bims-toxgon 2024-05-19 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2024–05–19
24 papers selected by
Lakesh Kumar, BITS Pilani

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency.
Immunoinformatics studies and design of a novel multi-epitope peptide vaccine against Toxoplasma gondii based on calcium-dependent protein kinases antigens through an in-silico analysis.
The role of microRNA-142a in Toxoplasma gondii infection-induced downregulation of Foxp3: implications for adverse pregnancy outcomes.
Bicyclic Pyrrolidine Inhibitors of Toxoplasma gondii Phenylalanine t-RNA Synthetase with Antiparasitic Potency In Vitro and Brain Exposure.
Toxoplasma replication is inhibited by MMV676477 without development of resistance.
A comparison of teleost rodlet cells with apicomplexan cells.
Toxoplasma gondii and Neospora caninum infection in epileptic dogs.
Unexpected roles for AMPK in the suppression of autophagy and the reactivation of MTORC1 signaling during prolonged amino acid deprivation.
Humane Endpoints in Swiss Webster Mice Infected with Toxoplasma gondii RH Strain.
Geoepidemiology, seroprevalence and factors associated with Toxoplasma gondii infection in domicilied cats from Paraíba (Brazil).
AMPK as a mediator of tissue preservation: time for a shift in dogma?
IPMK regulates HDAC3 activity and histone H4 acetylation in human cells.
TurboID mapping reveals the exportome of secreted intrinsically disordered proteins in the transforming parasite Theileria annulata.
Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.
2-APQC, a small-molecule activator of Sirtuin-3 (SIRT3), alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis.
An ApiAp2 Transcription Factor with a Dispensable Role in Plasmodium berghei Life Cycle.
Cryo-EM structures of the human Elongator complex at work.
MmpL3, Wag31 and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability.
SIRT1 Serum Concentrations in Lipodystrophic Syndromes.
Charting new territory: The Plasmodium falciparum tRNA modification landscape.
mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.
iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.
Extracellular signals induce dynamic ER remodeling through αTAT1-dependent microtubule acetylation.
Characterization of a novel HDAC2 pathogenetic variant: a missing puzzle piece for chromatinopathies.

mBio. 2024 May 15. e0295423

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency.

Michael J Holmes, Matheus S Bastos, Vishakha Dey, Vanessa Severo, Ronald C Wek, William J Sullivan.

  The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogeneous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.
IMPORTANCE: Toxoplasma gondii is an opportunistic pathogen important to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation is required. Here we show that the mRNA cap-binding protein, eIF4E1, regulates the encystation process. Encysted parasites reduce eIF4E1 levels, and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

Keywords:  Toxoplasma; bradyzoite; gene expression; parasite; translation

DOI:  https://doi.org/10.1128/mbio.02954-23
Clin Exp Vaccine Res. 2024 Apr;13(2): 146-154

Immunoinformatics studies and design of a novel multi-epitope peptide vaccine against Toxoplasma gondii based on calcium-dependent protein kinases antigens through an in-silico analysis.

Ali Dalir Ghaffari, Fardin Rahimi.

   Purpose: Infection by the intracellular apicomplexan parasite Toxoplasma gondii has serious clinical consequences in humans and veterinarians around the world. Although about a third of the world's population is infected with T. gondii, there is still no effective vaccine against this disease. The aim of this study was to develop and evaluate a multimeric vaccine against T. gondii using the proteins calcium-dependent protein kinase (CDPK)1, CDPK2, CDPK3, and CDPK5.
Materials and Methods: Top-ranked major histocompatibility complex (MHC)-I and MHC-II binding as well as shared, immunodominant linear B-cell epitopes were predicted and linked using appropriate linkers. Moreover, the 50S ribosomal protein L7/L12 (adjuvant) was mixed with the construct's N-terminal to increase the immunogenicity. Then, the vaccine's physicochemical characteristics, antigenicity, allergenicity, secondary and tertiary structure were predicted.
Results: The finally-engineered chimeric vaccine had a length of 680 amino acids with a molecular weight of 74.66 kDa. Analyses of immunogenicity, allergenicity, and multiple physiochemical parameters indicated that the constructed vaccine candidate was soluble, non-allergenic, and immunogenic, making it compatible with humans and hence, a potentially viable and safe vaccine candidate against T. gondii parasite.
Conclusion: In silico, the vaccine construct was able to trigger primary immune responses. However, further laboratory studies are needed to confirm its effectiveness and safety.

Keywords:  CDPK1; CDPK2; CDPK3; CDPK5; Immunoinformatics; In silico; Toxoplasma gondii

DOI:  https://doi.org/10.7774/cevr.2024.13.2.146
BMC Infect Dis. 2024 May 13. 24(1): 490

The role of microRNA-142a in Toxoplasma gondii infection-induced downregulation of Foxp3: implications for adverse pregnancy outcomes.

Yue Zhong, Yining Cao, Xiaoyu Geng, Shujin Yang, Tianmei Qian, Chun Liu, Jinling Chen.

   BACKGROUND: Toxoplasma gondii (T. gondii) is capable of infecting nearly all warm-blooded animals and approximately 30% of the global population. Though most infections are subclinical in immunocompetent individuals, congenital contraction can lead to severe consequences such as spontaneous abortion, stillbirth, and a range of cranio-cerebral and/or ocular abnormalities. Previous studies reported that T. gondii-infected pregnancy mice unveiled a deficit in both the amount and suppressive functions of regulatory T (Treg) cells, accompanied with reduced levels of forkhead box p3 (Foxp3). Recently, accumulative studies have demonstrated that microRNAs (miRNAs) are, to some extent, relevant to T. gondii infection. However, the link between alterations in miRNAs and downregulation of Foxp3 triggered by T. gondii has been only sporadically studied.
METHODS: Quantitative reverse transcription polymerase chain reaction (RT-qPCR), protein blotting and immunofluorescence were employed to evaluate the impact of T. gondii infection and antigens on miRNA transcription and Foxp3 expression. Dual-luciferase reporter gene assays were performed to examine the fluorescence activity in EL4 cells, which were transfected with recombinant plasmids containing full-length/truncated/mutant microRNA-142a-3p (miR-142a) promoter sequence or wild type/mutant of Foxp3 3' untranslated region (3' UTR).
RESULTS: We found a pronounced increase in miR-142a transcription, concurrent with a decrease in Foxp3 expression in T. gondii-infected mouse placental tissue. Similarly, comparable findings have been experimentally confirmed through the treatment of EL4 cells with T. gondii antigens (TgAg) in vitro. Simultaneously, miR-142a mimics attenuated Foxp3 expression, whereas its inhibitors markedly augmented Foxp3 expression. miR-142a promoter activity was elevated upon the stimulation of T. gondii antigens, which mitigated co-transfection of mutant miR-142a promoter lacking P53 target sites. miR-142a mimics deceased the fluorescence activity of Foxp3 3' untranslated region (3' UTR), but it did not affect the fluorescence activity upon the co-transfection of mutant Foxp3 3' UTR lacking miR-142a target site.
CONCLUSION: In both in vivo and in vitro studies, a negative correlation was discovered between Foxp3 expression and miR-142a transcription. TgAg enhanced miR-142a promoter activity to facilitate miR-142a transcription through a P53-dependent mechanism. Furthermore, miR-142a directly targeted Foxp3 3' UTR, resulting in the downregulation of Foxp3 expression. Therefore, harnessing miR-142a may be a possible therapeutic approach for adverse pregnancy caused by immune imbalances, particularly those induced by T. gondii infection.

Keywords:   Toxoplasma Gondii ; Forkhead box P3; P53; microRNA-142a-3p

DOI:  https://doi.org/10.1186/s12879-024-09375-0
ACS Infect Dis. 2024 May 14.

Bicyclic Pyrrolidine Inhibitors of Toxoplasma gondii Phenylalanine t-RNA Synthetase with Antiparasitic Potency In Vitro and Brain Exposure.

Chloe C Ence, Taher Uddin, Julien Borrel, Payal Mittal, Han Xie, Jochen Zoller, Amit Sharma, Eamon Comer, Stuart L Schreiber, Bruno Melillo, L David Sibley, Arnab K Chatterjee.

  Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular, to achieve optimal exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency and ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analogue series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.

Keywords:  anti-infective; central nervous system infection; small molecule inhibitor; toxoplasmosis

DOI:  https://doi.org/10.1021/acsinfecdis.4c00170
Cytoskeleton (Hoboken). 2024 May 16.

Toxoplasma replication is inhibited by MMV676477 without development of resistance.

Izra Abbaali, Danny Truong, Dawn M Wetzel, Naomi S Morrissette.

  Protozoan parasites cause life-threatening infections in both humans and animals, including agriculturally significant livestock. Available treatments are typically narrow spectrum and are complicated by drug toxicity and the development of resistant parasites. Protozoan tubulin is an attractive target for the development of broad-spectrum antimitotic agents. The Medicines for Malaria Pathogen Box compound MMV676477 was previously shown to inhibit replication of kinetoplastid parasites, such as Leishmania amazonensis and Trypanosoma brucei, and the apicomplexan parasite Plasmodium falciparum by selectively stabilizing protozoan microtubules. In this report, we show that MMV676477 inhibits intracellular growth of the human apicomplexan pathogen Toxoplasma gondii with an EC50 value of ~50 nM. MMV676477 does not stabilize vertebrate microtubules or cause other toxic effects in human fibroblasts. The availability of tools for genetic studies makes Toxoplasma a useful model for studies of the cytoskeleton. We conducted a forward genetics screen for MMV676477 resistance, anticipating that missense mutations would delineate the binding site on protozoan tubulin. Unfortunately, we were unable to use genetics to dissect target interactions because no resistant parasites emerged. This outcome suggests that future drugs based on the MMV676477 scaffold would be less likely to be undermined by the emergence of drug resistance.

Keywords:  isotype; microtubule‐targeting agent; mutagenesis; therapeutic index

DOI:  https://doi.org/10.1002/cm.21876
Acta Histochem. 2024 May 10. pii: S0065-1281(24)00035-7. [Epub ahead of print]126(4): 152167

A comparison of teleost rodlet cells with apicomplexan cells.

Joseph A DePasquale.

  Rodlet cells are unique pear-shaped cells found primarily in the epithelium of the teleost fishes. The rodlet cell was first identified by Thèlohan in 1892 who named it Rhabdospora thelohani as it was believed to be a protozoan parasite of the phylum Apicomplexa. The rodlet cell as parasite paradigm persisted for several decades afterwards but has since faded in the last 20 years or so. The rodlet cell is now generally believed to be an immune cell, functioning as an early responder to parasite intrusion. This short review makes a detailed comparison of apicomplexan structure and behavior with that of the rodlet cell to further strengthen the argument against a parasitic nature for the fish cell. It is then proposed that apical microvilli of the rodlet cell serve as a mechanical trigger for rodlet discharge as possible defense against larger ectoparasites.

Keywords:  Apicomplexa; Rodlet Cells; Toxoplasma gondii

DOI:  https://doi.org/10.1016/j.acthis.2024.152167
J Small Anim Pract. 2024 May 16.

Toxoplasma gondii and Neospora caninum infection in epileptic dogs.

G Morganti, G Rigamonti, M C Marchesi, G Maggi, G Angeli, I Moretta, L Brustenga, M Diaferia, F Veronesi.

OBJECTIVES: Studies in humans have demonstrated the role of Toxoplasma gondii, a protozoan parasite, in epileptic seizures. This study aimed to investigate the serological correlation between T. gondii and N. caninum and epilepsy in dogs.
MATERIALS AND METHODS: The medical record database of the Veterinary Teaching Hospital, University of Perugia, was searched for dogs serologically tested by IFAT for T. gondii and N. caninum and following specific inclusion criteria. Dogs were stratified by having a clinical diagnosis of epilepsy or suffering different conditions.
RESULTS: One-hundred and twenty-eight dogs were included, 64 with epilepsy and 64 without clinical signs of epilepsy. Seventeen of the 64 epileptic dogs (26.6%; 95% CI: 15.7% to 37.4%) and twenty-one of the 64 non-epileptic dogs (32.8%; 95% CI: 21.3% to 44.3%) tested positive for T. gondii. Eight of the epileptic dogs (12.5%; 95% CI: 4.4% to 20.6%) and three of the non-epileptic dogs (4.7%; 95% CI: 0% to 9.9%) tested positive for N. caninum. There was no statistically significant difference in the rate of T. gondii or N. caninum seroreactivity between epileptic and non-epileptic dogs.
CLINICAL SIGNIFICANCE: The results obtained do not seem to support the role of T. gondii and N. caninum as causative agents of dog epilepsy.

DOI: https://doi.org/10.1111/jsap.13735
Autophagy. 2024 May 14.

Unexpected roles for AMPK in the suppression of autophagy and the reactivation of MTORC1 signaling during prolonged amino acid deprivation.

Dubek Kazyken, Sydney G Dame, Claudia Wang, Maxwell Wadley, Diane C Fingar.

  AMPK promotes catabolic and suppresses anabolic cell metabolism to promote cell survival during energetic stress, in part by inhibiting MTORC1, an anabolic kinase requiring sufficient levels of amino acids. We found that cells lacking AMPK displayed increased apoptotic cell death during nutrient stress caused by prolonged amino acid deprivation. We presumed that impaired macroautophagy/autophagy explained this phenotype, as a prevailing view posits that AMPK initiates autophagy (often a pro-survival response) through phosphorylation of ULK1. Unexpectedly, however, autophagy remained unimpaired in cells lacking AMPK, as monitored by several autophagic readouts in several cell lines. More surprisingly, the absence of AMPK increased ULK1 signaling and MAP1LC3B/LC3B lipidation during amino acid deprivation while AMPK-mediated phosphorylation of ULK1 S555 (a site proposed to initiate autophagy) decreased upon amino acid withdrawal or pharmacological MTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted autophagy induced by amino acid withdrawal. These results demonstrate that AMPK activation and glucose deprivation suppress autophagy. As AMPK controlled autophagy in an unexpected direction, we examined how AMPK controls MTORC1 signaling. Paradoxically, we observed impaired reactivation of MTORC1 in cells lacking AMPK upon prolonged amino acid deprivation. Together these results oppose established views that AMPK promotes autophagy and inhibits MTORC1 universally. Moreover, they reveal unexpected roles for AMPK in the suppression of autophagy and the support of MTORC1 signaling in the context of prolonged amino acid deprivation. These findings prompt a reevaluation of how AMPK and its control of autophagy and MTORC1 affect health and disease.

Keywords:  ATG16L1; EIF4EBP1/4EBP1; LC3B; MTOR; RPS6KB1/S6K1; ULK1

DOI:  https://doi.org/10.1080/15548627.2024.2355074
Animals (Basel). 2024 Apr 29. pii: 1326. [Epub ahead of print]14(9):

Humane Endpoints in Swiss Webster Mice Infected with Toxoplasma gondii RH Strain.

Igor Falco Arruda, Maria Regina Reis Amendoeira, Thamires Francisco Bonifácio, Clarissa Nascimento da Silveira Raso, Hyago da Silva Medeiros Elidio, Jhônata Willy Rocha Coelho, Luiz Cesar Cavalcanti Pereira da Silva, Isabele Barbieri Dos Santos.

  The highly virulent Toxoplasma gondii RH strain is maintained through successive passages in mice, but there is still a lack of studies that refine these procedures from a 3Rs perspective, where humanitarian ideals aim to minimize the stress, pain, or suffering of the animals used in the research without the loss of results. The aim of this study was to establish humane endpoints in Swiss Webster mice inoculated with the T. gondii RH strain. A total of 52 mice were infected with 5 × 106 tachyzoites/mL and monitored for periods of up to 5 days. The parameters body weight; hair condition; higher than normal body temperature; hypothermia; respiratory function; pain; soft stools or diarrhea; bloody diarrhea; tense, nervous, or in distress during handling; and ascites were recorded daily in score tables. The results showed that prominent piloerection, respiratory function, pain parameters, and ascites are important clinical signs to be used as a cut-off point for implementing euthanasia. The application of this refinement method helped to avoid animal suffering and pain without compromising the number of parasites recovered. We therefore suggest adopting these parameters in research protocols that require the maintenance of the T. gondii RH strain in murine models to avoid and reduce animal suffering.

Keywords:  humane endpoints; murine model; refinement; toxoplasmosis

DOI:  https://doi.org/10.3390/ani14091326
Parasite. 2024 ;31 25

Geoepidemiology, seroprevalence and factors associated with Toxoplasma gondii infection in domicilied cats from Paraíba (Brazil).

Ana Letícia Pereira Fernandes, Mariana de Melo Alves, Jordania Oliveira Silva, Ividy Bison, Ariana de Castro Tavares Silva, Roberta Nunes Parentoni, Jose Romulo Soares Dos Santos, Thais Ferreira Feitosa, Vinícius Longo Ribeiro Vilela, Arthur Willian de Lima Brasil.

  Toxoplasma gondii is a parasite responsible for toxoplasmosis, an emerging and often neglected zoonosis in South America, particularly Brazil. Felines, the only definitive hosts, excrete oocysts in their feces, potentially infecting all homeotherms. Domestic cats are primarily responsible for contaminating human environments with these oocysts. Monitoring their populations is therefore essential to ensure proper toxoplasmosis prophylaxis. The aim of this study was to estimate the prevalence of T. gondii and exposure factors in a population of owner cats in the city of João Pessoa, Paraíba, Brazil. A total of 267 blood samples were collected from domestic cats aged between 1 and 15 years and tested with an immunofluorescence antibody test. The seroprevalence of antibodies against T. gondii was only 17.22% (46/267 individuals). This result therefore suggests a low contribution of domestic cats to T. gondii contamination of the urban environment. The cats' age and living environment were identified as risk factors for cat exposure to T. gondii.

Keywords:  Epidemiology; Feline; IFAT; One Health

DOI:  https://doi.org/10.1051/parasite/2024017
Nat Rev Endocrinol. 2024 May 17.

AMPK as a mediator of tissue preservation: time for a shift in dogma?

Henning Tim Langer, Maria Rohm, Marcus DaSilva Goncalves, Lykke Sylow.

Ground-breaking discoveries have established 5'-AMP-activated protein kinase (AMPK) as a central sensor of metabolic stress in cells and tissues. AMPK is activated through cellular starvation, exercise and drugs by either directly or indirectly affecting the intracellular AMP (or ADP) to ATP ratio. In turn, AMPK regulates multiple processes of cell metabolism, such as the maintenance of cellular ATP levels, via the regulation of fatty acid oxidation, glucose uptake, glycolysis, autophagy, mitochondrial biogenesis and degradation, and insulin sensitivity. Moreover, AMPK inhibits anabolic processes, such as lipogenesis and protein synthesis. These findings support the notion that AMPK is a crucial regulator of cell catabolism. However, studies have revealed that AMPK's role in cell homeostasis might not be as unidirectional as originally thought. This Review explores emerging evidence for AMPK as a promoter of cell survival and an enhancer of anabolic capacity in skeletal muscle and adipose tissue during catabolic crises. We discuss AMPK-activating interventions for tissue preservation during tissue wasting in cancer-associated cachexia and explore the clinical potential of AMPK activation in wasting conditions. Overall, we provide arguments that call for a shift in the current dogma of AMPK as a mere regulator of cell catabolism, concluding that AMPK has an unexpected role in tissue preservation.

DOI: https://doi.org/10.1038/s41574-024-00992-y
bioRxiv. 2024 Apr 29. pii: 2024.04.29.591660. [Epub ahead of print]

IPMK regulates HDAC3 activity and histone H4 acetylation in human cells.

Gregory A Sowd, Elizabeth A Stivison, Pratima Chapagain, Andrew T Hale, James C Poland, Lucia E Rameh, Raymond D Blind.

Histone deacetylases (HDACs) repress transcription by catalyzing the removal of acetyl groups from histones. Class 1 HDACs are activated by inositol phosphate signaling molecules in vitro , but it is unclear if this regulation occurs in human cells. Inositol Polyphosphate Multikinase (IPMK) is required for production of inositol hexakisphosphate (IP6), pentakisphosphate (IP5) and certain tetrakisphosphate (IP4) species, all known activators of Class 1 HDACs in vitro . Here, we generated IPMK knockout (IKO) human U251 glioblastoma cells, which decreased cellular inositol phosphate levels and increased histone H4-acetylation by mass spectrometry. ChIP-seq showed IKO increased H4-acetylation at IKO-upregulated genes, but H4-acetylation was unchanged at IKO-downregulated genes, suggesting gene-specific responses to IPMK knockout. HDAC deacetylase enzyme activity was decreased in HDAC3 immunoprecipitates from IKO vs . wild-type cells, while deacetylase activity of other Class 1 HDACs had no detectable changes in activity. Wild-type IPMK expression in IKO cells fully rescued HDAC3 deacetylase activity, while kinase-dead IPMK expression had no effect. Further, the deficiency in HDAC3 activity in immunoprecipitates from IKO cells could be fully rescued by addition of synthesized IP4 (Ins(1,4,5,6)P4) to the enzyme assay, while control inositol had no effect. These data suggest that cellular IPMK-dependent inositol phosphates are required for full HDAC3 enzyme activity and proper histone H4-acetylation. Implications for targeting IPMK in HDAC3-dependent diseases are discussed.

DOI: https://doi.org/10.1101/2024.04.29.591660
mBio. 2024 May 15. e0341223

TurboID mapping reveals the exportome of secreted intrinsically disordered proteins in the transforming parasite Theileria annulata.

Francis Brühlmann, Carmen Perry, Charlotte Griessen, Kapila Gunasekera, Jean-Louis Reymond, Arunasalam Naguleswaran, Sven Rottenberg, Kerry Woods, Philipp Olias.

  Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1-4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.

Keywords:  Babesia; BioID; Cryptosporidium; East Coast fever; Plasmodium; Toxoplasma; cancer; cattle; malaria; neglected tropical disease; protozoa; theileriosis

DOI:  https://doi.org/10.1128/mbio.03412-23
Protein Sci. 2024 Jun;33(6): e5019

Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

Yuting Yang, Ejaz Ahmad, Vidhya Premkumar, Alicen Liu, S M Ashikur Rahman, Zaneta Nikolovska-Coleska.

  AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the β-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

Keywords:  AF9; DOT1L; MBP fusion; X‐ray crystallography; disulfide bridge; intrinsically disordered proteins; peptidomimetics

DOI:  https://doi.org/10.1002/pro.5019
Signal Transduct Target Ther. 2024 May 15. 9(1): 133

2-APQC, a small-molecule activator of Sirtuin-3 (SIRT3), alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis.

Fu Peng, Minru Liao, Wenke Jin, Wei Liu, Zixiang Li, Zhichao Fan, Ling Zou, Siwei Chen, Lingjuan Zhu, Qian Zhao, Gu Zhan, Liang Ouyang, Cheng Peng, Bo Han, Jin Zhang, Leilei Fu.

Sirtuin 3 (SIRT3) is well known as a conserved nicotinamide adenine dinucleotide+ (NAD+)-dependent deacetylase located in the mitochondria that may regulate oxidative stress, catabolism and ATP production. Accumulating evidence has recently revealed that SIRT3 plays its critical roles in cardiac fibrosis, myocardial fibrosis and even heart failure (HF), through its deacetylation modifications. Accordingly, discovery of SIRT3 activators and elucidating their underlying mechanisms of HF should be urgently needed. Herein, we identified a new small-molecule activator of SIRT3 (named 2-APQC) by the structure-based drug designing strategy. 2-APQC was shown to alleviate isoproterenol (ISO)-induced cardiac hypertrophy and myocardial fibrosis in vitro and in vivo rat models. Importantly, in SIRT3 knockout mice, 2-APQC could not relieve HF, suggesting that 2-APQC is dependent on SIRT3 for its protective role. Mechanically, 2-APQC was found to inhibit the mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K), c-jun N-terminal kinase (JNK) and transforming growth factor-β (TGF-β)/ small mother against decapentaplegic 3 (Smad3) pathways to improve ISO-induced cardiac hypertrophy and myocardial fibrosis. Based upon RNA-seq analyses, we demonstrated that SIRT3-pyrroline-5-carboxylate reductase 1 (PYCR1) axis was closely assoiated with HF. By activating PYCR1, 2-APQC was shown to enhance mitochondrial proline metabolism, inhibited reactive oxygen species (ROS)-p38 mitogen activated protein kinase (p38MAPK) pathway and thereby protecting against ISO-induced mitochondrialoxidative damage. Moreover, activation of SIRT3 by 2-APQC could facilitate AMP-activated protein kinase (AMPK)-Parkin axis to inhibit ISO-induced necrosis. Together, our results demonstrate that 2-APQC is a targeted SIRT3 activator that alleviates myocardial hypertrophy and fibrosis by regulating mitochondrial homeostasis, which may provide a new clue on exploiting a promising drug candidate for the future HF therapeutics.

DOI: https://doi.org/10.1038/s41392-024-01816-1
ACS Infect Dis. 2024 May 16.

An ApiAp2 Transcription Factor with a Dispensable Role in Plasmodium berghei Life Cycle.

Nirdosh, Himadri Shukla, Satish Mishra.

  Malaria parasites have a complex life cycle and undergo replication and population expansion within vertebrate hosts and mosquito vectors. These developmental transitions rely on changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. The ApiAp2 family of DNA-binding proteins plays an important role in regulating gene expression in malaria parasites. Here, we characterized the ApiAp2 protein in Plasmodium berghei, which we termed Ap2-D. In silico analysis revealed that Ap2-D has three beta-sheets followed by a helix at the C-terminus for DNA binding. Using gene tagging with 3XHA-mCherry, we found that Ap2-D is expressed in Plasmodium blood stages and is present in the parasite cytoplasm and nucleus. Surprisingly, our gene deletion study revealed a completely dispensable role for Ap2-D in the entirety of the P. berghei life cycle. Ap2-D KO parasites were found to grow in the blood successfully and progress through the mosquito midgut and salivary glands. Sporozoites isolated from mosquito salivary glands were infective for hepatocytes and achieved similar patency as WT in mice. We emphasize the importance of genetic validation of antimalarial drug targets before progressing them to drug discovery.

Keywords:  ApiAp2; Liver stage; Malaria; Mosquito stage; Plasmodium; Transcription factor

DOI:  https://doi.org/10.1021/acsinfecdis.4c00240
Nat Commun. 2024 May 15. 15(1): 4094

Cryo-EM structures of the human Elongator complex at work.

Nour-El-Hana Abbassi, Marcin Jaciuk, David Scherf, Pauline Böhnert, Alexander Rau, Alexander Hammermeister, Michał Rawski, Paulina Indyka, Grzegorz Wazny, Andrzej Chramiec-Głąbik, Dominika Dobosz, Bozena Skupien-Rabian, Urszula Jankowska, Juri Rappsilber, Raffael Schaffrath, Ting-Yu Lin, Sebastian Glatt.

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.

DOI: https://doi.org/10.1038/s41467-024-48251-y
bioRxiv. 2024 Apr 30. pii: 2024.04.29.591792. [Epub ahead of print]

MmpL3, Wag31 and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability.

Neda Habibi Arejan, Desiree R Czapski, Joseph A Buonomo, Cara C Boutte.

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis , we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.

DOI: https://doi.org/10.1101/2024.04.29.591792
Int J Mol Sci. 2024 Apr 27. pii: 4785. [Epub ahead of print]25(9):

SIRT1 Serum Concentrations in Lipodystrophic Syndromes.

Luisa Salvatori, Silvia Magno, Giovanni Ceccarini, Rossella Tozzi, Savina Contini, Caterina Pelosini, Ferruccio Santini, Lucio Gnessi, Stefania Mariani.

  Lipodystrophies (LDs) are rare, complex disorders of the adipose tissue characterized by selective fat loss, altered adipokine profile and metabolic impairment. Sirtuins (SIRTs) are class III NAD+-dependent histone deacetylases linked to fat metabolism. SIRT1 plays a critical role in metabolic health by deacetylating target proteins in tissue types including liver, muscle, and adipose. Circulating SIRT1 levels have been found to be reduced in obesity and increased in anorexia nervosa and patients experiencing weight loss. We evaluated circulating SIRT1 levels in relation to fat levels in 32 lipodystrophic patients affected by congenital or acquired LDs compared to non-LD subjects (24 with anorexia nervosa, 22 normal weight, and 24 with obesity). SIRT1 serum levels were higher in LDs than normal weight subjects (mean ± SEM 4.18 ± 0.48 vs. 2.59 ± 0.20 ng/mL) and subjects with obesity (1.7 ± 0.39 ng/mL), whereas they were close to those measured in anorexia nervosa (3.44 ± 0.46 ng/mL). Our findings show that within the LD group, there was no relationship between SIRT1 levels and the amount of body fat. The mechanisms responsible for secretion and regulation of SIRT1 in LD deserve further investigation.

Keywords:  SIRT1; adipokines; adipose tissue; lipodystrophy

DOI:  https://doi.org/10.3390/ijms25094785
Biomed J. 2024 May 09. pii: S2319-4170(24)00048-9. [Epub ahead of print] 100745

Charting new territory: The Plasmodium falciparum tRNA modification landscape.

Benjamin Sian Teck Lee, Ameya Sinha, Peter Dedon, Peter Preiser.

  Ribonucleoside modifications comprising the epitranscriptome are present in all organisms and all forms of RNA, including mRNA, rRNA and tRNA, the three major RNA components of the translational machinery. Of these, tRNA is the most heavily modified and the tRNA epitranscriptome has the greatest diversity of modifications. In addition to their roles in tRNA biogenesis, quality control, structure, cleavage, and codon recognition, tRNA modifications have been shown to regulate gene expression post-transcriptionally in prokaryotes and eukaryotes, including humans. However, studies investigating the impact of tRNA modifications on gene expression in the malaria parasite Plasmodium falciparum are currently scarce. Current evidence shows that the parasite has a limited capacity for transcriptional control, which points to a heavier reliance on strategies for posttranscriptional regulation such as tRNA epitranscriptome reprogramming. This review addresses the known functions of tRNA modifications in the biology of P. falciparum while highlighting the potential therapeutic opportunities and the value of using P. falciparum as a model organism for addressing several open questions related to the tRNA epitranscriptome.

Keywords:  RNA modifications; developmental regulation; drug resistance; malaria parasites; stress response; tRNA

DOI:  https://doi.org/10.1016/j.bj.2024.100745
Nat Commun. 2024 May 14. 15(1): 4083

mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Tal Levy, Kai Voeltzke, Laura Hruby, Khawla Alasad, Zuelal Bas, Marteinn Snaebjörnsson, Ran Marciano, Katerina Scharov, Mélanie Planque, Kim Vriens, Stefan Christen, Cornelius M Funk, Christina Hassiepen, Alisa Kahler, Beate Heider, Daniel Picard, Jonathan K M Lim, Anja Stefanski, Katja Bendrin, Andres Vargas-Toscano, Ulf D Kahlert, Kai Stühler, Marc Remke, Moshe Elkabets, Thomas G P Grünewald, Andreas S Reichert, Sarah-Maria Fendt, Almut Schulze, Guido Reifenberger, Barak Rotblat, Gabriel Leprivier.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

DOI: https://doi.org/10.1038/s41467-024-48386-y
Proc Natl Acad Sci U S A. 2024 May 21. 121(21): e2314604121

iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.

Jonathan S Marvin, Alexandros C Kokotos, Mukesh Kumar, Camila Pulido, Ariana N Tkachuk, Jocelyn Shuxin Yao, Timothy A Brown, Timothy A Ryan.

  We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

Keywords:  ATP; fluorescent sensor; neuronal metabolism

DOI:  https://doi.org/10.1073/pnas.2314604121
Neoplasia. 2024 May 16. pii: S1476-5586(24)00045-9. [Epub ahead of print]53 101003

Extracellular signals induce dynamic ER remodeling through αTAT1-dependent microtubule acetylation.

Hannah R Ortiz, Paola Cruz Flores, Julia Podgorski, Aaron Ramonett, Tasmia Ahmed, Nadine Hempel, Pascale G Charest, Nathan A Ellis, Paul R Langlais, William R Montfort, Karthikeyan Mythreye, Sanjay Kumar, Nam Y Lee.

  Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to various growth factors and cytokines including TGF-β and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT1-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT1 pathway may be a key target in ER stress and dysfunction.

Keywords:  Alpha TAT1; BOK; Endoplasmic reticulum; Microtubules; TAK1; TGF-beta

DOI:  https://doi.org/10.1016/j.neo.2024.101003
Hum Genet. 2024 May 16.

Characterization of a novel HDAC2 pathogenetic variant: a missing puzzle piece for chromatinopathies.

Elisabetta Di Fede, Antonella Lettieri, Esi Taci, Silvia Castiglioni, Stefano Rebellato, Chiara Parodi, Elisa Adele Colombo, Paolo Grazioli, Federica Natacci, Paola Marchisio, Lidia Pezzani, Grazia Fazio, Donatella Milani, Valentina Massa, Cristina Gervasini.

Histone deacetylases (HDACs) are enzymes pivotal for histone modification (i.e. acetylation marks removal), chromatin accessibility and gene expression regulation. Class I HDACs (including HDAC1, 2, 3, 8) are ubiquitously expressed and they often participate in multi-molecular protein complexes. To date, three neurodevelopmental disorders caused by mutations in genes encoding for HDACs (HDAC4, HDAC6 and HDAC8) and thus belonging to the group of chromatinopathies, have been described. We performed whole exome sequencing (WES) for a patient (#249) clinically diagnosed with the chromatinopathy Rubinstein-Taybi syndrome (RSTS) but negative for mutations in RSTS genes, identifying a de novo frameshift variant in HDAC2 gene. We then investigated its molecular effects in lymphoblastoid cell lines (LCLs) derived from the patient compared to LCLs from healthy donors (HD). As the variant was predicted to be likely pathogenetic and to affect the sequence of nuclear localization signal, we performed immunocytochemistry and lysates fractionation, observing a nuclear mis-localization of HDAC2 compared to HD LCLs. In addition, HDAC2 total protein abundance resulted altered in patient, and we found that newly identified variant in HDAC2 affects also acetylation levels, with significant difference in acetylation pattern among patient #249, HD and RSTS cells and in expression of a known molecular target. Remarkably, RNA-seq performed on #249, HD and RSTS cells shows differentially expressed genes (DEGs) common to #249 and RSTS. Interestingly, our reported patient was clinically diagnosed with RSTS, a chromatinopathy which known causative genes encode for enzymes antagonizing HDACs. These results support the role of HDAC2 as causative gene for chromatinopathies, strengthening the genotype-phenotype correlations in this relevant group of disorders.

DOI: https://doi.org/10.1007/s00439-024-02675-0