bims-nenemi 2024-03-03 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2024‒03‒03
fifteen papers selected by
Marco Tigano, Thomas Jefferson University

mitoTALEN reduces the mutant mtDNA load in neurons.
Mitochondrial DNA leakage triggers inflammation in age-related cardiovascular diseases.
Translational response to mitochondrial stresses is orchestrated by tRNA modifications.
FBXL4: safeguarding against mitochondrial depletion through suppression of mitophagy.
ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.
Genetic and bioinformatic analyses reveal transcriptional networks underlying dual genomic coordination of mitochondrial biogenesis.
PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics.
Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.
Prophylactic nicotinamide treatment protects from rotenone-induced neurodegeneration by increasing mitochondrial content and volume.
Mitochondrial DNA integrity and metabolome profile are preserved in the human induced pluripotent stem cell reference line KOLF2.1J.
Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.
OrthoID: profiling dynamic proteomes through time and space using mutually orthogonal chemical tools.
The proteomic landscape of genotoxic stress-induced micronuclei.
Symmetry-breaking malachite green as a near-infrared light-activated fluorogenic photosensitizer for RNA proximity labeling.
A comparative study of apoptosis, pyroptosis, necroptosis, and PANoptosis components in mouse and human cells.

Mol Ther Nucleic Acids. 2024 Mar 12. 35(1): 102132

mitoTALEN reduces the mutant mtDNA load in neurons.

Sandra R Bacman, Jose Domingo Barrera-Paez, Milena Pinto, Derek Van Booven, James B Stewart, Anthony J Griswold, Carlos T Moraes.

  Mutations within mtDNA frequently give rise to severe encephalopathies. Given that a majority of these mtDNA defects exist in a heteroplasmic state, we harnessed the precision of mitochondrial-targeted TALEN (mitoTALEN) to selectively eliminate mutant mtDNA within the CNS of a murine model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T). This targeted approach was accomplished by the use of AAV-PHP.eB and a neuron-specific synapsin promoter for effective neuronal delivery and expression of mitoTALEN. We found that most CNS regions were effectively transduced and showed a significant reduction in mutant mtDNA. This reduction was accompanied by an increase in mitochondrial tRNA alanine levels, which are drastically reduced by the m.5024C>T mutation. These results showed that mitochondrial-targeted gene editing can be effective in reducing CNS-mutant mtDNA in vivo, paving the way for clinical trials in patients with mitochondrial encephalopathies.

Keywords:  AAV-PHPeB; CNS; MT: RNA/DNA editing; TALEN; gene therapy; heteroplasmy; mitochondria

DOI:  https://doi.org/10.1016/j.omtn.2024.102132
Front Cell Dev Biol. 2024 ;12 1287447

Mitochondrial DNA leakage triggers inflammation in age-related cardiovascular diseases.

Wanyue Ding, Jingyu Chen, Lei Zhao, Shuang Wu, Xiaomei Chen, Hong Chen.

  Mitochondrial dysfunction is one of the hallmarks of cardiovascular aging. The leakage of mitochondrial DNA (mtDNA) is increased in senescent cells, which are resistant to programmed cell death such as apoptosis. Due to its similarity to prokaryotic DNA, mtDNA could be recognized by cellular DNA sensors and trigger innate immune responses, resulting in chronic inflammatory conditions during aging. The mechanisms include cGAS-STING signaling, TLR-9 and inflammasomes activation. Mitochondrial quality controls such as mitophagy could prevent mitochondria from triggering harmful inflammatory responses, but when this homeostasis is out of balance, mtDNA-induced inflammation could become pathogenic and contribute to age-related cardiovascular diseases. Here, we summarize recent studies on mechanisms by which mtDNA promotes inflammation and aging-related cardiovascular diseases, and discuss the potential value of mtDNA in early screening and as therapeutic targets.

Keywords:  cardiovasuclar diseases; inflammation; innate immunity; mitochondrial DNA; senescence

DOI:  https://doi.org/10.3389/fcell.2024.1287447
bioRxiv. 2024 Feb 14. pii: 2024.02.14.580389. [Epub ahead of print]

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Sherif Rashad, Shadi Al-Mesitef, Abdulrahman Mousa, Yuan Zhou, Daisuke Ando, Guangxin Sun, Tomoko Fukuuchi, Yuko Iwasaki, Jingdong Xiang, Shane R Byrne, Jingjing Sun, Masamitsu Maekawa, Daisuke Saigusa, Thomas J Begley, Peter C Dedon, Kuniyasu Niizuma.

  Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

Keywords:  Codon usage; Mitochondrial stress; Oxidative stress; Queuine; Queuosine; RNA modifications; mRNA translation; tRNA; tRNA modifications

DOI:  https://doi.org/10.1101/2024.02.14.580389
Autophagy. 2024 Feb 29. 1-3

FBXL4: safeguarding against mitochondrial depletion through suppression of mitophagy.

Prajakta Kulkarni, Giang Thanh Nguyen-Dien, Keri-Lyn Kozul, Julia K Pagan.

  Mitophagy is a critical mitochondrial quality control process that selectively removes dysfunctional or excess mitochondria through the autophagy-lysosome system. The process is tightly controlled to ensure cellular and physiological homeostasis. Insufficient mitophagy can result in failure to remove damaged mitochondria and consequent cellular degeneration, but it is equally important to appropriately restrain mitophagy to prevent excessive mitochondrial depletion. Here, we discuss our recent discovery that the SKP1-CUL1-F-box (SCF)-FBXL4 (F-box and leucine-rich repeat protein 4) E3 ubiquitin ligase localizes to the mitochondrial outer membrane, where it constitutively mediates the ubiquitination and degradation of BNIP3L/NIX and BNIP3 mitophagy receptors to suppress mitophagy. The post-translational regulation of BNIP3L and BNIP3 is disrupted in mitochondrial DNA depletion syndrome 13 (MTDPS13), a multi-systemic disorder caused by mutations in the FBXL4 gene and characterized by elevated mitophagy and mitochondrial DNA/mtDNA depletion in patient fibroblasts. Our results demonstrate that mitophagy is not solely stimulated in response to specific conditions but is instead also actively suppressed through the continuous degradation of BNIP3L and BNIP3 mediated by the SCF-FBXL4 ubiquitin ligase. Thus, cellular conditions or signaling events that prevent the FBXL4-mediated turnover of BNIP3L and BNIP3 on specific mitochondria are expected to facilitate their selective removal.

Keywords:  BNIP3; BNIP3L/NIX; FBXL4; MTDPS13; mitophagy; ubiquitin ligase

DOI:  https://doi.org/10.1080/15548627.2024.2318077
J Cell Physiol. 2024 Feb 28.

ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.

Antentor Hinton, Prasanna Katti, Margaret Mungai, Duane D Hall, Olha Koval, Jianqiang Shao, Zer Vue, Edgar Garza Lopez, Rahmati Rostami, Kit Neikirk, Jessica Ponce, Jennifer Streeter, Brandon Schickling, Serif Bacevac, Chad Grueter, Andrea Marshall, Heather K Beasley, Young Do Koo, Sue C Bodine, Nayeli G Reyes Nava, Anita M Quintana, Long-Sheng Song, Isabella M Grumbach, Renata O Pereira, Brian Glancy, E Dale Abel.

  Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+ , lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

Keywords:  activating transcription factor 4; endoplasmic reticulum; integrated stress response; interorganelle communication; mitochondria

DOI:  https://doi.org/10.1002/jcp.31204
bioRxiv. 2024 Jan 29. pii: 2024.01.25.577217. [Epub ahead of print]

Genetic and bioinformatic analyses reveal transcriptional networks underlying dual genomic coordination of mitochondrial biogenesis.

Fan Zhang, Annie Lee, Anna Freitas, Jake Herb, Zongheng Wang, Snigdha Gupta, Zhe Chen, Hong Xu.

Mitochondrial genome encodes handful genes of respiratory chain complexes, whereas all the remaining mitochondrial proteins are encoded on the nuclear genome. However, the mechanisms coordinating these two genomes to control mitochondrial biogenesis remain largely unknown. To identify transcription circuits involved in these processes, we performed a candidate RNAi screen in developing eyes that had reduced mitochondrial DNA contents. We reasoned that impaired mitochondrial biogenesis would synergistically interact with mtDNA deficiency in disrupting tissue development. Over 638 transcription factors annotated in the fly genome, we identified 77 transcription factors that may be involved in mitochondrial genome maintenance and gene expression. Additional genetic and genomic analyses revealed that a novel transcription factor, CG1603, and its upstream factor YL-1 are essential for mitochondrial biogenesis. We constructed a regulator network among positive hits using the published CHIP-seq data. The network analysis revealed extensive connections, and complex hierarchical organization underlying the transcription regulation of mitochondrial biogenesis.

DOI: https://doi.org/10.1101/2024.01.25.577217
Nat Metab. 2024 Feb 27.

PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics.

Tsz-Leung To, Jason G McCoy, Naomi K Ostriker, Lev S Sandler, Carmen A Mannella, Vamsi K Mootha.

Our current understanding of mitochondrial organelle physiology has benefited from two broad approaches: classically, cuvette-based measurements with suspensions of isolated mitochondria, in which bioenergetic parameters are monitored acutely in response to respiratory chain substrates and inhibitors1-4, and more recently, highly scalable genetic screens for fitness phenotypes associated with coarse-grained properties of the mitochondrial state5-10. Here we introduce permeabilized-cell mitochondrial function sequencing (PMF-seq) to combine strengths of these two approaches to connect genes to detailed bioenergetic phenotypes. In PMF-seq, the plasma membranes within a pool of CRISPR mutagenized cells are gently permeabilized under conditions that preserve mitochondrial physiology, where detailed bioenergetics can be probed in the same way as with isolated organelles. Cells with desired bioenergetic parameters are selected optically using flow cytometry and subjected to next-generation sequencing. Using PMF-seq, we recover genes differentially required for mitochondrial respiratory chain branching and reversibility. We demonstrate that human D-lactate dehydrogenase specifically conveys electrons from D-lactate into cytochrome c to support mitochondrial membrane polarization. Finally, we screen for genetic modifiers of tBID, a pro-apoptotic protein that acts directly and acutely on mitochondria. We find the loss of the complex V assembly factor ATPAF2 acts as a genetic sensitizer of tBID's acute action. We anticipate that PMF-seq will be valuable for defining genes critical to the physiology of mitochondria and other organelles.

DOI: https://doi.org/10.1038/s42255-024-00994-0
Proc Natl Acad Sci U S A. 2024 Mar 05. 121(10): e2313540121

Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.

Mohamed A Eldeeb, Andrew N Bayne, Armaan Fallahi, Thomas Goiran, Emma J MacDougall, Andrea Soumbasis, Cornelia E Zorca, Jace-Jones Tabah, Rhalena A Thomas, Nathan Karpilovsky, Meghna Mathur, Thomas M Durcan, Jean-François Trempe, Edward A Fon.

  Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early-onset Parkinson's disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin (Ub) ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane forming a high-molecular-weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates Ub, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 N-terminal-C-terminal extension module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.

Keywords:  PINK1; mitochondrial import; mitochondrial quality control; mitophagy; proteolysis

DOI:  https://doi.org/10.1073/pnas.2313540121
Acta Neuropathol Commun. 2024 Mar 01. 12(1): 37

Prophylactic nicotinamide treatment protects from rotenone-induced neurodegeneration by increasing mitochondrial content and volume.

Amin Otmani, Gauti Jóhannesson, Rune Brautaset, James R Tribble, Pete A Williams.

  Leber's hereditary optic neuropathy (LHON) is driven by mtDNA mutations affecting Complex I presenting as progressive retinal ganglion cell dysfunction usually in the absence of extra-ophthalmic symptoms. There are no long-term neuroprotective agents for LHON. Oral nicotinamide provides a robust neuroprotective effect against mitochondrial and metabolic dysfunction in other retinal injuries. We explored the potential for nicotinamide to protect mitochondria in LHON by modelling the disease in mice through intravitreal injection of the Complex I inhibitor rotenone. Using MitoV mice expressing a mitochondrial-tagged YFP in retinal ganglion cells we assessed mitochondrial morphology through super-resolution imaging and digital reconstruction. Rotenone induced Complex I inhibition resulted in retinal ganglion cell wide mitochondrial loss and fragmentation. This was prevented by oral nicotinamide treatment. Mitochondrial ultrastructure was quantified by transition electron microscopy, demonstrating a loss of cristae density following rotenone injection, which was also prevented by nicotinamide treatment. These results demonstrate that nicotinamide protects mitochondria during Complex I dysfunction. Nicotinamide has the potential to be a useful treatment strategy for LHON to limit retinal ganglion cell degeneration.

Keywords:  Mitochondria; Neurodegeneration; Nicotinamide; Optic nerve; Retina; Retina ganglion cells

DOI:  https://doi.org/10.1186/s40478-024-01724-z
Stem Cell Reports. 2024 Feb 12. pii: S2213-6711(24)00012-2. [Epub ahead of print]

Mitochondrial DNA integrity and metabolome profile are preserved in the human induced pluripotent stem cell reference line KOLF2.1J.

Jochen Dobner, Thach Nguyen, Andreas Dunkel, Alessandro Prigione, Jean Krutmann, Andrea Rossi.

  Quality control of human induced pluripotent stem cells (iPSCs) is critical to ensure reproducibility of research. Recently, KOLF2.1J was characterized and published as a male iPSC reference line to study neurological disorders. Emerging evidence suggests potential negative effects of mtDNA mutations, but its integrity was not analyzed in the original publication. To assess mtDNA integrity, we conducted a targeted mtDNA analysis followed by untargeted metabolomics analysis. We found that KOLF2.1J mtDNA integrity was intact at the time of publication and is still preserved in the commercially distributed cell line. In addition, the basal KOLF2.1J metabolome profile was similar to that of the two commercially available iPSC lines IMR90 and iPSC12, but clearly distinct from an in-house-generated ERCC6R683X/R683X iPSC line modeling Cockayne syndrome. Conclusively, we validate KOLF2.1J as a reference iPSC line, and encourage scientists to conduct mtDNA analysis and unbiased metabolomics whenever feasible.

Keywords:  Cockayne syndrome; ERCC6; KOLF2.1J; Oxford Nanopore sequencing; iPSC reference line; iPSCs quality control; mtDNA analysis; mtDNA integrity

DOI:  https://doi.org/10.1016/j.stemcr.2024.01.009
bioRxiv. 2024 Feb 13. pii: 2024.02.12.579972. [Epub ahead of print]

Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

Yusuke Hirabayashi, Tommy L Lewis, Yudan Du, Daniel M Virga, Aubrianna M Decker, Giovanna Coceano, Jonatan Alvelid, Maëla A Paul, Stevie Hamilton, Parker Kneis, Yasufumi Takahashi, Jellert T Gaublomme, Ilaria Testa, Franck Polleux.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, in vitro and in vivo approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results demonstrate that in mammalian CPNs, axonal mitochondria do not play a major role in ATP supply, despite playing other functions critical to regulating neurotransmission such as Ca2+ buffering.

DOI: https://doi.org/10.1101/2024.02.12.579972
Nat Commun. 2024 Feb 29. 15(1): 1851

OrthoID: profiling dynamic proteomes through time and space using mutually orthogonal chemical tools.

Ara Lee, Gihyun Sung, Sanghee Shin, Song-Yi Lee, Jaehwan Sim, Truong Thi My Nhung, Tran Diem Nghi, Sang Ki Park, Ponnusamy Pon Sathieshkumar, Imkyeung Kang, Ji Young Mun, Jong-Seo Kim, Hyun-Woo Rhee, Kyeng Min Park, Kimoon Kim.

Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report "OrthoID", a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins. This approach effectively identifies protein candidates associated with the ER-mitochondria contact, including LRC59, whose roles at the contact site were-to the best of our knowledge-previously unknown, and tracks multiple protein sets undergoing structural and locational changes at MAM during mitophagy. These findings demonstrate that OrthoID could be a powerful proteomics tool for the identification and analysis of spatiotemporal proteins at organelle contact sites and revealing their dynamic behaviors in vital cellular processes.

DOI: https://doi.org/10.1038/s41467-024-46034-z
Mol Cell. 2024 Feb 22. pii: S1097-2765(24)00097-2. [Epub ahead of print]

The proteomic landscape of genotoxic stress-induced micronuclei.

Kate M MacDonald, Shahbaz Khan, Brian Lin, Rose Hurren, Aaron D Schimmer, Thomas Kislinger, Shane M Harding.

  Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signaling cascades. Here, we measured the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, depleted of spliceosome and DNA damage repair components while enriched for a subset of the replisome. We show that the depletion of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. The presence of transcription machinery in MN is stress-dependent, causing a contextual induction of MN DNA damage through spliceosome deficiency. This dataset represents a unique resource detailing the global proteome of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.

Keywords:  DNA damage; chromothripsis; genotoxin; mass spectrometry; micronuclei; mitotic errors; proteomics; radiation; spliceosome

DOI:  https://doi.org/10.1016/j.molcel.2024.02.001
Nucleic Acids Res. 2024 Feb 26. pii: gkae125. [Epub ahead of print]

Symmetry-breaking malachite green as a near-infrared light-activated fluorogenic photosensitizer for RNA proximity labeling.

Lan Li, Jinghua Han, Hei-Yong G Lo, Winnie Wai Ling Tam, Han Jia, Edmund Chun Ming Tse, J Matthew Taliaferro, Ying Li.

Cellular RNA is asymmetrically distributed in cells and the regulation of RNA localization is crucial for proper cellular functions. However, limited chemical tools are available to capture dynamic RNA localization in complex biological systems with high spatiotemporal resolution. Here, we developed a new method for RNA proximity labeling activated by near-infrared (NIR) light, which holds the potential for deep penetration. Our method, termed FAP-seq, utilizes a genetically encoded fluorogen activating protein (FAP) that selectively binds to a set of substrates known as malachite green (MG). FAP binding restricts the rotation of MG and rapidly activates its fluorescence in a wash-free manner. By introducing a monoiodo modification to MG, we created a photosensitizer (MG-HI) with the highest singlet oxygen generation ability among various MG derivatives, enabling both protein and RNA proximity labeling in live cells. New insights are provided in the transcriptome analysis with FAP-seq, while a deeper understanding of the symmetry-breaking structural arrangement of FAP-MG-HI was obtained through molecular dynamics simulations. Overall, our wash-free and NIR light-inducible RNA proximity labeling method (FAP-seq) offers a powerful and versatile approach for investigating complex mechanisms underlying RNA-related biological processes.

DOI: https://doi.org/10.1093/nar/gkae125
PLoS One. 2024 ;19(2): e0299577

A comparative study of apoptosis, pyroptosis, necroptosis, and PANoptosis components in mouse and human cells.

Sk Mohiuddin Choudhury, Roman Sarkar, Rajendra Karki, Thirumala-Devi Kanneganti.

Regulated cell death is a key component of the innate immune response, which provides the first line of defense against infection and homeostatic perturbations. However, cell death can also drive pathogenesis. The most well-defined cell death pathways can be categorized as nonlytic (apoptosis) and lytic (pyroptosis, necroptosis, and PANoptosis). While specific triggers are known to induce each of these cell death pathways, it is unclear whether all cell types express the cell death proteins required to activate these pathways. Here, we assessed the protein expression and compared the responses of immune and non-immune cells of human and mouse origin to canonical pyroptotic (LPS plus ATP), apoptotic (staurosporine), necroptotic (TNF-α plus z-VAD), and PANoptotic (influenza A virus infection) stimuli. When compared to fibroblasts, both mouse and human innate immune cells, macrophages, expressed higher levels of cell death proteins and activated cell death effectors more robustly, including caspase-1, gasdermins, caspase-8, and RIPKs, in response to specific stimuli. Our findings highlight the importance of considering the cell type when examining the mechanisms regulating inflammation and cell death. Improved understanding of the cell types that contain the machinery to execute different forms of cell death and their link to innate immune responses is critical to identify new strategies to target these pathways in specific cellular populations for the treatment of infectious diseases, inflammatory disorders, and cancer.

DOI: https://doi.org/10.1371/journal.pone.0299577