bims-toxgon 2024-08-04 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2024‒08‒04
seventeen papers selected by
Lakesh Kumar, BITS Pilani

The initiation and early development of apical-basal polarity in Toxoplasma gondii.
Toxoplasma gondii bradyzoite-specific BAG1 is nonessential for cyst formation due to compensation by other heat-shock proteins.
Cytoskeleton Organization in Formation and Motility of Apicomplexan Parasites.
Acetyl-CoA: An interplay between metabolism and epigenetics in cancer.
CRISPR screens identify genes essential for in vivo virulence among proteins of hyperLOPIT-unassigned subcellular localization in Toxoplasma.
Deciphering the dynamics: Exploring the impact of mechanical forces on histone acetylation.
Functionality of the V-type ATPase during asexual growth and development of Plasmodium falciparum.
Apicomplexan Pore-Forming Toxins.
Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons.
Plant responses to abiotic stress regulated by histone acetylation.
Overview of class I HDAC modulators: Inhibitors and degraders.
The pyruvate dehydrogenase complex at the epigenetic crossroads of acetylation and lactylation.
Emerging role of sirtuins in non‑small cell lung cancer (Review).
Acetyl-CoA metabolism maintains histone acetylation for syncytialization of human placental trophoblast stem cells.
Recent advances in fluorescent probes for ATP imaging.
Auto-Regulatory mechanism of enzyme activity by the nuclear localization signal of lysine specific demethylase 1.
Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas.

bioRxiv. 2024 Jul 16. pii: 2024.07.14.603470. [Epub ahead of print]

The initiation and early development of apical-basal polarity in Toxoplasma gondii.

Luisa F Arias Padilla, Jonathan Munera Lopez, Aika Shibata, John M Murray, Ke Hu.

The human parasite Toxoplasma gondii has a distinctive body plan with a well-defined polarity. In the apical complex, the minus ends of the 22 cortical microtubules are anchored to the apical polar ring, a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end, and is critical for cytokinesis. How this apical-basal polarity axis is initiated was unknown. Here we examined the development of the apical polar ring and the basal complex in nascent daughters using expansion microscopy. We found that different substructures in the apical polar ring have different sensitivity to stress. In addition, apical-basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the construction of the daughter framework progresses towards the centrioles, the apical and the basal arcs co-develop in striking synchrony ahead of the microtubule array, and close into a ring-form before all the microtubules are nucleated. We also found that two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of each protein individually has modest to no impact on the lytic cycle. However, the loss of both results in abnormalities in the microtubule array and highly reduced plaquing and invasion efficiency.

DOI: https://doi.org/10.1101/2024.07.14.603470
Parasit Vectors. 2024 Jul 30. 17(1): 322

Toxoplasma gondii bradyzoite-specific BAG1 is nonessential for cyst formation due to compensation by other heat-shock proteins.

Weiling Wu, Qiqi Chen, Weihao Zou, Jiating Chen, Di Zhu, Huijing Yang, Lishan Ouyang, Xiaojun Liu, Hongjuan Peng.

  BACKGROUND: Toxoplasma gondii is an opportunistic pathogenic protozoan that infects all warm-blooded animals, including humans, and causes zoonotic toxoplasmosis. The bradyzoite antigen 1 (BAG1), known as heat-shock protein (HSP)30, is a specific antigen expressed during the early stage of T. gondii tachyzoite-bradyzoite conversion.METHODS: A bag1 gene knockout strain based on the T. gondii type II ME49 was constructed and designated as ME49Δbag1. The invasion, proliferation, and cyst formation efficiency in the cell model and survival in the mouse model were compared between the ME49 and ME49Δbag1 strains after infection. Quantitative polymerase chain reaction (qPCR) was used to detect the transcriptional level of important genes, and western-blot was used to detect protein levels.
RESULTS: ME49Δbag1 displayed significantly inhibited cyst formation, although it was not completely blocked. During early differentiation induced by alkaline and starvation conditions in vitro, the proliferation of ME49Δbag1 was significantly accelerated relative to the ME49 strain. Meanwhile, the transcription of the HSP family and bradyzoite formation deficient 1 (bfd1) were significantly enhanced. The observed upregulation suggests a compensatory mechanism to counterbalance the impaired stress responses of T. gondii following bag1 knockout. On the other hand, the elevated transcription levels of several HSP family members, including HSP20, HSP21, HSP40, HSP60, HSP70, and HSP90, along with BFD1, implied the involvement of alternative regulatory factors in bradyzoite differentiation aside from BAG1.
CONCLUSIONS: The data suggested that when bag1 was absent, the stress response of T. gondii was partially compensated by increased levels of other HSPs, resulting in the formation of fewer cysts. This highlighted a complex regulatory network beyond BAG1 influencing the parasite's transformation into bradyzoites, emphasizing the vital compensatory function of HSPs in the T. gondii life cycle adaptation.

Keywords:   Toxoplasma gondii ; bag1 ; CRISPR/Cas9; Heat-shock protein; Tachyzoite and bradyzoite transformation

DOI:  https://doi.org/10.1186/s13071-024-06339-w
Annu Rev Microbiol. 2024 Aug 02.

Cytoskeleton Organization in Formation and Motility of Apicomplexan Parasites.

Ross G Douglas, Robert W Moon, Friedrich Frischknecht.

Apicomplexan parasites are a group of eukaryotic protozoans with diverse biology that have affected human health like no other group of parasites. These obligate intracellular parasites rely on their cytoskeletal structures for giving them form, enabling them to replicate in unique ways and to migrate across tissue barriers. Recent progress in transgenesis and imaging tools allowed detailed insights into the components making up and regulating the actin and microtubule cytoskeleton as well as the alveolate-specific intermediate filament-like cytoskeletal network. These studies revealed interesting details that deviate from the cell biology of canonical model organisms. Here we review the latest developments in the field and point to a number of open questions covering the most experimentally tractable parasites: Plasmodium, the causative agent of malaria; Toxoplasma gondii, the causative agent of toxoplasmosis; and Cryptosporidium, a major cause of diarrhea.

DOI: https://doi.org/10.1146/annurev-micro-041222-011539
Front Mol Med. 2022 ;2 1044585

Acetyl-CoA: An interplay between metabolism and epigenetics in cancer.

Yang Hao, Qin Yi, Xu XiaoWu, Chen WeiBo, Zu GuangChen, Chen XueMin.

  Due to its high mortality and severe economic burden, cancer has become one of the most difficult medical problems to solve today. As a key node in metabolism and the main producer of energy, acetyl-coenzyme A (acetyl-CoA) plays an important role in the invasion and migration of cancer. In this review, we discuss metabolic pathways involving acetyl-CoA, the targeted therapy of cancer through acetyl-CoA metabolic pathways and the roles of epigenetic modifications in cancer. In particular, we emphasize that the metabolic pathway of acetyl-CoA exerts a great impact in cancer; this process is very different from normal cells due to the "Warburg effect". The concentration of acetyl-CoA is increased in the mitochondria of cancer cells to provide ATP for survival, hindering the growth of normal cells. Therefore, it may be possible to explore new feasible and more effective treatments through the acetyl-CoA metabolic pathway. In addition, a growing number of studies have shown that abnormal epigenetic modifications have been shown to play contributing roles in cancer formation and development. In most cancers, acetyl-CoA mediated acetylation promotes the growth of cancer cells. Thus, acetylation biomarkers can also be detected and serve as potential cancer prediction and prognostic markers.

Keywords:  acetyl-CoA; cancer; epigenetics; histone acetylation; metabolism

DOI:  https://doi.org/10.3389/fmmed.2022.1044585
mBio. 2024 Jul 31. e0172824

CRISPR screens identify genes essential for in vivo virulence among proteins of hyperLOPIT-unassigned subcellular localization in Toxoplasma.

Yuta Tachibana, Miwa Sasai, Masahiro Yamamoto.

  The research field to identify and characterize genes essential for in vivo virulence in Toxoplasma gondii has been dramatically advanced by a series of in vivo clustered regularly interspaced short palindromic repeats (CRISPR) screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1,000 proteins remained unassigned, and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed in vivo CRISPR screens. As a result, we identified several genes essential for in vivo virulence that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the in vivo essentiality of Toxoplasma proteins with previously unknown localizations.IMPORTANCEToxoplasma gondii is a protozoan parasite that causes severe infection in immunocompromised patients or newborns. Toxoplasma possesses more than 8,000 genes; however, the genes essential for in vivo virulence were not fully identified. The apicomplexan parasites, including Toxoplasma, developed unique organelles that do not exist in other model organisms; thus, determining the subcellular location of parasite proteins is important for understanding their functions. Here, we used in vivo genetic screens that enabled us to investigate hundreds of genes in Toxoplasma during mouse infection. We screened approximately 600 parasite proteins with previously unknown subcellular localizations. We identified many novel genes that confer parasite virulence in mice. Among the top hits, we characterized two genes essential for in vivo virulence, TgGTPase and TgRimM, which are widely conserved in the phylum Apicomplexa. Our findings will contribute to understanding how apicomplexans adapt to the host environment and cause disease.

Keywords:  CRISPR screen; Toxoplasma; hyperLOPIT-unassigned virulence factors

DOI:  https://doi.org/10.1128/mbio.01728-24
FASEB J. 2024 Aug 15. 38(15): e23849

Deciphering the dynamics: Exploring the impact of mechanical forces on histone acetylation.

Jingyi Cai, Yudi Deng, Ziyang Min, Chaoyuan Li, Zhihe Zhao, Dian Jing.

  Living cells navigate a complex landscape of mechanical cues that influence their behavior and fate, originating from both internal and external sources. At the molecular level, the translation of these physical stimuli into cellular responses relies on the intricate coordination of mechanosensors and transducers, ultimately impacting chromatin compaction and gene expression. Notably, epigenetic modifications on histone tails govern the accessibility of gene-regulatory sites, thereby regulating gene expression. Among these modifications, histone acetylation emerges as particularly responsive to the mechanical microenvironment, exerting significant control over cellular activities. However, the precise role of histone acetylation in mechanosensing and transduction remains elusive due to the complexity of the acetylation network. To address this gap, our aim is to systematically explore the key regulators of histone acetylation and their multifaceted roles in response to biomechanical stimuli. In this review, we initially introduce the ubiquitous force experienced by cells and then explore the dynamic alterations in histone acetylation and its associated co-factors, including HDACs, HATs, and acetyl-CoA, in response to these biomechanical cues. Furthermore, we delve into the intricate interactions between histone acetylation and mechanosensors/mechanotransducers, offering a comprehensive analysis. Ultimately, this review aims to provide a holistic understanding of the nuanced interplay between histone acetylation and mechanical forces within an academic framework.

Keywords:  biomechanics; epigenetics; histone acetylases; histone acetylation; histone deacetylases

DOI:  https://doi.org/10.1096/fj.202400907RR
J Biol Chem. 2024 Jul 29. pii: S0021-9258(24)02109-4. [Epub ahead of print] 107608

Functionality of the V-type ATPase during asexual growth and development of Plasmodium falciparum.

Neeta Shadija, Swati Dass, Wei Xu, Liying Wang, Hangjun Ke.

  V-type ATPases are highly conserved hetero-multi-subunit proton pumping machineries found in all eukaryotes. They utilize ATP hydrolysis to pump protons, acidifying intracellular or extracellular compartments, and are thus crucial for various biological processes. Despite their evolutionary conservation in malaria parasites, this proton pump remains understudied. To understand the localization and biological functions of Plasmodium falciparum V-type ATPase, we employed CRISPR/Cas9 to endogenously tag the subunit A of the V1 domain. V1A (PF3D7_1311900) was tagged with a triple hemagglutinin epitope and the TetR-DOZI-aptamer system for conditional expression under the regulation of anhydrotetracycline. Via immunofluorescence assays, we identified that V-type ATPase is expressed throughout the intraerythrocytic developmental cycle and is mainly localized on the digestive vacuole and parasite plasma membrane. Immuno-electron microscopy further revealed that V-type ATPase is also localized on secretory organelles in merozoites. Knockdown of V1A led to cytosolic pH imbalance and blockage of hemoglobin digestion in the digestive vacuole, resulting in an arrest of parasite development in the trophozoite stage and, ultimately, parasite demise. Using Bafilomycin A1, a specific inhibitor of V-type ATPases, we found that the P. falciparum V-type ATPase is likely involved in parasite invasion but is not critical for ring stage development. Further, we detected a large molecular weight complex in BN-PAGE (∼ 1.0 MDa), corresponding to the total molecular weights of V1 and Vo domains. Together, we show that V-type ATPase is localized on multiple subcellular compartments in P. falciparum, and its functionality throughout the asexual cycle varies depending on the parasite developmental stages.

Keywords:  Malaria; Plasmodium; Plasmodium falciparum; V-type ATPase; digestive vacuole; hemoglobin digestion; pH regulation; proton pump; ring stage; schizont stage; trophozoite stage

DOI:  https://doi.org/10.1016/j.jbc.2024.107608
Annu Rev Microbiol. 2024 Aug 01.

Apicomplexan Pore-Forming Toxins.

Vern B Carruthers.

Pore-forming toxins (PFTs) are released by one cell to directly inflict damage on another cell. Hosts use PFTs, including members of the membrane attack complex/perforin protein family, to fight bacterial infections and cancer, while bacteria and parasites deploy PFTs to promote infection. Apicomplexan parasites secrete perforin-like proteins as PFTs to egress from infected cells and traverse tissue barriers. Other protozoa, along with helminth parasites, utilize saposin-like PFTs prospectively for nutrient acquisition during infection. This review discusses seminal and more recent advances in understanding how parasite PFTs promote infection and describes how they are regulated and fulfill their roles without causing parasite self-harm. Although exciting progress has been made in defining mechanisms of pore formation by PFTs, many open questions remain to be addressed to gain additional key insights into these remarkable determinants of parasitic infections.

DOI: https://doi.org/10.1146/annurev-micro-041222-025939
Nat Microbiol. 2024 Jul 29.

Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons.

Shahar Bracha, Hannah J Johnson, Nicole A Pranckevicius, Francesca Catto, Athena E Economides, Sergey Litvinov, Karoliina Hassi, Marco Tullio Rigoli, Cristina Cheroni, Matteo Bonfanti, Alessia Valenti, Sarah Stucchi, Shruti Attreya, Paul D Ross, Daniel Walsh, Nati Malachi, Hagay Livne, Reut Eshel, Vladislav Krupalnik, Doron Levin, Stuart Cobb, Petros Koumoutsakos, Nicolò Caporale, Giuseppe Testa, Adriano Aguzzi, Anita A Koshy, Lilach Sheiner, Oded Rechavi.

Delivering macromolecules across biological barriers such as the blood-brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii, a parasite that naturally travels from the human gut to the central nervous system (CNS), can deliver proteins to host cells. Here we engineered T. gondii's endogenous secretion systems, the rhoptries and dense granules, to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells, brain organoids and in vivo, and probe protein activity using imaging, pull-down assays, scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept, we demonstrate GRA16-mediated brain delivery of the MeCP2 protein, a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system, we aim to guide future improvements that will be required for broader application.

DOI: https://doi.org/10.1038/s41564-024-01750-6
Front Plant Sci. 2024 ;15 1404977

Plant responses to abiotic stress regulated by histone acetylation.

Fei Wang, Chong-Hua Li, Ying Liu, Ling-Feng He, Ping Li, Jun-Xin Guo, Na Zhang, Bing Zhao, Yang-Dong Guo.

  In eukaryotes, histone acetylation and deacetylation play an important role in the regulation of gene expression. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increasing evidence highlights histone acetylation plays essential roles in the regulation of gene expression in plant response to environmental stress. In this review, we discussed the recent advance of histone acetylation in the regulation of abiotic stress responses including temperature, light, salt and drought stress. This information will contribute to our understanding of how plants adapt to environmental changes. As the mechanisms of epigenetic regulation are conserved in many plants, research in this field has potential applications in improvement of agricultural productivity.

Keywords:  abiotic stress; epigenetic regulation; histone acetylation; histone acetyltransferase; histone deacetylase

DOI:  https://doi.org/10.3389/fpls.2024.1404977
Eur J Med Chem. 2024 Jul 30. pii: S0223-5234(24)00576-2. [Epub ahead of print]276 116696

Overview of class I HDAC modulators: Inhibitors and degraders.

Ziqian Huang, Limei Zeng, Binbin Cheng, Deping Li.

  Class I histone deacetylases (HDACs) are closely associated with the development of a diverse array of diseases, including cancer, neurodegenerative disorders, HIV, and inflammatory diseases. Considering the essential roles in tumorigenesis, class I HDACs have emerged as highly desirable targets for therapeutic strategies, particularly in the field of anticancer drug development. However, the conventional class I HDAC inhibitors faced several challenges such as acquired resistance, inherent toxicities, and limited efficacy in inhibiting non-enzymatic functions of HDAC. To address these problems, novel strategies have emerged, including the development of class I HDAC dual-acting inhibitors, targeted protein degradation (TPD) technologies such as PROTACs, molecular glues, and HyT degraders, as well as covalent inhibitors. This review provides a comprehensive overview of class I HDAC enzymes and inhibitors, by initially introducing their structure and biological roles. Subsequently, we focus on the recent advancements of class I HDAC modulators, including isoform-selective class I inhibitors, dual-target inhibitors, TPDs, and covalent inhibitors, from the perspectives of rational design principles, pharmacodynamics, pharmacokinetics, and clinical progress. Finally, we also provide the challenges and outlines future prospects in the realm of class I HDAC-targeted drug discovery for cancer therapeutics.

Keywords:  Covalent inhibitors; Degraders; Histone deacetylases (HDACs); Modulators; PROTACs

DOI:  https://doi.org/10.1016/j.ejmech.2024.116696
Mol Genet Metab. 2024 Jul 16. pii: S1096-7192(24)00424-4. [Epub ahead of print]143(1-2): 108540

The pyruvate dehydrogenase complex at the epigenetic crossroads of acetylation and lactylation.

Peter W Stacpoole, Carolyn O Dirain.

  The pyruvate dehydrogenase complex (PDC) is remarkable for its size and structure as well as for its physiological and pathological importance. Its canonical location is in the mitochondrial matrix, where it primes the tricarboxylic acid (TCA) cycle by decarboxylating glycolytically-derived pyruvate to acetyl-CoA. Less well appreciated is its role in helping to shape the epigenetic landscape, from early development throughout mammalian life by its ability to "moonlight" in the nucleus, with major repercussions for human healthspan and lifespan. The PDC's influence on two crucial modifiers of the epigenome, acetylation and lactylation, is the focus of this brief review.

Keywords:  Acetylation; Epigenetics; Glycolysis; Histones; Inborn metabolic disorders; Lactylation; Nuclear translocation; Pyruvate dehydrogenase complex; Tricarboxylic acid cycle

DOI:  https://doi.org/10.1016/j.ymgme.2024.108540
Oncol Rep. 2024 Oct;pii: 127. [Epub ahead of print]52(4):

Emerging role of sirtuins in non‑small cell lung cancer (Review).

Min Zhou, Lin Wei, Renfu Lu.

  Non‑small cell lung cancer (NSCLC) is a highly prevalent lung malignancy characterized by insidious onset, rapid progression and advanced stage at the time of diagnosis, making radical surgery impossible. Sirtuin (SIRT) is a histone deacetylase that relies on NAD+ for its function, regulating the aging process through modifications in protein activity and stability. It is intricately linked to various processes, including glycolipid metabolism, inflammation, lifespan regulation, tumor formation and stress response. An increasing number of studies indicate that SIRTs significantly contribute to the progression of NSCLC by regulating pathophysiological processes such as energy metabolism, autophagy and apoptosis in tumor cells through the deacetylation of histones or non‑histone proteins. The present review elaborates on the roles of different SIRTs and their mechanisms in NSCLC, while also summarizing novel therapeutic agents based on SIRTs. It aims to present new ideas and a theoretical basis for NSCLC treatment.

Keywords:  NAD+; apoptosis; autophagy; histone deacetylase; non‑small cell lung cancer; sirtuins

DOI:  https://doi.org/10.3892/or.2024.8786
Cell Stem Cell. 2024 Jul 23. pii: S1934-5909(24)00253-4. [Epub ahead of print]

Acetyl-CoA metabolism maintains histone acetylation for syncytialization of human placental trophoblast stem cells.

Xin Yu, Hao Wu, Jiali Su, Xupeng Liu, Kun Liang, Qianqian Li, Ruoxuan Yu, Xuan Shao, Hongmei Wang, Yan-Ling Wang, Ng Shyh-Chang.

  During pregnancy, placental-fetal nutrient allocation is crucial for fetal and maternal health. However, the regulatory mechanisms for nutrient metabolism and allocation in placental trophoblasts have remained unclear. Here, we used human first-trimester placenta samples and human trophoblast stem cells (hTSCs) to discover that glucose metabolism is highly active in hTSCs and cytotrophoblasts, but during syncytialization, it decreases to basal levels, remaining necessary for fueling acetyl-CoA and differentiation potential. Acetate supplementation could rescue syncytiotrophoblast fusion from glycolysis deficiency by replenishing acetyl-CoA and maintaining histone acetylation, thus rescuing the activation of syncytialization genes. Even brief glycolysis deficiency could permanently inhibit differentiation potential and promote inflammation, which could also be permanently rescued by brief acetate supplementation in vivo. These results suggest that hTSCs retain only basal glycolytic acetyl-CoA metabolism during syncytialization to regulate cell fates via nutrient-responsive histone acetylation, with implications for our understanding of the balance between placental and fetal nutrition.

Keywords:  acetate; epigenetics; glucose; histone acetylation; placenta; pregnancy; trophoblast stem cell

DOI:  https://doi.org/10.1016/j.stem.2024.07.003
Talanta. 2024 Jul 28. pii: S0039-9140(24)01001-4. [Epub ahead of print]279 126622

Recent advances in fluorescent probes for ATP imaging.

Chen Zhang, Guanzhao Wu.

  Adenosine-5'-triphosphate (ATP) is a critical biological molecule that functions as the primary energy currency within cells. ATP synthesis occurs in the mitochondria, and variations in its concentration can significantly influence mitochondrial and cellular performance. Prior studies have established a link between ATP levels and a variety of diseases, such as cancer, neurodegenerative conditions, ischemia, and hypoglycemia. Consequently, researchers have developed many fluorescent probes for ATP detection, recognizing the importance of monitoring intracellular ATP levels to understand cellular processes. These probes have been effectively utilized for visualizing ATP in living cells and biological samples. In this comprehensive review, we categorize fluorescent sensors developed in the last five years for ATP detection. We base our classification on fluorophores, structure, multi-response channels, and application. We also evaluate the challenges and potential for advancing new generations of fluorescence imaging probes for monitoring ATP in living cells. We hope this summary motivates researchers to design innovative and effective probes tailored to ATP sensing. We foresee imminent progress in the development of highly sophisticated ATP probes.

Keywords:  ATP detection; Adenosine-5′-triphosphate; Fluorescence imaging; Nanoprobes; Small-molecule probes

DOI:  https://doi.org/10.1016/j.talanta.2024.126622
J Biol Chem. 2024 Jul 29. pii: S0021-9258(24)02108-2. [Epub ahead of print] 107607

Auto-Regulatory mechanism of enzyme activity by the nuclear localization signal of lysine specific demethylase 1.

Dulmi Senanayaka, Danyun Zeng, Sahar Alishiri, William J Martin, Khadijah I Moore, Roshni Patel, Zigmund Luka, Alexander Hirschi, Nicholas J Reiter.

  The N-terminal region of the human Lysine Specific Demethylase 1 (LSD1) has no predicted structural elements, contains a nuclear localization signal (NLS), undergoes multiple post-translational modifications (PTMs), and acts as a protein-protein interaction hub. This intrinsically disordered region (IDR) extends from core LSD1 structure, resides atop the catalytic active site, and is known to be dispensable for catalysis. Here, we show differential nucleosome binding between the full-length and an N-terminus deleted LSD1 and identify that a conserved NLS and PTM containing element of the N-terminus contains an alpha helical structure, and that this conserved element impacts demethylation. Enzyme assays reveal that LSD1's own electropositive NLS amino acids 107-120 inhibit demethylation activity on a model Histone 3 lysine 4 di-methyl (H3K4me2) peptide (Kiapp ∼ 3.3 μM) and H3K4me2 nucleosome substrates (IC50 ∼ 30.4 μM), likely mimicking the histone H3 tail. Further, when the identical, inhibitory NLS region contains phosphomimetic modifications, inhibition is partially relieved. Based upon these results and biophysical data, a regulatory mechanism for the LSD1-catalyzed demethylation reaction is proposed whereby NLS-mediated autoinhibition can occur through electrostatic interactions, and be partially relieved through phosphorylation that occurs proximal to the NLS. Taken together, the results highlight a dynamic and synergistic role for PTMs, IDRs, and structured regions near LSD1 active site and introduces the notion that phosphorylated mediated NLS regions can function to fine-tune chromatin modifying enzyme activity.

Keywords:  NMR; autoinhibition; histone demethylase; inhibition mechanism; intrinsically disordered protein; nuclear localization signal (NLS); nucleosome; structural biology

DOI:  https://doi.org/10.1016/j.jbc.2024.107607
Elife. 2024 Aug 02. pii: RP96257. [Epub ahead of print]13

Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas.

Danielle Algranati, Roni Oren, Bareket Dassa, Liat Fellus-Alyagor, Alexander Plotnikov, Haim Barr, Alon Harmelin, Nir London, Guy Ron, Noa Furth, Efrat Shema.

  Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

Keywords:  DMG; cancer biology; epigenetics; human; oncogene

DOI:  https://doi.org/10.7554/eLife.96257