possible intervention points to overcome artemisinin resistance. Here we report the activity of small molecule inhibitors targeting mammalian DUBs in malaria parasites. We show that generic DUB inhibitors can block intraerythrocytic development of malaria parasites in vitro and possess antiparasitic activity in vivo and can be used in combination with additive effect. We also show that inhibition of these upstream components of the UPS can potentiate the activity of artemisinin in vitro as well as in vivo to the extent that ART resistance can be overcome. Combinations of DUB inhibitors anticipated to target different DUB activities and downstream 20s proteasome inhibitors are even more effective at improving the potency of artemisinins than either inhibitors alone providing proof that targeting multiple UPS activities simultaneously could be an attractive approach to overcoming artemisinin resistance. These data further validate the parasite UPS as a target to both enhance artemisinin action and potentially overcome resistance. Lastly, we confirm that DUB inhibitors can be developed into in vivo antimalarial drugs with promise for activity against all of human malaria and could thus further exploit their current pursuit as anticancer agents in rapid drug repurposing programs.Graphical abstract

Download Full-text

IN SILICO ANTIMALARIAL TARGET SELECTION CONSERVED IN FOUR PLASMODIUM SPECIES

Universal Journal of Pharmaceutical Research ◽

10.22270/ujpr.v5i5.483 ◽

2020 ◽

Author(s):

Oladoja AWofisayo

Keyword(s):

Comparative Genomics ◽

Peer Review ◽

In Silico ◽

Drug Targets ◽

Homo Sapiens ◽

Mammalian Species ◽

Target Selection ◽

Plasmodium Species ◽

Data Bank ◽

Sequence Motifs

Objectives: The need for new antimalarials drugs and drug targets is pertinent due to the emergence of drug resistant strains of the parasites. Improper target selection has resulted in therapeutic failure. The genomic/post genomic era has made possible the deciphering of the 3D crystal structures of proteins and DNA which are drug targets and are deposited in the protein data bank. Methods: Novel antimalarial targets obtained from evolutionary conserved short sequence motifs are utilised and are essential in transcription processes in the parasite. The motifs TGCATGCA, GTGCAC and GTGCGTGC were curated from experimental work, validated and analysed via phylogenomics genomics and comparative genomics. PlasmoDB blastn was applied to determine their similarity in Plasmodium vivax, knowlesi, Ovale and yoeli. The complete genome of Plasmodium falciparum vivax, knowlesi, Ovale and yoeli was downloaded from the plasmoDB and their positions determined. Results: The targets are essential, conserved in rodent and mammalian species via phylogenomics with percentage identity and similarity greater than 80%, have no similar genes in the same genome and also found to be selective in the parasites vis-à-vis the Homo sapiens via comparative genomics with 0% identity and similarity in the human genome. Conclusion: The targets reveal at the molecular and biochemical level, the vulnerable regions in the parasite while safe in human hence their choices in subsequent rationale drug discovery and design protocols. Peer Review History: Received: 18 July 2020; Revised: 1 October; Accepted: 12 October, Available online: 15 November 2020 UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file Average Peer review marks at initial stage: 5.5/10 Average Peer review marks at publication stage: 7.0/10 Reviewer(s) detail: Dr. Tamer ELHABIBI, ERU University, Egypt, [email protected] Dr. Soroush Sardari, Biotech Pasteur Institute of Iran, Tehran, Iran, [email protected] Comments of reviewer(s): Similar Articles: IN SILICO LIGAND-BASED 2D PHARMACOPHORE GENERATION FOR H+/K+ ATPASE INHIBITORS

Download Full-text

OVERVIEW OF MALARIA PARASITE AND ITS PREVENTION IN INDIA

Journal of Biomedical and Pharmaceutical Research ◽

10.32553/jbpr.v8i1.575 ◽

2019 ◽

Vol 8 (1) ◽

Author(s):

Adil Raza ◽

Megha Chaudhary ◽

Sonika Devi

Keyword(s):

Red Blood Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Malaria Parasite ◽

Plasmodium Species ◽

Peripheral Blood Smear ◽

Infected Mosquito ◽

Protozoan Parasites ◽

Malaria Parasites ◽

Genus Plasmodium

Background: Malaria is a systematic disease caused by a parasite called Plasmodium which is transmitted into the human blood via female Anopheles mosquito. Malaria in humans is caused by four species of protozoan parasites of the genus Plasmodium: P. falciparum, P. vivax, P. ovale, and P. malariae. The parasite enters the human body through a mosquito bite and travel to the very crucial organ, the liver, where they multiply and come back to the bloodstream and destroy red blood cells. Malaria causes symptoms that typically include fever, tiredness, vomiting, and headaches. In severe cases it can cause yellow skin, seizures, coma, or death. Symptoms usually begin ten to fifteen days after being bitten by an infected mosquito. In those who have recently survived an infection, reinfection usually causes milder symptoms. Objectives: Isolation of different species of malaria parasites. The prevalence of malaria parasite in India. Methods: The procedure follows these steps: collection of peripheral blood, staining of smear with Leishman’s stain and examination of red blood cells for malaria parasites under the microscope. Results: We observed the plasmodium species in peripheral blood smear. Conclusion: Worldwide, the number of cases of malaria caused by Plasmodium falciparum, the most dangerous species of the parasite, is on the rise.

Download Full-text

Queuine is a nutritional regulator of Entamoeba histolytica response to oxidative stress and a virulence attenuator

10.1101/2020.04.30.070276 ◽

2020 ◽

Author(s):

Shruti Nagaraja ◽

Maggi W. Cai ◽

Jingjing Sun ◽

Hugo Varet ◽

Lotem Sarid ◽

...

Keyword(s):

Oxidative Stress ◽

Entamoeba Histolytica ◽

Cysteine Proteases ◽

Small Gtpases ◽

Cytoskeletal Proteins ◽

Transfer Rnas ◽

Amebic Dysentery ◽

Naturally Occurring ◽

Expression Of Genes ◽

Negative Effect

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here we describe the effects of queuine on the physiology of the eukaryotic parasite, Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in tRNAAspGUC. Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in OS response, such as heat shock protein 70 (Hsp 70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in tRNAAspGUC, parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.

Download Full-text

Identification of a large complex containing the integrin alpha 6 beta 1 laminin receptor in neural retinal cells

Journal of Cell Science ◽

10.1242/jcs.107.11.3165 ◽

1994 ◽

Vol 107 (11) ◽

pp. 3165-3172 ◽

Cited By ~ 1

Author(s):

I. de Curtis ◽

G. Gatti

Keyword(s):

Biochemical Properties ◽

Cytoskeletal Proteins ◽

Laminin Receptor ◽

Retinal Neurons ◽

Differential Distribution ◽

Gradient Distribution ◽

Large Complex ◽

Beta 1 Integrin ◽

Integrin Alpha 6 ◽

And Function

Integrin alpha 6 beta 1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to identify potential interactions between the alpha 6 beta 1 receptor and cellular proteins that may be involved in integrin signaling and function. For this purpose we have used a biochemical approach based on the solubilization of retinal neurons cultured on laminin with nonionic detergents, followed by centrifugation on sucrose velocity gradients. Analysis of the distribution of the alpha 6 and beta 1 integrin subunits in the gradients showed that they migrate as a large complex after extraction of cells with octylglucoside, but not after Triton X-100 extraction. Cytoskeletal proteins known to localize in adhesion plaques did not comigrate with alpha 6 beta 1 in octylglucoside gradients, while a set of polypeptides whose tyrosine phosphorylation was enhanced by culture on laminin colocalized with alpha 6 beta 1 on the gradients after octylglucoside solubilization. Culture of retinal neurons on bovine serum albumin, a nonadhesive substratum, partially affected the gradient distribution of the receptor after octylglucoside extraction. Furthermore, analysis of the gradient distribution of two alternatively spliced isoforms of the alpha 6 subunit, alpha 6-cytoA and alpha 6-cytoB, showed that the effect of non-adhesion on the sedimentation properties of the two integrin alpha 6 isoforms was more dramatic for alpha 6-cytoB than alpha 6-cytoA. These differences in the sedimentation behaviour indicate distinct biochemical properties of the two alpha 6 isoforms that, together with previous observations on their differential distribution in the developing retina, may reflect functional specificities.

Download Full-text