bims-toxgon 2024-08-11 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2024‒08‒11
nine papers selected by
Lakesh Kumar, BITS Pilani

1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) as target for anti Toxoplasma gondii agents: crystal structure, biochemical characterisation and biological evaluation of inhibitors.
In Silico Analysis of the ROP29 Protein as a Vaccine Candidate Against Toxoplasma gondii.
TgMIC6 inhibition of autophagy is partially responsible for the phenotypic differences between Chinese 1 Toxoplasma gondii strains.
Synthesis and In vitro evaluation of bichalcones as novel anti-toxoplasma agents.
The structural biology and dynamics of malate dehydrogenases.
Ca2+ as an essential signaling molecule controlling Snf1-mediated Atg1 activation.
Interplay between DOT1L and HDAC1 regulates Leishmania donovani infection in human THP-1 cells.
A bright red fluorescent genetically encoded sensor for lactate imaging.
Evaluation of AlphaFold 3's Protein-Protein Complexes for Predicting Binding Free Energy Changes upon Mutation.

Biochem J. 2024 Aug 06. pii: BCJ20240110. [Epub ahead of print]

1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) as target for anti Toxoplasma gondii agents: crystal structure, biochemical characterisation and biological evaluation of inhibitors.

Flaminia Mazzone, Astrid Hoeppner, Jens Reiners, Christoph G W Gertzen, Violetta Applegate, Mona Abdullah Abdullaziz, Julia Gottstein, Daniel Degrandi, Martina Wesemann, Thomas Kurz, Sander Hj Smits, Klaus Pfeffer.

  Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and E. coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize α-phenyl-ß-thia and -oxa reverse fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast to fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

Keywords:  DXR; DXR inhibitors; Toxoplasma gondii; antibiotics; crystallography; small-angle scattering

DOI:  https://doi.org/10.1042/BCJ20240110
J Parasitol Res. 2024 ;2024 1918202

In Silico Analysis of the ROP29 Protein as a Vaccine Candidate Against Toxoplasma gondii.

Amir Karimipour-Saryazdi, Fatemeh Ghaffarifar, Abdolhossein Dalimi, Masoud Foroutan, John Horton, Javid Sadraei.

  The progression of Toxoplasma gondii (T. gondii) invasion is aided by rhoptry proteins (ROPs), which are also crucial for the parasite's survival in host cells. In this study, in silico analysis was performed to examine the various aspects of the ROP29 protein, such as physicochemical properties, potential T- and B-cell epitopes, and other significant features. The research revealed that there were 55 possible sites for posttranslational modification in the ROP29 protein. The secondary structure of the ROP29 protein consists of a random coil, an alpha-helix, and an extended strand, which account for 49.69%, 36.81%, and 13.50%, respectively. Moreover, a number of putative T- and B-cell epitopes for ROP29 were found. The Ramachandran plot showed that 88.91% (crude model) and 97.54% (refine model) of the amino acid residues were located in the favored regions. Also, the testing of this protein's antigenicity and allergenicity showed that it was nonallergenic and immunogenic. Our results suggested that employing in silico tools to apply structural and functional predictions to the ROP29 protein can lower the likelihood that laboratory investigations will fail. This research served as a crucial foundation for further research. More research is required in the future in suitable animal model employing ROP29 alone or in combination with other antigens.

Keywords:  ROP29; Toxoplasma gondii; bioinformatic analysis; vaccine

DOI:  https://doi.org/10.1155/2024/1918202
Int Immunopharmacol. 2024 Aug 07. pii: S1567-5769(24)01378-X. [Epub ahead of print]140 112857

TgMIC6 inhibition of autophagy is partially responsible for the phenotypic differences between Chinese 1 Toxoplasma gondii strains.

Yang Wang, Jingyang Li, Jinjin Zhu, Haiyang Ma, Baocan Zhuang, Ji Zhao, Famin Zhang, Li Yu.

  Chinese1 is the predominant Toxoplasma gondii lineage in China, and significant phenotypic differences are observed within the lineage. WH3 and WH6 are two representative strains of Chinese 1, which exhibit divergent virulence and pathogenicity in mice. However, virulence determinants and their modulating mechanisms remain elusive. A global genome expression analysis of the WH3 and WH6 transcriptional profiles identified microneme secretory protein 6 (MIC6), which may be associated with the phenotypic difference observed in WH3. In the present study, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technique was used to generate a T. gondii microneme secretory protein (TgMIC6) knockout in WH3. Wild-type mice and different mouse and human cell lines were infected with the WH3, WH3-Δmic6, and WH6 strains. The survival rate of mice, related cytokine levels in serum, and the proliferation of parasites were observed. These results suggested that TgMIC6 is an important effector molecule that determines the differential virulence of WH3 in vivo and in vitro. Furthermore, MIC6 may enhance WH3 virulence via inhibition of host cell autophagy and activation of key molecules in the epidermal growth factor receptor (EGFR)-Akt-mammalian target of rapamycin (mTOR) classical autophagy pathway. CD40L was cleared in vivo by i.p injection of CD40L monoclonal antibody, and it was found that the virulence of WH3-Δmic6 to mice was restored to a certain extent in the absence of CD40L. This study elucidates the virulence determinants and immune escape strategies of Toxoplasma gondii in China. Moreover, these data will aid the development of effective strategies for the prevention and control of toxoplasmosis.

Keywords:  Autophagy; CD40L-CD40; Chinese 1; MIC6; Transcriptome sequencing

DOI:  https://doi.org/10.1016/j.intimp.2024.112857
Front Chem. 2024 ;12 1406307

Synthesis and In vitro evaluation of bichalcones as novel anti-toxoplasma agents.

Flaminia Mazzone, Moritz K T Klischan, Julian Greb, Sander H J Smits, Jörg Pietruszka, Klaus Pfeffer.

  Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii, an apicomplexan parasite that infects approximately a third of the world's human population. This disease can cause serious complications during pregnancy and can be fatal in immunocompromised hosts. The current treatment options for toxoplasmosis face several limitations. Thus, to address the urgent medical need for the discovery of novel anti-toxoplasma potential drug candidates, our research focused on exploring a series of monomeric and dimeric chalcones, polyphenolic molecules belonging to the class of flavonoids. Chalcones 1aa-1bg and axially chiral A-A'-connected bichalcones 2aa-2bg were evaluated in vitro against the proliferation of the parasite in a cell-based assay. A comparison of the efficacy demonstrated that, in several cases, bichalcones exhibited increased bioactivity compared to their corresponding monomeric counterparts. Among these compounds, a bichalcone with a phenyl substituent and a methyl moiety 2ab showed the most potent and selective inhibitory activity in the nanomolar range. Both enantiomers of this bichalcone were synthesized using an axially chiral biphenol building block. The biaryl bond was forged using Suzuki cross-coupling in water under micellar catalysis conditions. Separation of the atropisomers of this biphenol building block was conducted by chiral HPLC on a preparative scale. The biological evaluation of the enantiomers revealed that the (R a)-enantiomer (R a)-2ab is the eutomer. These studies suggest that bichalcones may be important drug candidates for further in vivo evaluations for the discovery of anti-toxoplasma drugs.

Keywords:  Toxoplasma gondii; anti-infective; anti-toxoplasma; biaryl; bichalcones; bioactivity; stereoisomers

DOI:  https://doi.org/10.3389/fchem.2024.1406307
Essays Biochem. 2024 Aug 08. pii: EBC20230082. [Epub ahead of print]

The structural biology and dynamics of malate dehydrogenases.

Christopher E Berndsen, Jessica K Bell.

  Malate dehydrogenase (MDH) enzymes catalyze the reversible oxidoreduction of malate to oxaloacetate using NAD(P) as a cofactor. This reaction is vital for metabolism and the exchange of reducing equivalents between cellular compartments. There are more than 100 structures of MDH in the Protein Data Bank, representing species from archaea, bacteria, and eukaryotes. This conserved family of enzymes shares a common nucleotide-binding domain, substrate-binding domain, and subunits associate to form a dimeric or a tetrameric enzyme. Despite the variety of crystallization conditions and ligands in the experimental structures, the conformation and configuration of MDH are similar. The quaternary structure and active site dynamics account for most conformational differences in the experimental MDH structures. Oligomerization appears essential for activity despite each subunit having a structurally independent active site. There are two dynamic regions within the active site that influence substrate binding and possibly catalysis, with one of these regions adjoining the subunit interface. In this review, we introduce the reader to the general structural framework of MDH highlighting the conservation of certain features and pointing out unique differences that regulate MDH enzyme activity.

Keywords:  crystallography; protein dynamics; protein structure

DOI:  https://doi.org/10.1042/EBC20230082
Autophagy. 2024 Aug 05.

Ca2+ as an essential signaling molecule controlling Snf1-mediated Atg1 activation.

Yanyang Wu, Cong Yi.

  Macroautophagy/autophagy is essential for maintaining glucose homeostasis, but the mechanisms by which cells sense glucose starvation and initiate autophagy are not yet fully understood. Recently, we reported that the assembly of a Ca2+-triggered Snf1-Bmh1/Bmh2-Atg11 complex initiates autophagy in response to glucose starvation. Our research reveals that during glucose starvation, the efflux of vacuolar Ca2+ increases cytoplasmic Ca2+ levels, which activates the protein kinase Rck2. Rck2-mediated phosphorylation of Atg11 enhances its interaction with Bmh1 and Bmh2. This interaction recruits the Snf1-Sip1-Snf4 complex, which is located on the vacuolar membrane, to the phagophore assembly site (PAS), leading to the activation of Atg1 and the initiation of autophagy. In summary, we have identified a previously unrecognized signaling pathway involved in glucose starvation-induced autophagy, where Ca2+ acts as a fundamental signaling molecule that links energy stress to the formation of the autophagy initiation complex.

Keywords:  Atg11-Bmh1/2-Snf1 complex; Ca2+; Rck2; autophagy; glucose starvation

DOI:  https://doi.org/10.1080/15548627.2024.2389483
Acta Trop. 2024 Aug 03. pii: S0001-706X(24)00234-1. [Epub ahead of print] 107352

Interplay between DOT1L and HDAC1 regulates Leishmania donovani infection in human THP-1 cells.

Akanksha Kanojia, Gargi Roy, Rentala Madhubala, Rohini Muthuswami.

  Leishmania donovani, a protozoan parasite, causes visceral leishmaniasis. The parasite modifies the global gene expressions of the host genome, facilitating its survival within the host. Thus, the host epigenetic modulators play important roles in host-pathogen interaction and host epigenetic modification in response to infection. Previously, we had reported that the host epigenetic modulator, histone deacetylase 1 (HDAC1) expression was upregulated on Leishmania donovani infection. This upregulation led to the repression of host defensin genes in response to the infection. In this paper, we have investigated the interplay between the host DOT1L, a histone methyltransferase, and HDAC1 in response to Leishmania donovani infection. We show that the expression of DOT1L is upregulated both at transcript and protein level following infection leading to increase in H3K79me, H3K79me2, and H3K79me3 levels. ChIP experiments showed that DOT1L regulated the expression of HDAC1. Downregulation of DOT1L using siRNA resulted in decreased expression of HDAC1 and increased transcription of defensin genes and thereby, lower parasite load. In turn, HDAC1 regulates the expression of DOT1L on Leishmania donovani infection as downregulation of HDAC1 using siRNA led to reduced expression of DOT1L. Thus, during Leishmania donovani infection, an interplay between DOT1L and HDAC1 regulates the expression of these two histone modifiers leading to downregulation of defensin gene expression.

Keywords:  DOT1L; L. donovani.; defensins; histone deacetylation; host-pathogen interaction

DOI:  https://doi.org/10.1016/j.actatropica.2024.107352
Biochem Biophys Res Commun. 2024 Jul 25. pii: S0006-291X(24)00985-9. [Epub ahead of print]734 150449

A bright red fluorescent genetically encoded sensor for lactate imaging.

Xuanming Chang, Xiaoqian Chen, Xiuze Zhang, Nian Chen, Weitao Tang, Zhuo Zhang, Sulin Zheng, Jiong Huang, Yihan Ji, Yuzheng Zhao, Yi Yang, Xie Li.

  Lactate plays a crucial role in energy metabolism and greatly impacts protein activities, exerting diverse physiological and pathological effects. Therefore, convenient lactate assays for tracking spatiotemporal dynamics in living cells are desirable. In this paper, we engineered and optimized a red fluorescent protein sensor for l-lactate named FiLa-Red. This indicator exhibited a maximal fluorescence change of 730 % and an apparent dissociation constant (Kd) of approximately 460 μM. By utilizing FiLa-Red and other sensors, we monitored energy metabolism in a multiplex manner by simultaneously tracking lactate and NAD+/NADH abundance in the cytoplasm, nucleus, and mitochondria. The FiLa-Red sensor is expected to be a useful tool for performing metabolic analysis in vitro, in living cells and in vivo.

Keywords:  Fluorescence imaging; Genetically encoded fluorescent sensor; Lactate metabolism; Real-time monitoring; Spatiotemporal dynamics

DOI:  https://doi.org/10.1016/j.bbrc.2024.150449
J Chem Inf Model. 2024 Aug 08.

Evaluation of AlphaFold 3's Protein-Protein Complexes for Predicting Binding Free Energy Changes upon Mutation.

JunJie Wee, Guo-Wei Wei.

AlphaFold 3 (AF3), the latest version of protein structure prediction software, goes beyond its predecessors by predicting protein-protein complexes. It could revolutionize drug discovery and protein engineering, marking a major step toward comprehensive, automated protein structure prediction. However, independent validation of AF3's predictions is necessary. In this work, we evaluate AF3 complex structures using the SKEMPI 2.0 database which involves 317 protein-protein complexes and 8338 mutations. AF3 complex structures when applied to the most advanced TDL model, MT-TopLap (MultiTask-Topological Laplacian), give rise to a very good Pearson correlation coefficient of 0.86 for predicting protein-protein binding free energy changes upon mutation, which is slightly less than the 0.88 achieved earlier with the Protein Data Bank (PDB) structures. Nonetheless, AF3 complex structures led to a 8.6% increase in the prediction RMSE compared to original PDB complex structures. Additionally, some of AF3's complex structures have large errors, which were not captured in its ipTM performance metric. Finally, it is found that AF3's complex structures are not reliable for intrinsically flexible regions or domains.

DOI: https://doi.org/10.1021/acs.jcim.4c00976