bims-toxgon 2022-10-16 papers

Issue of 2022–10–16
five papers selected by
Lakesh Kumar, BITS Pilani

Microbiol Spectr. 2022 Oct 10. e0136322

Essential Functions of Calmodulin and Identification of Its Proximal Interacting Proteins in Tachyzoite-Stage Toxoplasma gondii via BioID Technology.

Yongle Song, Longjiao Li, Xinyu Mo, Ming Pan, Bang Shen, Rui Fang, Min Hu, Junlong Zhao, Yanqin Zhou.

Toxoplasma gondii (T. gondii) is a pathogen belonging to the apicomplexan phylum, and it threatens human and animal health. Calcium ions, a critical second messenger in cells, can regulate important biological processes, including parasite invasion and egress. Calmodulin (CaM) is a small, highly conserved, Ca2+-binding protein found in all eukaryotic cells. After binding to Ca2+, CaM can be activated to interact with various proteins. However, little is known about CaM's function and its interacting proteins in T. gondii. In this study, we successfully knocked down CaM in the T. gondii parent strain TATI using a tetracycline-off system with the Toxoplasma CaM promoter. The results indicated that CaM was required for tachyzoite proliferation, invasion, and egress and that CaM depletion resulted in apicoplast loss, thus threatening parasite survival in the next lytic cycle. In the tachyzoite stage, CaM loss caused significant anomalies in the parasite's basal constriction, motility, and parasite rosette-like arrangement in the parasitophorous vacuole (PV). These phenotypic defects caused by CaM depletion indicate the importance of CaM in T. gondii. Therefore, it is important to identify the CaM-interacting proteins in T. gondii. Applying BioID technology, more than 300 CaM's proximal interacting proteins were identified from T. gondii. These CaM partners were broadly distributed throughout the parasite. Furthermore, the protein interactome and transcriptome analyses indicated the potential role of CaM in ion binding, cation binding, metal ion binding, calcium ion binding, and oxidation-reduction. Our findings shed light on the CaM function and CaM-interactome in T. gondii and other eukaryotes. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that threatens human and animal health. This unicellular parasite is active in many biological processes, such as egress and invasion. The implementation efficiency of T. gondii biological processes is dependent on signal transmission. Ca2+, as a second messenger, is essential for the parasite's life cycle. Calmodulin, a ubiquitous Ca2+ receptor protein, is highly conserved and mediates numerous Ca2+-dependent events in eukaryotes. Few CaM functions or regulated partners have been characterized in T. gondii tachyzoites. Here, we reported the essential functions of calmodulin in T. gondii tachyzoite and the identification of its interacting partners using BioID technology, shedding light on the CaM function and CaM-interactome in Toxoplasma gondii and other eukaryotes.

Keywords: Toxoplasma gondii; calmodulin; protein interaction; tetracycline-off; transcriptome

DOI: https://doi.org/10.1128/spectrum.01363-22

J Eukaryot Microbiol. 2022 Oct 11. e12951

The Toxoplasma Plant-Like Vacuolar Compartment (PLVAC).

Andrew J Stasic, Silvia N J Moreno, Vern B Carruthers, Zhicheng Dou.

Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. T. gondii shares several characteristics with plants including a non-photosynthetic plastid termed apicoplast and a multi-vesicular organelle that was named the plant-like vacuole (PLV) or vacuolar compartment (VAC). The name plant-like vacuole was selected based on its resemblance in composition and function to plant vacuoles. The name VAC represents its general vacuolar characteristics. We will refer to the organelle as PLVAC in this review. New findings in recent years have revealed that the PLVAC represents the lysosomal compartment of T. gondii which has adapted peculiarities to fulfill specific Toxoplasma needs. In this review, we discuss the composition and functions of the PLVAC highlighting its roles in ion storage and homeostasis, endocytosis, exocytosis, and autophagy.

Keywords: Toxoplasma gondii; apicomplexan; calcium; digestive; lysosome; plant-like vacuole; vacuolar compartment; zinc

DOI: https://doi.org/10.1111/jeu.12951

Genomics Proteomics Bioinformatics. 2022 Oct 07. pii: S1672-0229(22)00126-7. [Epub ahead of print]

Protein Lactylation and Metabolic Regulation of the Zoonotic Parasite Toxoplasma gondii.

Deqi Yin, Ning Jiang, Chang Cheng, Xiaoyu Sang, Ying Feng, Ran Chen, Qijun Chen.

The biology of Toxoplasma gondii, the causative pathogen of one of the most widespread parasitic diseases (toxoplasmosis), remains poorly understood. Lactate, which is derived glucose metabolism, is not only an energy source in a variety of organisms, including T. gondii, but also a regulatory molecule that participates in gene activation and protein function. Lysine lactylation (Kla) is a type of posttranslational modification (PTM) that has been recently associated with chromatin remodeling; however, Kla of histone and non-histone proteins has not yet been studied in T. gondii. To examine the prevalence and function of lactylation in T. gondii parasites, we mapped thelactylome of proliferating tachyzoite cells and identified 1964 lactylation sites on 955 proteins in the T. gondii RH strain. Lactylated proteins are distributed in multiple subcellular compartments and are closely related to a wide variety of biological processes, including mRNA splicing, glycolysis, aminoacyl-tRNA biosynthesis, RNA transport, and many signaling pathways. We also performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis using a lactylation-specific antibody and found that the histones H4K12la and H3K14la were enriched in the promoter and exon regions of T. gondii associated with microtubule-based movement and cell invasion. We further confirmed the delactylase activity of histone deacetylases TgHDACs 2, 3, and 4, and found that treatment with anti-histone acetyltransferase (TgMYST-A) antibodies profoundly reduced protein lactylation in T. gondii. This study offers the first dataset of the global lactylation proteome and provides a basis for further dissecting the functional biology of T. gondii.

Keywords: ChIP-seq; Lactylation; Metabolism; Protein posttranslational modification; Toxoplasma gondii

DOI: https://doi.org/10.1016/j.gpb.2022.09.010

Protein Expr Purif. 2022 Oct 07. pii: S1046-5928(22)00144-9. [Epub ahead of print] 106187

Heterologous expression, purification, and partial characterisation of the apicoplast protein 3-oxoacyl-[acyl-carrier-protein] reductase from Toxoplasma gondii.

Can Aygün, Sinem Kocer, Özkan Daniş, Soner Cubuk, Ozal Mutlu.

Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.

Keywords: 3-Oxoacyl-[acyl-carrier-protein] reductase; Apicoplast protein; FabG; Protein expression and purification; Toxoplasma gondii

DOI: https://doi.org/10.1016/j.pep.2022.106187

Acta Pharm Sin B. 2022 Oct;12(10): 3743-3782

Autophagy and beyond: Unraveling the complexity of UNC-51-like kinase 1 (ULK1) from biological functions to therapeutic implications.

Ling Zou, Minru Liao, Yongqi Zhen, Shiou Zhu, Xiya Chen, Jin Zhang, Yue Hao, Bo Liu.

UNC-51-like kinase 1 (ULK1), as a serine/threonine kinase, is an autophagic initiator in mammals and a homologous protein of autophagy related protein (Atg) 1 in yeast and of UNC-51 in Caenorhabditis elegans. ULK1 is well-known for autophagy activation, which is evolutionarily conserved in protein transport and indispensable to maintain cell homeostasis. As the direct target of energy and nutrition-sensing kinase, ULK1 may contribute to the distribution and utilization of cellular resources in response to metabolism and is closely associated with multiple pathophysiological processes. Moreover, ULK1 has been widely reported to play a crucial role in human diseases, including cancer, neurodegenerative diseases, cardiovascular disease, and infections, and subsequently targeted small-molecule inhibitors or activators are also demonstrated. Interestingly, the non-autophagy function of ULK1 has been emerging, indicating that non-autophagy-relevant ULK1 signaling network is also linked with diseases under some specific contexts. Therefore, in this review, we summarized the structure and functions of ULK1 as an autophagic initiator, with a focus on some new approaches, and further elucidated the key roles of ULK1 in autophagy and non-autophagy. Additionally, we also discussed the relationships between ULK1 and human diseases, as well as illustrated a rapid progress for better understanding of the discovery of more candidate small-molecule drugs targeting ULK1, which will provide a clue on novel ULK1-targeted therapeutics in the future.

Keywords: Autophagy; Biological function; Human diseases; Non-autophagy; Small-molecule drug; ULK1-targeted therapy; UNC-51-like kinase 1 (ULK1)

DOI: https://doi.org/10.1016/j.apsb.2022.06.004