bims-nenemi 2023-12-31 papers

Issue of 2023–12–31
seven papers selected by
Marco Tigano, Thomas Jefferson University

Front Cell Dev Biol. 2023 ;11 1322816

An optimized protocol for expression and purification of monomeric full-length BAX protein for functional interrogations.

Yiyang Chen, Jesse D Gelles, Jarvier N Mohammed, Jerry Edward Chipuk.

Diverse developmental signals and pro-death stresses converge on the regulation of the mitochondrial pathway of apoptosis. BAX, a proapoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial membrane to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX while identifying small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive protocol for expressing, purifying, and storing functional monomeric recombinant BAX protein. We use an intein-chitin binding domain-tagged BAX-expressing construct and employ a two-step chromatography strategy to capture and purify BAX. We also provide examples of standard assays to observe BAX activation, and highlight the best practices for handling and storing BAX to effectively preserve its quality, shelf life, and function.

Keywords: BAX; BCL-2 family; apoptosis; mitochondria; mitochondrial outer membrane permeabilization; recombinant protein production

DOI: https://doi.org/10.3389/fcell.2023.1322816

Front Cell Dev Biol. 2023 ;11 1271141

Signaling plasticity in the integrated stress response.

Morgane Boone, Francesca Zappa.

The Integrated Stress Response (ISR) is an essential homeostatic signaling network that controls the cell's biosynthetic capacity. Four ISR sensor kinases detect multiple stressors and relay this information to downstream effectors by phosphorylating a common node: the alpha subunit of the eukaryotic initiation factor eIF2. As a result, general protein synthesis is repressed while select transcripts are preferentially translated, thus remodeling the proteome and transcriptome. Mounting evidence supports a view of the ISR as a dynamic signaling network with multiple modulators and feedback regulatory features that vary across cell and tissue types. Here, we discuss updated views on ISR sensor kinase mechanisms, how the subcellular localization of ISR components impacts signaling, and highlight ISR signaling differences across cells and tissues. Finally, we consider crosstalk between the ISR and other signaling pathways as a determinant of cell health.

Keywords: homeostasis; integrated stress response; sensor kinase; signal crosstalk; signal heterogeneity; signaling network; subcellular compartmentalization

DOI: https://doi.org/10.3389/fcell.2023.1271141

Front Immunol. 2023 ;14 1235936

Cellular RNA and DNA sensing pathways are essential for the dose-dependent response of human monocytes to ionizing radiation.

Natallia Mikhalkevich, Eric Russ, Sergey Iordanskiy.

Circulating monocytes are important players of the inflammatory response to ionizing radiation (IR). These IR-resistant immune cells migrate to radiation-damaged tissues and differentiate into macrophages that phagocytize dying cells, but also facilitate inflammation. Besides the effect of damage-associated molecular patterns, released from irradiated tissues, the inflammatory activation of monocytes and macrophages is largely dependent on IR-induced DNA damage and aberrant transcriptional activity, which may facilitate expression of type I interferons (IFN-I) and numerous inflammation-related genes. We analyzed the accumulation of dsRNA, dsDNA fragments, and RNA:DNA hybrids in the context of induction of RNA-triggered MAVS-mediated and DNA-triggered STING-mediated signaling pathways, in primary human monocytes and a monocytic cell line, THP1, in response to various doses of gamma IR. We found that exposure to lower doses (<7.5 Gy) led to the accumulation of dsRNA, along with dsDNA and RNA:DNA hybrids and activated both MAVS and STING pathway-induced gene expression and signaling activity of IFN-I. Higher doses of IR resulted in the reduced dsRNA level, degradation of RNA-sensing mediators involved in MAVS signaling and coincided with an increased accumulation of dsDNA and RNA:DNA hybrids that correlated with elevated STING signaling and NF-κB-dependent gene expression. While both pathways activate IFN-I expression, using MAVS- and STING-knockout THP1 cells, we identified differences in the spectra of interferon-stimulated genes (ISGs) that are associated with each specific signaling pathway and outlined a large group of STING signaling-associated genes. Using the RNAi technique, we found that increasing the dose of IR activates STING signaling through the DNA sensor cGAS, along with suppression of the DDX41 helicase, which is known to reduce the accumulation of RNA:DNA hybrids and thereby limit cGAS/STING signaling activity. Together, these results indicate that depending on the applied dose, IR leads to the activation of either dsRNA-induced MAVS signaling, which predominantly leads to the expression of both pro- and anti-inflammatory markers, or dsDNA-induced STING signaling that contributes to pro-inflammatory activation of the cells. While RNA:DNA hybrids boost both MAVS- and STING-mediated signaling pathways, these structures being accumulated upon high IR doses promote type I interferon expression and appear to be potent enhancers of radiation dose-dependent pro-inflammatory activation of monocytes.

Keywords: MAVS; RIG-I-like receptors (RLRs); RNA:DNA hybrids; gamma radiation; macrophages; melanoma differentiation-associated protein 5 (MDA-5); monocytes; stimulator of interferon genes (STING)

DOI: https://doi.org/10.3389/fimmu.2023.1235936

Mol Cell. 2023 Dec 21. pii: S1097-2765(23)01014-6. [Epub ahead of print]

A mitophagy sensor PPTC7 controls BNIP3 and NIX degradation to regulate mitochondrial mass.

Yuqiu Sun, Yu Cao, Huayun Wan, Adalet Memetimin, Yang Cao, Lin Li, Chongyang Wu, Meng Wang, She Chen, Qi Li, Yan Ma, Mengqiu Dong, Hui Jiang.

Mitophagy mediated by BNIP3 and NIX critically regulates mitochondrial mass. Cellular BNIP3 and NIX levels are tightly controlled by SCFFBXL4-mediated ubiquitination to prevent excessive mitochondrial loss and lethal disease. Here, we report that knockout of PPTC7, a mitochondrial matrix protein, hyperactivates BNIP3-/NIX-mediated mitophagy and causes perinatal lethality that is rescued by NIX knockout in mice. Biochemically, the PPTC7 precursor is trapped by BNIP3 and NIX to the mitochondrial outer membrane, where PPTC7 scaffolds assembly of a substrate-PPTC7-SCFFBXL4 holocomplex to degrade BNIP3 and NIX, forming a homeostatic regulatory loop. PPTC7 possesses an unusually weak mitochondrial targeting sequence to facilitate its outer membrane retention and mitophagy control. Starvation upregulates PPPTC7 expression in mouse liver to repress mitophagy, which critically maintains hepatic mitochondrial mass, bioenergetics, and gluconeogenesis. Collectively, PPTC7 functions as a mitophagy sensor that integrates homeostatic and physiological signals to dynamically control BNIP3 and NIX degradation, thereby maintaining mitochondrial mass and cellular homeostasis.

Keywords: Cullin; FBXL4; PPTC7; metabolism; mitochondrial mass; mitophagy receptors BNIP3 and NIX; ubiquitin

DOI: https://doi.org/10.1016/j.molcel.2023.11.038

Nat Commun. 2023 Dec 27. 14(1): 8501

Structural basis for DNA proofreading.

Gina Buchel, Ashok R Nayak, Karl Herbine, Azadeh Sarfallah, Viktoriia O Sokolova, Angelica Zamudio-Ochoa, Dmitry Temiakov.

DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved 'bolt-action' mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.

DOI: https://doi.org/10.1038/s41467-023-44198-8

Inflammation. 2023 Dec 26.

cGAS Mediates the Inflammatory Responses of Human Microglial Cells to Genotoxic DNA Damage.

Alexander J Suptela, Yasmine Radwan, Christine Richardson, Shan Yan, Kirill A Afonin, Ian Marriott.

Genomic instability is a key driving force for the development and progression of many age-related neurodegenerative diseases and central nervous system (CNS) cancers. Recently, the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), has been shown to detect and respond to self-DNA accumulation resulting from DNA damaging insults in peripheral cell types. cGAS has been shown to be important in the responses of microglia to DNA viruses and amyloid beta, and we have reported that it underlies the responses of human microglia to exogenous DNA. However, the role of this cytosolic sensor in the detection of self-DNA by glia is poorly understood and its ability to mediate the cellular responses of human microglia to genotoxic DNA damage has not been established. Here, we describe the ability of ionizing radiation and oxidative stress to elicit genomic DNA damage in human microglial cells and to stimulate the production of key inflammatory mediators by these cells in an NF-kB dependent manner. Importantly, we have utilized CRISPR/Cas9 and siRNA-mediated knockdown approaches and a pharmacological inhibitor of the cGAS adaptor protein stimulator of interferon genes (STING) to demonstrate that the cGAS-STING pathway plays a critical role in the generation of these microglial immune responses to such genotoxic insults. Together, these studies support the notion that cGAS mediates the detection of cytosolic self-DNA by microglia, providing a potential mechanism linking genomic instability to the development of CNS cancers and neurodegenerative disorders.

Keywords: DNA damage; cGAS; genomic instability; inflammation.; microglia

DOI: https://doi.org/10.1007/s10753-023-01946-8

Mol Metab. 2023 Dec 23. pii: S2212-8778(23)00193-X. [Epub ahead of print] 101859

Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes.

BACKGROUND: Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy.
METHODS: We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca2+ kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tvHeLa).
RESULTS: Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca2+ concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to β-adrenergic stimulation.
CONCLUSIONS: Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and mitochondrial function, which suggests that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca2+ kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.

Keywords: Contractility; Dilated cardiomyopathy with ataxia; Genetics; Metabolism; Mitochondria; OXPHOS; ROS

DOI: https://doi.org/10.1016/j.molmet.2023.101859