bims-toxgon 2024-12-22 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2024–12–22
27 papers selected by
Lakesh Kumar, BITS Pilani

Importin α inhibitors act against the differentiated stages of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii.
Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism.
Glabridin exhibits potent inhibitory effects against Toxoplasma gondii in vitro and in vivo.
Apicomplexan mitoribosome from highly fragmented rRNAs to a functional machine.
Sirtuins as Key Regulators in Pancreatic Cancer: Insights into Signaling Mechanisms and Therapeutic Implications.
Protective humoral immunity induced by virus-like particles expressing Toxoplasma gondii CST1 or MIC8.
Salmonella-induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis.
Key Regulators of Parasite Biology Viewed Through a Post-Translational Modification Repertoire.
Mitochondrial metabolism and epigenetic crosstalk drive the SASP.
The F204S mutation in adrenodoxin oxidoreductase drives salinomycin resistance in Eimeria tenella.
SIRT2 Inhibition by AGK2 Promotes Perinuclear Cytoskeletal Organisation and Reduces Invasiveness of MDA-MB-231 Triple-Negative Breast Cancer Cells in Confined In Vitro Models.
Aldehyde dehydrogenases as drug targets for cancer: SAR and structural biology aspects for inhibitor design.
Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing.
Epigenetic modifications and emerging therapeutic targets in cardiovascular aging and diseases.
Fantastic proteins and where to find them - histones, in the nucleus and beyond.
Rapid degradation of histone deacetylase 1 (HDAC1) reveals essential roles in both gene repression and active transcription.
ACSL4-mediated H3K9 and H3K27 hyperacetylation upregulates SNAIL to drive TNBC metastasis.
Diacylglycerol Kinases and Its Role in Lipid Metabolism and Related Diseases.
Stage-specific function of sphingolipid synthases in African trypanosomes.
Inhibition of L-Threonine Dehydrogenase from Trypanosoma cruzi reduces glycine and acetate production and interferes with parasite growth and viability.
A novel SUN1-ALLAN complex coordinates segregation of the bipartite MTOC across the nuclear envelope during rapid closed mitosis in Plasmodium.
NAD+ Boosting Strategies.
Optogenetically Induced Microtubule Acetylation Unveils the Molecular Dynamics of Actin-Microtubule Crosstalk in Directed Cell Migration.
Lysine and arginine methylation of transcription factors.
Co-crystal structure of Helicobacter pylori biotin protein ligase with biotinyl-5-ATP.
SLMO transfers phosphatidylserine between the outer and inner mitochondrial membrane in Drosophila.
Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.

J Antimicrob Chemother. 2024 Dec 18. pii: dkae434. [Epub ahead of print]

Importin α inhibitors act against the differentiated stages of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii.

Manasi Bhambid, Sujata B Walunj, C A Anupama, Shilpi Jain, Diksha Mehta, Anjali Arya, Kylie M Wagstaff, Ashutosh Panda, David A Jans, Asif Mohmmed, Swati Patankar.

BACKGROUND: Nuclear import, dependent on the transporter importin α (IMPα), is a drug target for apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Indeed, a panel of small molecule inhibit interactions between IMPα and nuclear localization signals (NLSs) in vitro and the growth of rapidly dividing stages (P. falciparum blood stages and T. gondii tachyzoites) in culture.
OBJECTIVES: As new drugs targeting multiple life cycle stages of both parasites are required, the panel of IMPα inhibitors was tested for their ability to inhibit nuclear transport in the rapidly dividing stages and the maturation of differentiated stages (P. falciparum gametocytes and T. gondii bradyzoites).
METHODS: Using biophysical assays, Bay 11-7082, a Bay 11-7085 structural analogue, was tested for inhibition of IMPα:NLS interactions. The effect of the panel of inhibitors on the nuclear localization of reporter proteins was analysed in both parasites using transfections and microscopy. Also, using microscopy, the effect of inhibitors on differentiated stages of both parasites was tested.
RESULTS: Bay 11-7085 can inhibit nuclear transport in tachyzoites, while GW5074 and Caffeic Acid Phenethyl Ester (CAPE) can inhibit nuclear transport in the blood stages. Interestingly, CAPE can strongly inhibit gametocyte maturation, and Bay 11-7082 and Bay 11-7085 weakly inhibit bradyzoite differentiation.
CONCLUSIONS: As differentiation of gametocytes and bradyzoites is dependent on the activation of gene expression triggered by the nuclear translocation of transcription factors, our work provides a 'proof of concept' that targeting nuclear import is a viable strategy for the development of therapeutics against multiple stages of apicomplexan parasites, some of which are recalcitrant to existing drugs.

DOI: https://doi.org/10.1093/jac/dkae434
Commun Biol. 2024 Dec 16. 7(1): 1637

Toxoplasma gondii infection induces early host cell cycle arrest and DNA damage in primary human host cells by a MYR1-dependent mechanism.

Zahady D Velásquez, Lisbeth Rojas-Baron, Iván Conejeros, Carlos Hermosilla, Anja Taubert.

Toxoplasma gondii, an obligate intracellular parasite, control its host cell cycle through mechanisms that are not fully understood. Key effector molecules, including MYR1 and HCE1, play roles in translocating parasite proteins and inducing host cellular cyclin E1 overexpression, respectively. We investigated the early role of MYR1- and HCE1-driven host cell cycle arrest and DNA damage (up to 3 h p.i.). Our findings showed that T. gondii-infected cells experienced S-phase arrest and displayed double-strand DNA breaks as soon as 15 min p.i. This condition persisted until 3 h p.i., at which point we also observed increased host cell binucleation and micronuclei formation, both hallmarks of genomic instability. Furthermore, host cells responded to DNA damage by activating the ATM branch of the homologous recombination repair pathway. MYR1 was shown to be crucial, as TgΔmyr1 tachyzoites failed to induce S-phase arrest and DNA damage foci. In contrast, the absence of HCE1 did not produce these effects, suggesting that cyclin E1 expression was not involved. Also, DNA damage was demonstrated to be ROS-independent, suggesting that ROS did not trigger DNA damage. Our results suggest that T. gondii compromises host cellular DNA integrity depending on MYR1 shortly after infection, maintaining it over time.

DOI: https://doi.org/10.1038/s42003-024-07374-0
Parasit Vectors. 2024 Dec 18. 17(1): 522

Glabridin exhibits potent inhibitory effects against Toxoplasma gondii in vitro and in vivo.

Lu Wang, Bintao Zhai, Chen Wang, Hany M Elsheikha, Haiting Guo, Xiao-Nan Zheng, Chun-Xue Zhou, Xing-Quan Zhu.

   BACKGROUND: Toxoplasma gondii is an obligate protozoan parasite capable of infecting a wide range of warm-blooded animals and humans. Current treatment options, primarily pyrimethamine and sulfadiazine, have limitations, such as high recurrence rates, long treatment durations, and limited effectiveness against T. gondii. There is an unmet need for novel, safe, low-toxicity, and highly effective treatments. This study aimed to evaluate the anti-T. gondii effects of glabridin, a natural compound derived from the roots of a widely used medicinal plant.
METHODS: The cytotoxicity of glabridin in Vero cells was assessed using a CCK-8 cell viability assay. Quantitative polymerase chain reaction (qPCR) targeting the Tg-529 gene was developed to quantify T. gondii and assess the inhibitory effects of glabridin on parasite proliferation. Ultrastructural changes in T. gondii after treatment were examined using electron microscopy. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were examined to assess the effects of glabridin on ROS levels and ΔΨm in T. gondii tachyzoites. Additionally, metabolomics and transcriptomics analyses were conducted to investigate the mechanisms underlying glabridin's anti-T. gondii effects.
RESULTS: Glabridin exhibited low toxicity to host cells and effectively inhibited T. gondii invasion and proliferation in vitro in a time-dependent manner. Glabridin-treated tachyzoites exhibited significant structural alterations, along with increased ROS production and a reduction in ΔΨm. Metabolomic analysis indicated that glabridin significantly affected amino acid metabolism pathways in T. gondii. In vivo, glabridin treatment significantly improved survival rates in T. gondii-infected BALB/c mice at a dosage of 100 mg/kg.
CONCLUSIONS: This study demonstrates that glabridin has potent anti-T. gondii effects in vitro and in vivo, likely through disruption of amino acid metabolism in the parasite. These findings highlight glabridin's potential as a promising therapeutic agent for toxoplasmosis.

Keywords:   Toxoplasma gondii ; Glabridin; Metabolomics; Survival rate; Transcriptomics

DOI:  https://doi.org/10.1186/s13071-024-06610-0
Nat Commun. 2024 Dec 17. 15(1): 10689

Apicomplexan mitoribosome from highly fragmented rRNAs to a functional machine.

Chaoyue Wang, Sari Kassem, Rafael Eduardo Oliveira Rocha, Pei Sun, Tan-Trung Nguyen, Joachim Kloehn, Xianyong Liu, Lorenzo Brusini, Alessandro Bonavoglia, Sramona Barua, Fanny Boissier, Mayara Lucia Del Cistia, Hongjuan Peng, Xinming Tang, Fujie Xie, Zixuan Wang, Oscar Vadas, Xun Suo, Yaser Hashem, Dominique Soldati-Favre, Yonggen Jia.

The phylum Apicomplexa comprises eukaryotic parasites that cause fatal diseases affecting millions of people and animals worldwide. Their mitochondrial genomes have been significantly reduced, leaving only three protein-coding genes and highly fragmented mitoribosomal rRNAs, raising challenging questions about mitoribosome composition, assembly and structure. Our study reveals how Toxoplasma gondii assembles over 40 mt-rRNA fragments using exclusively nuclear-encoded mitoribosomal proteins and three lineage-specific families of RNA-binding proteins. Among these are four proteins from the Apetala2/Ethylene Response Factor (AP2/ERF) family, originally known as transcription factors in plants and Apicomplexa, now repurposed as essential mitoribosome components. Cryo-EM analysis of the mitoribosome structure demonstrates how these AP2 proteins function as RNA binders to maintain mitoribosome integrity. The mitoribosome is also decorated with members of lineage-specific RNA-binding proteins belonging to RAP (RNA-binding domain abundant in Apicomplexa) proteins and HPR (heptatricopeptide repeat) families, highlighting the unique adaptations of these parasites. Solving the molecular puzzle of apicomplexan mitoribosome could inform the development of therapeutic strategies targeting organellar translation.

DOI: https://doi.org/10.1038/s41467-024-55033-z
Cancers (Basel). 2024 Dec 06. pii: 4095. [Epub ahead of print]16(23):

Sirtuins as Key Regulators in Pancreatic Cancer: Insights into Signaling Mechanisms and Therapeutic Implications.

Surbhi Chouhan, Anil Kumar, Naoshad Muhammad, Darksha Usmani, Tabish H Khan.

  Pancreatic ductal adenocarcinoma (PDAC) stands as one of the most lethal cancers, marked by rapid progression, pronounced chemoresistance, and a complex network of genetic and epigenetic dysregulation. Within this challenging context, sirtuins, NAD+-dependent deacetylases, have emerged as pivotal modulators of key cellular processes that drive pancreatic cancer progression. Each sirtuin contributes uniquely to PDAC pathogenesis. SIRT1 influences apoptosis and chemoresistance through hypoxia, enhancing glycolytic metabolism and HIF-1α signaling, which sustain tumor survival against drugs like gemcitabine. SIRT2, conversely, disrupts cancer cell proliferation by inhibiting eIF5A, while SIRT3 exerts tumor-suppressive effects by regulating mitochondrial ROS and glycolysis. SIRT4 inhibits aerobic glycolysis, and its therapeutic upregulation has shown promise in curbing PDAC progression. Furthermore, SIRT5 modulates glutamine and glutathione metabolism, offering an avenue to disrupt PDAC's metabolic dependencies. SIRT6 and SIRT7, through their roles in angiogenesis, EMT, and metastasis, represent additional targets, with modulators of SIRT6, such as JYQ-42, showing potential to reduce tumor invasiveness. This review aims to provide a comprehensive exploration of the emerging roles of sirtuins, a family of NAD+-dependent enzymes, as critical regulators within the oncogenic landscape of pancreatic cancer. This review meticulously explores the nuanced involvement of sirtuins in pancreatic cancer, elucidating their contributions to tumorigenesis and suppression through mechanisms such as metabolic reprogramming, the maintenance of genomic integrity and epigenetic modulation. Furthermore, it emphasizes the urgent need for the development of targeted therapeutic interventions aimed at precisely modulating sirtuin activity, thereby enhancing therapeutic efficacy and optimizing patient outcomes in the context of pancreatic malignancies.

Keywords:  SIRT; cancer signaling pathways; deacetylase; pancreatic cancer; pancreatic ductal adenocarcinoma (PDAC); sirtuins; therapeutic interventions

DOI:  https://doi.org/10.3390/cancers16234095
Acta Trop. 2024 Dec 16. pii: S0001-706X(24)00385-1. [Epub ahead of print] 107501

Protective humoral immunity induced by virus-like particles expressing Toxoplasma gondii CST1 or MIC8.

Jie Mao, Gi-Deok Eom, Keon-Woong Yoon, Su In Heo, Hae-Ji Kang, Ki Back Chu, Eun-Kyung Moon, Fu-Shi Quan.

  Protective efficacy assessment of toxoplasmosis vaccines, at least at the preclinical level, frequently involves lethal dose challenge infection. Nonetheless, their efficacies remain largely unexplored against low infection doses which better reflects how humans become infected in the real world. In this study, we compared the immunity elicited in mice that were heterologously immunized with recombinant baculovirus and virus-like particles expressing either the cyst wall protein (CST1) or microneme protein 8 (MIC8) of Toxoplasma gondii (T. gondii). We also investigated how these vaccines fared against both light and heavy infection intensities of T. gondii ME49. Interestingly, under light infection intensity, vaccines expressing CST1 induced significantly higher mucosal antibody responses than MIC8. Germinal center B (GC B) cell responses were elicited to a greater extent following immunization with either antigen, regardless of the infection dose. Similarly, both antigens suppressed IFN-γ production in the brains upon heavy infection. The overall vaccine-induced protection was also similar for the two vaccine antigens under heavy infection. However, in lightly infected mice, CST1 conferred improved GC B cell induction and further inhibited IFN-γ and cyst burden than those elicited by MIC8, thereby contributing to better protection. These findings indicated that light infection could be used to identify optimal vaccine candidates, thus highlighting the impact of infection intensity in vaccine efficacy evaluations.

Keywords:  Toxoplasma gondii; heavy infection; light infection; vaccine

DOI:  https://doi.org/10.1016/j.actatropica.2024.107501
Elife. 2024 Dec 18. pii: RP93125. [Epub ahead of print]13

Salmonella-induced SIRT1 and SIRT3 are crucial for maintaining the metabolic switch in bacteria and host for successful pathogenesis.

Dipasree Hajra, Raju S Rajmani, Ayushi Devendrasingh Chaudhary, Shashi Kumar Gupta, Dipshikha Chakravortty.

  Sirtuins are the major players in host immunometabolic regulation. However, the role of sirtuins in the modulation of the immune metabolism pertaining to salmonellosis is largely unknown. Here, our investigation focussed on the role of two important sirtuins, SIRT1 and SIRT3, shedding light on their impact on intracellular Salmonella's metabolic switch and pathogenesis establishment. Our study indicated the ability of the live Salmonella Typhimurium to differentially regulate the levels of SIRT1 and SIRT3 for maintaining the high glycolytic metabolism and low fatty acid metabolism in Salmonella. Perturbing SIRT1 or SIRT3 through knockdown or inhibition resulted in a remarkable shift in the host metabolism to low fatty acid oxidation and high glycolysis. This switch led to decreased proliferation of Salmonella in the macrophages. Further, Salmonella-induced higher levels of SIRT1 and SIRT3 led to a skewed polarization state of the macrophages from a pro-inflammatory M1 state toward an immunosuppressive M2, making it more conducive for the intracellular life of Salmonella. Alongside, governing immunological functions by modulating p65 NF-κB acetylation, SIRT1, and SIRT3 also skew Salmonella-induced host metabolic switch by regulating the acetylation status of HIF-1α and PDHA1. Interestingly, though knockdown of SIRT1/3 attenuated Salmonella proliferation in macrophages, in in vivo mice model of infection, inhibition or knockdown of SIRT1/3 led to more dissemination and higher organ burden, which can be attributed to enhanced ROS and IL-6 production. Our study hence reports for the first time that Salmonella modulates SIRT1/3 levels to maintain its own metabolism for successful pathogenesis.

Keywords:  SIRT1; SIRT3; Salmonella enterica serovar Typhimurium; Salmonella metabolism; bacterial dissemination; immune regulation; infectious disease; metabolic shift; microbiology

DOI:  https://doi.org/10.7554/eLife.93125
Proteomics. 2024 Dec 17. e202400120

Key Regulators of Parasite Biology Viewed Through a Post-Translational Modification Repertoire.

Naiwen Zhang, Ning Jiang, Qijun Chen.

  Parasites are the leading causes of morbidity and mortality in both humans and animals, imposing substantial socioeconomic burdens worldwide. Controlling parasitic diseases has become one of the key issues in achieving "One Health". Most parasites have sophisticated life cycles exhibiting progressive developmental stages, morphologies, and host-switching, which are controlled by various regulatory machineries including protein post-translational modifications (PTMs). PTMs have emerged as a key mechanism by which parasites modulate their virulence, developmental transitions, and environmental adaptations. PTMs are enzyme-mediated additions or removals of chemical groups that dynamically regulate the stability and functions of proteins and confer novel properties, playing vital roles in a variety of biological processes and cellular functions. In this review, we circumscribe how parasites utilize various PTMs to regulate their intricate lives, with a focus on the biological role of PTMs in parasite biology and pathogenesis.

Keywords:  epigenetic regulation; parasites; post‐translational modification; proteomics

DOI:  https://doi.org/10.1002/pmic.202400120
Res Sq. 2024 Dec 05. pii: rs.3.rs-5278203. [Epub ahead of print]

Mitochondrial metabolism and epigenetic crosstalk drive the SASP.

Joao Passos, Helene Martini, Jodie Birch, Francisco Marques, Stella Victorelli, Anthony Lagnado, Nicholas Pirius, Ana Franco, Gung Lee, Yeaeun Han, Jennifer Rowsey, Alexandre Gaspar-Maia, Aaron Havas, Rabi Murad, Xue Lei, Rebecca Porritt, Oliver Maddocks, Diana Jurk, Sundeep Khosla, Peter Adams.

Senescent cells drive tissue dysfunction through the senescence-associated secretory phenotype (SASP). We uncovered a central role for mitochondria in the epigenetic regulation of the SASP, where mitochondrial-derived metabolites, specifically citrate and acetyl-CoA, fuel histone acetylation at SASP gene loci, promoting their expression. We identified the mitochondrial citrate carrier (SLC25A1) and ATP-citrate lyase (ACLY) as critical for this process. Inhibiting these pathways selectively suppresses SASP without affecting cell cycle arrest, highlighting their potential as therapeutic targets for age-related inflammation. Notably, SLC25A1 inhibition reduces systemic inflammation and extends healthspan in aged mice, establishing mitochondrial metabolism as pivotal to the epigenetic control of aging.

DOI: https://doi.org/10.21203/rs.3.rs-5278203/v1
Vet Res. 2024 Dec 18. 55(1): 170

The F204S mutation in adrenodoxin oxidoreductase drives salinomycin resistance in Eimeria tenella.

Pei Sun, Chaoyue Wang, Fujie Xie, Linlin Chen, Yuanyuan Zhang, Xinming Tang, Dandan Hu, Yang Gao, Ning Zhang, Zhenkai Hao, Yonglan Yu, Jingxia Suo, Xun Suo, Xianyong Liu.

  Salinomycin is a polyether ionophore widely used for the treatment of coccidiosis in poultry. However, the emergence of coccidia strains resistant to salinomycin presents challenges for control efforts, and the mechanisms underlying this resistance in Eimeria remain inadequately understood. In this study, 78 stable salinomycin-resistant strains were generated through experimental evolution approaches. Whole-genome sequencing of salinomycin-resistant Eimeria tenella isolates revealed single nucleotide polymorphisms (SNPs), with 12 candidate genes harboring nonsynonymous mutations identified. To confirm the candidate gene responsible for conferring salinomycin resistance, we leveraged reverse genetic strategies and identified a key amino acid substitution (F204S) in adrenodoxin oxidoreductase (EtADR), which markedly reduced susceptibility to salinomycin. Our results elucidate the complex interactions among salinomycin resistance, parasite fitness, point mutations, and the structure of EtADR, laying the foundation for future studies on drug resistance in Eimeria and contributing to the development of targeted control strategies.

Keywords:   Eimeria tenella ; drug resistance; point mutation; salinomycin

DOI:  https://doi.org/10.1186/s13567-024-01431-6
Cells. 2024 Dec 07. pii: 2023. [Epub ahead of print]13(23):

SIRT2 Inhibition by AGK2 Promotes Perinuclear Cytoskeletal Organisation and Reduces Invasiveness of MDA-MB-231 Triple-Negative Breast Cancer Cells in Confined In Vitro Models.

Emily Jessop, Natalie Young, Beatriz Garcia-Del-Valle, Jack T Crusher, Boguslaw Obara, Iakowos Karakesisoglou.

  Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterised by the absence of targetable hormone receptors and increased metastatic rates. As nuclear softening strongly contributes to TNBC's enhanced metastatic capacity, increasing the nuclear stiffness of TNBC cells may present a promising therapeutic avenue. Previous evidence has demonstrated the ability of Sirtuin 2 (SIRT2) inhibition to induce cytoskeletal reorganisation, a key factor in regulating nuclear mechanics. Thus, our study aimed to investigate the effect of SIRT2 inhibition on the nuclear mechanics and migratory behaviour of TNBC cells. To achieve this, SIRT2 was pharmacologically inhibited in MDA-MB-231 cells using AGK2, a SIRT2-specific inhibitor. Although SIRT2 inhibition had no effect on LINC complex composition, the AGK2-treated MDA-MB-231 cells displayed more prominent perinuclear organisations of acetylated α-tubulin, vimentin, and F-actin. Additionally, the nuclei of the AGK2-treated MDA-MB-231 cells exhibited greater resistance to collapse under osmotic shock. Scratch-wound assays also revealed that SIRT2 inhibition led to polarity defects in the MDA-MB-231 cells, while in vitro space-restrictive invasion assays highlighted their reduced migratory capacity upon AGK2 treatment. Taken together, our findings suggest that SIRT2 inhibition promotes a perinuclear cytoskeletal organisation in MDA-MB-231 cells, which enhances their nuclear rigidity and impedes their invasion through confined spaces in vitro.

Keywords:  AGK2; LINC complex; SIRT2; lamin; microtubules; nesprins; nuclear mechanics; nucleus; vimentin

DOI:  https://doi.org/10.3390/cells13232023
Bioorg Chem. 2024 Dec 04. pii: S0045-2068(24)00924-6. [Epub ahead of print]154 108019

Aldehyde dehydrogenases as drug targets for cancer: SAR and structural biology aspects for inhibitor design.

Himanshu Tahiliani, Arunkumar Dhayalan, Mu-Chun Li, Hsing-Pang Hsieh, Mohane Selvaraj Coumar.

  Aldehydes are organic compounds containing a carbonyl group found exogenously or produced by normal metabolic processes and their accumulation can lead to toxicity if not cleared. Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent enzymes that catalyze the oxidation of such aldehydes and prevent their accumulation. Along with this primary detoxification function, the known 19 human isoforms of ALDHs, which act on different substrates, are also involved in various physiological and developmental processes. Functional alterations of ALDHs via mutations or expression levels cause various disease conditions, including many different cancer types like lung, ovarian, etc. These properties make this family of enzymes an ideal therapeutic and prognostic target for drug development. However, sequence similarities between the ALDH isoforms force the need to design inhibitors for a specific isoform using the differences in the substrate-binding sites of each protein. This has resulted in developing isoform-specific inhibitors, especially for ALDH1A1, ALDH2, and ALDH3A1, which are implicated in various cancers. In this review, we briefly outline the functional roles of the different isoforms of the ALDH family members, their role in cancer and discuss the various selective inhibitors that have been developed for the ALDH1A1 and ALDH3A1 enzymes, along with a detailed examination of the respective structure-activity relationship (SAR) studies available. From the available SAR and structural biology data, insights into the functional groups and interactions necessary to develop selective inhibitors for ALDH1A1 and ALDH3A1 are highlighted, which can act as a guide for developing more potent and selective inhibitors of ALDH isoforms.

Keywords:  ALDH inhibitors; Aldehyde dehydrogenase; Cancer; Selectivity; Structural biology; Structure–activity relationship

DOI:  https://doi.org/10.1016/j.bioorg.2024.108019
Nature. 2024 Dec 18.

Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing.

Qi Qu, Yan Chen, Yu Wang, Weiche Wang, Shating Long, Heng-Ye Yang, Jianfeng Wu, Mengqi Li, Xiao Tian, Xiaoyan Wei, Yan-Hui Liu, Shengrong Xu, Jinye Xiong, Chunyan Yang, Zhenhua Wu, Xi Huang, Changchuan Xie, Yaying Wu, Zheni Xu, Cixiong Zhang, Baoding Zhang, Jin-Wei Feng, Junjie Chen, Yuanji Feng, Huapan Fang, Liyun Lin, Z K Xie, Beibei Sun, Huayu Tian, Yong Yu, Hai-Long Piao, Xiao-Song Xie, Xianming Deng, Chen-Song Zhang, Sheng-Cai Lin.

Lithocholic acid (LCA) is accumulated in mammals during calorie restriction and it can activate AMP-activated protein kinase (AMPK) to slow down ageing1. However, the molecular details of how LCA activates AMPK and induces these biological effects are unclear. Here we show that LCA enhances the activity of sirtuins to deacetylate and subsequently inhibit vacuolar H+-ATPase (v-ATPase), which leads to AMPK activation through the lysosomal glucose-sensing pathway. Proteomics analyses of proteins that co-immunoprecipitated with sirtuin 1 (SIRT1) identified TUB-like protein 3 (TULP3), a sirtuin-interacting protein2, as a LCA receptor. In detail, LCA-bound TULP3 allosterically activates sirtuins, which then deacetylate the V1E1 subunit of v-ATPase on residues K52, K99 and K191. Muscle-specific expression of a V1E1 mutant (3KR), which mimics the deacetylated state, strongly activates AMPK and rejuvenates muscles in aged mice. In nematodes and flies, LCA depends on the TULP3 homologues tub-1 and ktub, respectively, to activate AMPK and extend lifespan and healthspan. Our study demonstrates that activation of the TULP3-sirtuin-v-ATPase-AMPK pathway by LCA reproduces the benefits of calorie restriction.

DOI: https://doi.org/10.1038/s41586-024-08348-2
Pharmacol Res. 2024 Dec 13. pii: S1043-6618(24)00491-2. [Epub ahead of print]211 107546

Epigenetic modifications and emerging therapeutic targets in cardiovascular aging and diseases.

Yurou Qiu, Qing Xu, Peichen Xie, Chenshuang He, Qiuchan Li, Xin Yao, Yang Mao, Xiaoqian Wu, Tiejun Zhang.

  The complex mechanisms underlying the development of cardiovascular diseases remain not fully elucidated. Epigenetics, which modulates gene expression without DNA sequence changes, is shedding light on these mechanisms and their heritable effects. This review focus on epigenetic regulation in cardiovascular aging and diseases, detailing specific epigenetic enzymes such as DNA methyltransferases (DNMTs), histone acetyltransferases (HATs), and histone deacetylases (HDACs), which serve as writers or erasers that modify the epigenetic landscape. We also discuss the readers of these modifications, such as the 5-methylcytosine binding domain proteins, and the erasers ten-eleven translocation (TET) proteins. The emerging role of RNA methylation, particularly N6-methyladenosine (m6A), in cardiovascular pathogenesis is also discussed. We summarize potential therapeutic targets, such as key enzymes and their inhibitors, including DNMT inhibitors like 5-azacytidine and decitabine, HDAC inhibitors like belinostat and givinotide, some of which have been approved by the FDA for various malignancies, suggesting their potential in treating cardiovascular diseases. Furthermore, we highlight the role of novel histone modifications and their associated enzymes, which are emerging as potential therapeutic targets in cardiovascular diseases. Thus, by incorporating the recent studies involving patients with cardiovascular aging and diseases, we aim to provide a more detailed and updated review that reflects the advancements in the field of epigenetic modification in cardiovascular diseases.

Keywords:  Cardiovascular diseases; Epigenetic modification; Epigenetics; Epigenetics-related regulatory mechanisms; Potential targets

DOI:  https://doi.org/10.1016/j.phrs.2024.107546
J Cell Sci. 2024 Dec 15. pii: jcs262071. [Epub ahead of print]137(24):

Fantastic proteins and where to find them - histones, in the nucleus and beyond.

Johanna Grinat, Noah P Shriever, Maria A Christophorou.

  Animal genomes are packaged into chromatin, a highly dynamic macromolecular structure of DNA and histone proteins organised into nucleosomes. This accommodates packaging of lengthy genomic sequences within the physical confines of the nucleus while also enabling precise regulation of access to genetic information. However, histones existed before chromatin and have lesser-known functions beyond genome regulation. Most notably, histones are potent antimicrobial agents, and the release of chromatin to the extracellular space is a defence mechanism nearly as ancient and widespread as chromatin itself. Histone sequences have changed very little throughout evolution, suggesting the possibility that some of their 'non-canonical' functions are at play in parallel or in concert with their genome regulatory functions. In this Review, we take an evolutionary perspective of histone, nuclear chromatin and extracellular chromatin biology and describe the known extranuclear and extracellular functions of histones. We detail molecular mechanisms of chromatin release and extracellular chromatin sensing, and we discuss their roles in physiology and disease. Finally, we present evidence and give a perspective on the potential of extracellular histones to act as bioactive, cell modulatory factors.

Keywords:  Chromatin; Evolution; Extracellular; Histones; Post-translational modifications; Signalling

DOI:  https://doi.org/10.1242/jcs.262071
Nucleic Acids Res. 2024 Dec 19. pii: gkae1223. [Epub ahead of print]

Rapid degradation of histone deacetylase 1 (HDAC1) reveals essential roles in both gene repression and active transcription.

David M English, Samuel N Lee, Khadija A Sabat, India M Baker, Trong Khoa Pham, Mark O Collins, Shaun M Cowley.

Histone Deacetylase 1 (HDAC1) removes acetyl groups from lysine residues on core histones, a critical step in regulating chromatin accessibility. Despite histone deacetylation being an apparently repressive activity, suppression of HDACs causes both up- and downregulation of gene expression. Here we exploited the degradation tag (dTAG) system to rapidly degrade HDAC1 in mouse embryonic stem cells (ESCs) lacking its paralog, HDAC2. The dTAG system allowed specific degradation and removal of HDAC1 in <1 h (100x faster than genetic knockouts). This rapid degradation caused increased histone acetylation in as little as 2 h, with H2BK5 and H2BK11 being the most sensitive. The majority of differentially expressed genes following 2 h of HDAC1 degradation were upregulated (275 genes up versus 15 down) with increased proportions of downregulated genes observed at 6 h (1153 up versus 443 down) and 24 h (1146 up versus 967 down), respectively. Upregulated genes showed increased H2BK5ac and H3K27ac around their transcriptional start site (TSS). In contrast, decreased acetylation and chromatin accessibility of super-enhancers was linked to the most strongly downregulated genes. These findings suggest a paradoxical role for HDAC1 in the maintenance of histone acetylation levels at critical enhancer regions required for the pluripotency-associated gene network.

DOI: https://doi.org/10.1093/nar/gkae1223
Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2408049121

ACSL4-mediated H3K9 and H3K27 hyperacetylation upregulates SNAIL to drive TNBC metastasis.

Abhipsa Sinha, Krishan Kumar Saini, Aakash Chandramouli, Kiran Tripathi, Muqtada Ali Khan, Saumya Ranjan Satrusal, Ayushi Verma, Biswajit Mandal, Priyanka Rai, Sanjeev Meena, Mushtaq Ahmad Nengroo, Manish Pratap Singh, Namratha Shashi Bhushan, Madavan Vasudevan, Atin Singhai, Kulranjan Singh, Anand Kumar Mishra, Siddhesh S Kamat, Dipak Datta.

  Triple-negative breast cancer (TNBC) has profound unmet medical need globally for its devastating clinical outcome associated with rapid metastasis and lack of targeted therapies. Recently, lipid metabolic reprogramming especially fatty acid oxidation (FAO) has emerged as a major driver of breast cancer metastasis. Analyzing the expression of major FAO regulatory genes in breast cancer, we found selective overexpression of acyl-CoA synthetase 4 (ACSL4) in TNBC, which is primarily attributed to the absence of progesterone receptor. Loss of ACSL4 function, by genetic ablation or pharmacological inhibition significantly reduces metastatic potential of TNBC. Global transcriptome analysis reveals that ACSL4 activity positively influences the gene expression related to TNBC migration and invasion. Mechanistically, ACSL4 modulates FAO and intracellular acetyl-CoA levels, leading to hyperacetylation of particularly H3K9ac and H3K27ac marks resulting in overexpression of SNAIL during the course of TNBC metastatic spread to lymph node and lung. Further, human TNBC metastasis exhibits positive correlation among ACSL4, H3K9ac, H3K27ac, and SNAIL expression. Altogether, our findings provide molecular insights regarding the intricate interplay between metabolic alterations and epigenetic modifications, intertwined to orchestrate TNBC metastasis, and posit a rational understanding for the development of ACSL4 inhibitors as a targeted therapy against TNBC.

Keywords:  ACSL4; SNAIL; TNBC; histone acetylation; metastasis

DOI:  https://doi.org/10.1073/pnas.2408049121
Int J Mol Sci. 2024 Dec 09. pii: 13207. [Epub ahead of print]25(23):

Diacylglycerol Kinases and Its Role in Lipid Metabolism and Related Diseases.

Yishi Liu, Zehui Yang, Xiaoman Zhou, Zijie Li, Nakanishi Hideki.

  Lipids are essential components of eukaryotic membranes, playing crucial roles in membrane structure, energy storage, and signaling. They are predominantly synthesized in the endoplasmic reticulum (ER) and subsequently transported to other organelles. Diacylglycerol kinases (DGKs) are a conserved enzyme family that phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), both of which are key intermediates in lipid metabolism and second messengers involved in numerous cellular processes. Dysregulation of DGK activity is associated with several diseases, including cancer and metabolic disorders. In this review, we provide a comprehensive overview of DGK types, functions, cellular localization, and their potential as therapeutic targets. We also discuss DGKs' roles in lipid metabolism and their physiological functions and related diseases.

Keywords:  diacylglycerol kinase; lipid; phosphatidic acid; phosphorylate diacylglycerol

DOI:  https://doi.org/10.3390/ijms252313207
mBio. 2024 Dec 16. e0350124

Stage-specific function of sphingolipid synthases in African trypanosomes.

Norton Heise, Carolina M Koeller, Mohamed Sharif, James D Bangs.

  The protozoan parasite Trypanosoma brucei is the only known eukaryote capable of synthesizing the three main phosphosphingolipids: sphingomyelin (SM), inositol phosphorylceramide (IPC), and ethanolamine phosphorylceramide (EPC). It has four paralogous genes encoding sphingolipid synthases (TbSLS1-4). TbSLS1 is a dedicated IPC synthase, TbSLS2 is a dedicated EPC synthase, and TbSLS3 and TbSLS4 are bifunctional SM/EPC synthases. IPC synthesis occurs exclusively in the procyclic insect stage (PCF), EPC is limited to the mammalian bloodstream form (BSF), and SM is synthesized throughout the life cycle. TbSLSs are indispensable for the viability of BSF and are, thus, potential drug targets. The relative stage-specific expression of each TbSLS paralog was compared, and the results match phosphosphingolipid content. Induction of pan-specific RNAi silencing was lethal in both BSF and PCF. To investigate individual TbSLS functions, separate HA-tagged genes, recoded to be RNAi-resistant (RNAiR), were engineered to replace a single allele of the entire TbSLS locus within parental BSF and PCF RNAi cell lines. RNAiR TbSLS3 and TbSLS4 both rescued BSF growth under silencing. Expression of RNAiR TbSLS1, normally repressed in BSF, did not rescue BSF viability but was not detrimental to normal in vitro growth. RNAiR TbSLS1, TbSLS3, and TbSLS4 were each sufficient to rescue PCF growth, indicating IPC is not essential for PCF viability in vitro. All TbSLSs localize to distal Golgi compartments in both BSF and PCF cells. These findings raise interesting questions about the roles of individual phosphosphingolipids in in vivo infection of the mammalian and tsetse hosts.
IMPORTANCE: African trypanosomes are eukaryotic pathogens that cause human and veterinary African trypanosomaisis. Uniquely, they synthesize all three major phosphosphingolipid species using four distinct sphingolipid synthases (SLS). This work details the function of each SLS in both bloodstream and insect form parasites. Novel and unexpected sphingolipid dependences are found in each stage. These results are consistent with this metabolic pathway being a valid target for chemotherapeutic intervention.

Keywords:  inositolphosphorylceramide; sphingolipid; sphingolipid synthase; sphingomyelin; trypanosome

DOI:  https://doi.org/10.1128/mbio.03501-24
J Biol Chem. 2024 Dec 13. pii: S0021-9258(24)02582-1. [Epub ahead of print] 108080

Inhibition of L-Threonine Dehydrogenase from Trypanosoma cruzi reduces glycine and acetate production and interferes with parasite growth and viability.

Jessica do Nascimento Faria, Amanda G Eufrásio, Michelle Fagundes, Angel Lobo-Rojas, Letícia Marchese, Caio Cesar de Lima Silva, Eduardo H S Bezerra, Gustavo F Mercaldi, Marcos R Alborghetti, Mauricio L Sforca, Artur T Cordeiro.

  Trypanosoma cruzi is a flagellated protozoan and the etiological agent of Chagas Disease, a neglected tropical disease described by Carlos Chagas in 1909 that remains without appropriate diagnostics and treatment. Throughout its life cycle, T. cruzi undergoes through many different environments, requiring adaptation of its metabolism to different nutrition sources. Recent studies have confirmed the adaptability of T. cruzi metabolism to different carbon sources and encouraged a deeper investigation of related metabolic pathways. In the present study, we investigated the catabolism of threonine in T. cruzi epimastigotes cultivated in LIT medium and following 24h of starvation in PBS. In LIT medium, threonine, serine and histidine were rapidly consumed concomitantly with carbohydrates during parasite exponential growth. When threonine was provided as the only carbon source to starved parasites, they excreted acetate and glycine, corroborating the activity of a mitochondrial threonine degradation pathway. Subsequently, we used a recombinant T. cruzi L-threonine dehydrogrenase (TcTDH) to screen the ChagasBox, an open-source collection of phenotypic hits and identified compound TCMDC-143160 as a low micromolar TcTDH inhibitor (IC50 = 3.5 μM). When TCDMC-143160 was administrated to starved parasites, it inhibited the threonine degradation pathway. Finally, we report the crystal structure of TcTDH and characterize its allosteric activation by potassium. Collectively, these data demonstrate the relevance of threonine catabolism in T. cruzi metabolism and provide a set of tools to further investigate TcTDH as a potential drug target for Chagas disease.

Keywords:  Chagasbox; drug discovery; metabolism; potassium binding; substrate-level phosphorylation; target identification

DOI:  https://doi.org/10.1016/j.jbc.2024.108080
bioRxiv. 2024 Dec 05. pii: 2024.12.04.625416. [Epub ahead of print]

A novel SUN1-ALLAN complex coordinates segregation of the bipartite MTOC across the nuclear envelope during rapid closed mitosis in Plasmodium.

Mohammad Zeeshan, Igor Blatov, Ryuji Yanase, David J P Ferguson, Sarah L Pashley, Zeinab Chahine, Yoshiki Yamaryo Botté, Akancha Mishra, Baptiste Marché, Suhani Bhanvadia, Molly Hair, Sagar Batra, Robert Markus, Declan Brady, Andrew Bottrill, Sue Vaughan, Cyrille Y Botté, Karine Le Roch, Anthony A Holder, Eelco C Tromer, Rita Tewari.

Mitosis in eukaryotes involves reorganization of the nuclear envelope (NE) and microtubule-organizing centres (MTOCs). In Plasmodium , the causative agent of malaria, male gametogenesis mitosis is exceptionally rapid and divergent. Within 8 minutes, the haploid male gametocyte genome undergoes three replication cycles (1N to 8N), while maintaining an intact NE. Axonemes assemble in the cytoplasm and connect to a bipartite MTOC-containing nuclear pole and cytoplasmic basal body, producing eight flagellated gametes. The mechanisms coordinating NE remodelling, MTOC dynamics, and flagellum assembly remain poorly understood. Here, we identify the SUN1-ALLAN complex as a novel mediator of NE remodelling and bipartite MTOC coordination during Plasmodium male gametogenesis. SUN1, a conserved NE protein, localizes to dynamic loops and focal points near nuclear spindle poles. ALLAN, a divergent Allantoicase-like protein, has a location like that of SUN1 at nuclear MTOCs. SUN1 and ALLAN form a unique complex, detected by live-cell imaging, ultrastructural expansion microscopy, and interactomics. Deletion of either SUN1 or ALLAN gene disrupts nuclear MTOC organization, leading to basal body mis-segregation, defective spindle assembly, and impaired kinetochore attachment, but axoneme formation remains intact. Ultrastructural analysis revealed nuclear and cytoplasmic MTOC miscoordination, producing aberrant flagellated gametes lacking nuclear material. Sun1 deletion also alters parasite lipid composition, underscoring its role in NE homeostasis. These defects block parasite development in the mosquito and transmission, highlighting the essential functions of this complex. This study reveals a bipartite MTOC and a highly divergent mechanism of NE remodelling during Plasmodium male gametogenesis. The SUN1-ALLAN complex is an unusual adaptation of the LINC complex, in absence of canonical KASH-domain proteins in Plasmodium , providing new insights into the evolution of closed mitosis and highlighting potential targets for blocking malaria transmission.

DOI: https://doi.org/10.1101/2024.12.04.625416
Subcell Biochem. 2024 ;107 63-90

NAD+ Boosting Strategies.

Jared Rice, Sofie Lautrup, Evandro F Fang.

  Nicotinamide adenine dinucleotide (oxidized form, NAD+) serves as a co-substrate and co-enzyme in cells to execute its key roles in cell signalling pathways and energetic metabolism, arbitrating cell survival and death. It was discovered in 1906 by Arthur Harden and William John Young in yeast extract which could accelerate alcohol fermentation. NAD acts as an electron acceptor and cofactor throughout the processes of glycolysis, Tricarboxylic Acid Cycle (TCA), β oxidation, and oxidative phosphorylation (OXPHOS). NAD has two forms: NAD+ and NADH. NAD+ is the oxidising coenzyme that is reduced when it picks up electrons. NAD+ levels steadily decline with age, resulting in an increase in vulnerability to chronic illness and perturbed cellular metabolism. Boosting NAD+ levels in various model organisms have resulted in improvements in healthspan and lifespan extension. These results have prompted a search for means by which NAD+ levels in the body can be augmented by both internal and external means. The aim of this chapter is to provide an overview of NAD+, appraise clinical evidence of its importance and success in potentially extending health- and lifespan, as well as to explore NAD+ boosting strategies.

Keywords:  Ageing; Caloric restriction; NAD+; Neurodegeneration; Nicotinamide mononucleotide; Nicotinamide riboside; Oxidative stress; Supplementation

DOI:  https://doi.org/10.1007/978-3-031-66768-8_4
bioRxiv. 2024 Dec 02. pii: 2024.12.01.626286. [Epub ahead of print]

Optogenetically Induced Microtubule Acetylation Unveils the Molecular Dynamics of Actin-Microtubule Crosstalk in Directed Cell Migration.

Abhijit Deb Roy, Cristian Saez Gonzalez, Farid Shahid, Eesha Yadav, Takanari Inoue.

Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise and rapid induction of microtubule acetylation within minutes in live cells. Using optoTAT, we observed striking and rapid responses at both molecular and cellular level. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional cell migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation in the regulation of directed cell migration, revealing a dynamic crosstalk between the actin and microtubule cytoskeletal networks.

DOI: https://doi.org/10.1101/2024.12.01.626286
Cell Mol Life Sci. 2024 Dec 16. 82(1): 5

Lysine and arginine methylation of transcription factors.

Benedetto Daniele Giaimo, Francesca Ferrante, Tilman Borggrefe.

  Post-translational modifications (PTMs) are implicated in many biological processes including receptor activation, signal transduction, transcriptional regulation and protein turnover. Lysine's side chain is particularly notable, as it can undergo methylation, acetylation, SUMOylation and ubiquitination. Methylation affects not only lysine but also arginine residues, both of which are implicated in epigenetic regulation. Beyond histone-tails as substrates, dynamic methylation of transcription factors has been described. The focus of this review is on these non-histone substrates providing a detailed discussion of what is currently known about methylation of hypoxia-inducible factor (HIF), P53, nuclear receptors (NRs) and RELA. The role of methylation in regulating protein stability and function by acting as docking sites for methyl-reader proteins and via their crosstalk with other PTMs is explored.

Keywords:  Cell cycle; Hypoxia; KDM; KMT; PRMT; PTM

DOI:  https://doi.org/10.1007/s00018-024-05531-6
Acta Crystallogr F Struct Biol Commun. 2025 Jan 01.

Co-crystal structure of Helicobacter pylori biotin protein ligase with biotinyl-5-ATP.

Jesuferanmi P Ayanlade, Dylan E Davis, Sandhya Subramanian, David M Dranow, Donald D Lorimer, Brad Hammerson, Peter J Myler, Oluwatoyin A Asojo.

  Helicobacter pylori, a type 1 carcinogen that causes human gastric ulcers and cancer, is a priority target of the Seattle Structural Genomics Center for Infectious Disease (SSGCID). These efforts include determining the structures of potential H. pylori therapeutic targets. Here, the purification, crystallization and X-ray structure of one such target, H. pylori biotin protein ligase (HpBPL), are reported. HpBPL catalyzes the activation of various biotin-dependent metabolic pathways, including fatty-acid synthesis, gluconeogenesis and amino-acid catabolism, and may facilitate the survival of H. pylori in the high-pH gastric mucosa. HpBPL is a prototypical bacterial biotin protein ligase, despite having less than 35% sequence identity to any reported structure in the Protein Data Bank. A biotinyl-5-ATP molecule sits in a well conserved cavity. HpBPL shares extensive tertiary-structural similarity with Mycobacterium tuberculosis biotin protein ligase (MtBPL), despite having less than 22% sequence identity. The active site of HpBPL is very similar to that of MtBPL and has the necessary residues to bind inhibitors developed for MtBPL.

Keywords:  SSGCID; cancer; gastric ulcers; infectious diseases; undergraduate education and training

DOI:  https://doi.org/10.1107/S2053230X24012056
PLoS Biol. 2024 Dec;22(12): e3002941

SLMO transfers phosphatidylserine between the outer and inner mitochondrial membrane in Drosophila.

Siwen Zhao, Xuguang Jiang, Ning Li, Tao Wang.

Phospholipids are critical building blocks of mitochondria, and proper mitochondrial function and architecture rely on phospholipids that are primarily transported from the endoplasmic reticulum (ER). Here, we show that mitochondrial form and function rely on synthesis of phosphatidylserine (PS) in the ER through phosphatidylserine synthase (PSS), trafficking of PS from ER to mitochondria (and within mitochondria), and the conversion of PS to phosphatidylethanolamine (PE) by phosphatidylserine decarboxylase (PISD) in the inner mitochondrial membrane (IMM). Using a forward genetic screen in Drosophila, we found that Slowmo (SLMO) specifically transfers PS from the outer mitochondrial membrane (OMM) to the IMM within the inner boundary membrane (IBM) domain. Thus, SLMO is required for shaping mitochondrial morphology, but its putative conserved binding partner, dTRIAP, is not. Importantly, SLMO's role in maintaining mitochondrial morphology is conserved in humans via the SLMO2 protein and is independent of mitochondrial dynamics. Our results highlight the importance of a conserved PSS-SLMO-PISD pathway in maintaining the structure and function of mitochondria.

DOI: https://doi.org/10.1371/journal.pbio.3002941
Methods Mol Biol. 2025 ;2888 193-200

Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.

Shenliang Yu.

  This chapter describes two imaging-based approaches for examining the localization of bridge-like lipid transfer proteins at membrane contact sites during native biological processes. These approaches use multi-color fluorescence imaging, enabling high spatial and temporal resolution and overcoming the limitations of biochemical methods. The first approach involves immunofluorescence in fixed cells, while the second utilizes time-lapse imaging in live cells. These methods are showcased through the example of ATG2, an essential autophagy-related protein, and demonstrate the ability to overcome technical difficulties such as large protein size, lack of high-quality antibodies, and imaging highly dynamic subcellular structures. These described methods provide a powerful tool for understanding protein function and biological processes and can be widely applied to various research questions in cell biology.

Keywords:  ATG2; Autophagy; Fluorescence microscopy; Lipid transport; Membrane contact site

DOI:  https://doi.org/10.1007/978-1-0716-4318-1_13