bims-minimp 2021-09-26 papers

on Mitochondria, innate immunity, proteostasis

Issue of 2021‒09‒26
thirty-four papers selected by
Hanna Salmonowicz
International Institute of Molecular Mechanisms and Machines of the Polish Academy of Sciences

Protein structure and aggregation: a marriage of necessity ruled by aggregation gatekeepers.
Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.
Controlling the topology of mammalian mitochondrial DNA.
Mitochondrial-derived compartments: A "fusel alcohol generator" in yeast?
Autophagy and Mitochondrial Homeostasis During Infection: A Double-Edged Sword.
Mitophagy in Antiviral Immunity.
Mitofusin 2: A Link Between Mitochondrial Function and Substrate Metabolism?
Enhanced glycolysis and GSK3 inactivation promote brain metabolic adaptations following neuronal mitochondrial stress.
Creld2 function during unfolded protein response is essential for liver metabolism homeostasis.
Persistent DNA damage alters the neuronal transcriptome suggesting cell cycle dysregulation and altered mitochondrial function.
Transposon-triggered innate immune response confers cancer resistance to the blind mole rat.
Mitochondrial tRNA-Derived Fragments and Their Contribution to Gene Expression Regulation.
Skeletal muscle type-specific mitochondrial adaptation to high-fat diet relies on differential autophagy modulation.
Heritable shifts in redox metabolites during mitochondrial quiescence reprogramme progeny metabolism.
A killer metamorphosis: catching BAK in action at the membrane.
RBC mitochondria trigger interferon release.
Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage.
STING nuclear partners contribute to innate immune signaling responses.
Metformin alleviates stress-induced cellular senescence of aging human adipose stromal cells and the ensuing adipocyte dysfunction.
Biomarkers of cellular aging during a controlled human malaria infection.
The protein arginine methyltransferase PRMT1 promotes TBK1 activation through asymmetric arginine methylation.
Subcellular proteomics.
Blood cell respiration rates and mtDNA copy number: a promising tool for the diagnosis of mitochondrial disease.
Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria.
Controllable membrane remodeling by a modified fragment of the apoptotic protein Bax.
In situ observation of mitochondrial biogenesis as the early event of apoptosis.
Metabolic drift in the aging nervous system is reflected in human cerebrospinal fluid.
Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma.
Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.
A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance.
High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes.
Brown adipocyte ATF4 activation improves thermoregulation and systemic metabolism.
Topological principles of protein folding.

Trends Biochem Sci. 2021 Sep 21. pii: S0968-0004(21)00190-0. [Epub ahead of print]

Protein structure and aggregation: a marriage of necessity ruled by aggregation gatekeepers.

Bert Houben, Frederic Rousseau, Joost Schymkowitz.

  Protein aggregation propensity is a pervasive and seemingly inescapable property of proteomes. Strikingly, a significant fraction of the proteome is supersaturated, meaning that, for these proteins, their native conformation is less stable than the aggregated state. Maintaining the integrity of a proteome under such conditions is precarious and requires energy-consuming proteostatic regulation. Why then is aggregation propensity maintained at such high levels over long evolutionary timescales? Here, we argue that the conformational stability of the native and aggregated states are correlated thermodynamically and that codon usage strengthens this correlation. As a result, the folding of stable proteins requires kinetic control to avoid aggregation, provided by aggregation gatekeepers. These unique residues are evolutionarily selected to kinetically favor native folding, either on their own or by coopting chaperones.

Keywords:  amyloid; kinetic partitioning; protein aggregation; protein stability; protein structure; proteostasis

DOI:  https://doi.org/10.1016/j.tibs.2021.08.010
Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00688-2. [Epub ahead of print]81(18): 3786-3802.e13

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.

Max-Hinderk Schuler, Alyssa M English, Tianyao Xiao, Thane J Campbell, Janet M Shaw, Adam L Hughes.

  Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

Keywords:  MDC; Tom70; amino acid; lysosome; mitochondria; nutrient carrier; vacuole

DOI:  https://doi.org/10.1016/j.molcel.2021.08.021
EMBO J. 2021 Sep 20. e108648

Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.

Sergio Guerrero-Castillo, Joeri van Strien, Ulrich Brandt, Susanne Arnold.

  So-called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in-depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.

Keywords:  OXPHOS; complexome profiling; mitochondria; mtDNA; rho0 cells

DOI:  https://doi.org/10.15252/embj.2021108648
Open Biol. 2021 Sep;11(9): 210168

Controlling the topology of mammalian mitochondrial DNA.

Katja E Menger, Alejandro Rodríguez-Luis, James Chapman, Thomas J Nicholls.

  The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.

Keywords:  DNA topology; mitochondria; mitochondrial DNA; mitochondrial disease; topoisomerases

DOI:  https://doi.org/10.1098/rsob.210168
Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00712-7. [Epub ahead of print]81(18): 3670-3671

Mitochondrial-derived compartments: A "fusel alcohol generator" in yeast?

Tim König, Heidi M McBride.

Schuler et al. (2021) demonstrate that mitochondrial-derived compartments protect cells from amino acid toxicity by activation of amino acid catabolism through the Ehrlich pathway, thus highlighting the incredible plasticity of mitochondria in rewiring cellular metabolism.

DOI: https://doi.org/10.1016/j.molcel.2021.08.032
Front Cell Dev Biol. 2021 ;9 738932

Autophagy and Mitochondrial Homeostasis During Infection: A Double-Edged Sword.

Sutian Wang, Kunli Zhang, Yuchang Yao, Jianhao Li.

  Autophagy, an essential biological process that affects immunity, is a powerful tool that host cells can use to defend against infections caused by pathogenic microorganisms. Autophagy can not only initiate innate immune responses but also degrade the cellular components that provide the conditions for removing the invaders. However, hyperactivated or inhibited autophagy leads to mitochondrial dysfunction, which is harmful to the host itself and is involved in many types of diseases. Mitochondria perform the functions of biological oxidation and energy exchange. In addition, mitochondrial functions are closely related to cell death, oxygen radical formation, and disease. Accumulation of mitochondrial metabolites affects survival of intracellular pathogens. In this mini-review, we focus on the crosstalk between autophagy and mitochondrial homeostasis during infection.

Keywords:  autophagy; dual role; homeostasis; mitochondria; pathogen infection

DOI:  https://doi.org/10.3389/fcell.2021.738932
Front Cell Dev Biol. 2021 ;9 723108

Mitophagy in Antiviral Immunity.

Hongna Wang, Yongfeng Zheng, Jieru Huang, Jin Li.

  Mitochondria are important organelles whose primary function is energy production; in addition, they serve as signaling platforms for apoptosis and antiviral immunity. The central role of mitochondria in oxidative phosphorylation and apoptosis requires their quality to be tightly regulated. Mitophagy is the main cellular process responsible for mitochondrial quality control. It selectively sends damaged or excess mitochondria to the lysosomes for degradation and plays a critical role in maintaining cellular homeostasis. However, increasing evidence shows that viruses utilize mitophagy to promote their survival. Viruses use various strategies to manipulate mitophagy to eliminate critical, mitochondria-localized immune molecules in order to escape host immune attacks. In this article, we will review the scientific advances in mitophagy in viral infections and summarize how the host immune system responds to viral infection and how viruses manipulate host mitophagy to evade the host immune system.

Keywords:  autophagy; immune; infection; mitochondria; mitophagy; virus

DOI:  https://doi.org/10.3389/fcell.2021.723108
Mitochondrion. 2021 Sep 15. pii: S1567-7249(21)00121-5. [Epub ahead of print]

Mitofusin 2: A Link Between Mitochondrial Function and Substrate Metabolism?

Janna M Emery, Rudy M Ortiz.

  Mitochondria are dynamic, interactive organelles that connect cellular signaling and whole-cell homeostasis. This "mitochatting" allows the cell to receive information about the mitochondria's condition before accommodating energy demands. Mitofusin 2 (Mfn2), an outer mitochondrial membrane fusion protein specializes in mediating mitochondrial homeostasis. Early studies defined the biological significance of Mfn2, latter studies highlighted its role in substrate metabolism. However, determining Mfn2 potential to contribute to energy homeostasis needs study. This review summarizes current literature on mitochondrial metabolic processes, dynamics, and evidence of interactions among Mfn2 and regulatory processes that may link Mfn2's role in maintaining mitochondrial function and substrate metabolism.

Keywords:  fatty acid oxidation; fission; fusion; glycolysis; mitochondrial dynamics; mitophagy

DOI:  https://doi.org/10.1016/j.mito.2021.09.003
Hum Mol Genet. 2021 Sep 24. pii: ddab282. [Epub ahead of print]

Enhanced glycolysis and GSK3 inactivation promote brain metabolic adaptations following neuronal mitochondrial stress.

Sofia Garcia, Amy Saldana-Caboverde, Mir Anwar, Ami Pravinkant Raval, Nadee Nissanka, Milena Pinto, Carlos Torres Moraes, Francisca Diaz.

  We analyzed early brain metabolic adaptations in response to mitochondrial dysfunction in a mouse model of mitochondrial encephalopathy with complex IV deficiency (neuron specific COX10 KO). In this mouse model the onset of the mitochondrial defect did not coincide with immediate cell death suggesting early adaptive metabolic responses to compensate for the energetic deficit. Metabolomic analysis in the knockout mice revealed increased levels of glycolytic and pentose phosphate pathway intermediates, amino acids and lysolipids. Glycolysis was modulated by enhanced activity of glycolytic enzymes, and not by their overexpression, suggesting the importance of post-translational modifications in the adaptive response. GSK3 inactivation was the most upstream regulation identified, implying that it is a key event in this adaptive mechanism. Because neurons are thought not to rely on glycolysis for ATP production in normal conditions, our results indicate that neurons still maintain their ability to upregulate this pathway when under mitochondrial respiration stress.

Keywords:  mitochondrial diseasescomplex IV deficiencyneuron specific COX10 KOmetabolic adaptationposttranslational modificationsglycolysisGSK3

DOI:  https://doi.org/10.1093/hmg/ddab282
FASEB J. 2021 Oct;35(10): e21939

Creld2 function during unfolded protein response is essential for liver metabolism homeostasis.

Paul Kern, Nora R Balzer, Nelli Blank, Cornelia Cygon, Klaus Wunderling, Franziska Bender, Alex Frolov, Jan-Peter Sowa, Lorenzo Bonaguro, Thomas Ulas, Mirka Homrich, Eva Kiermaier, Christoph Thiele, Joachim L Schultze, Ali Canbay, Reinhard Bauer, Elvira Mass.

  The unfolded protein response (UPR) is associated with hepatic metabolic function, yet it is not well understood how endoplasmic reticulum (ER) disturbance might influence metabolic homeostasis. Here, we describe the physiological function of Cysteine-rich with EGF-like domains 2 (Creld2), previously characterized as a downstream target of the ER-stress signal transducer Atf6. To this end, we generated Creld2-deficient mice and induced UPR by injection of tunicamycin. Creld2 augments protein folding and creates an interlink between the UPR axes through its interaction with proteins involved in the cellular stress response. Thereby, Creld2 promotes tolerance to ER stress and recovery from acute stress. Creld2-deficiency leads to a dysregulated UPR and causes the development of hepatic steatosis during ER stress conditions. Moreover, Creld2-dependent enhancement of the UPR assists in the regulation of energy expenditure. Furthermore, we observed a sex dimorphism in human and mouse livers with only male patients showing an accumulation of CRELD2 protein during the progression from non-alcoholic fatty liver disease to non-alcoholic steatohepatitis and only male Creld2-deficient mice developing hepatic steatosis upon aging. These results reveal a Creld2 function at the intersection between UPR and metabolic homeostasis and suggest a mechanism in which chronic ER stress underlies fatty liver disease in males.

Keywords:  Creld2; ER stress; NASH; UPR; liver steatosis

DOI:  https://doi.org/10.1096/fj.202002713RR
Eur J Neurosci. 2021 Sep 18.

Persistent DNA damage alters the neuronal transcriptome suggesting cell cycle dysregulation and altered mitochondrial function.

Irina Vazquez-Villasenor, Claire J Garwood, Julie E Simpson, Paul R Heath, Heather Mortiboys, Stephen B Wharton.

  Oxidative DNA damage induces changes in the neuronal cell cycle and activates a DNA damage response to promote repair, but these processes may be altered under a chronic oxidative environment, leading to the accumulation of unrepaired DNA damage and continued activation of a DNA damage response. Failure to repair DNA damage can lead to apoptosis or senescence, which is characterized by a permanent cell-cycle arrest. Increased oxidative stress and accumulation of oxidative DNA damage are features of brain ageing and neurodegeneration but the effects of persistent DNA damage in neurons are not well-characterized. We developed a model of persistent oxidative DNA damage in immortalized post-mitotic neurons in vitro by exposing them to a sub-lethal concentration of hydrogen peroxide following a "double stress" protocol, and performed a detailed characterization of the neuronal transcriptome using microarray analysis. Persistent DNA damage significantly altered the expression of genes involved in cell cycle regulation, DNA damage response and repair mechanisms, and mitochondrial function, suggesting an active DDR response to replication stress and alterations in mitochondrial electron transport chain. qPCR and functional validation experiments confirmed hyperactivation of mitochondrial Complex I in response to persistent DNA damage. These changes in response to persistent oxidative DNA damage may lead to further oxidative stress, contributing to neuronal dysfunction and ultimately neurodegeneration.

Keywords:  DNA damage response; cell cycle re-entry; mitochondrial complex I; oxidative stress; post-mitotic

DOI:  https://doi.org/10.1111/ejn.15466
Nat Immunol. 2021 Sep 23.

Transposon-triggered innate immune response confers cancer resistance to the blind mole rat.

Yang Zhao, Ena Oreskovic, Quanwei Zhang, Quan Lu, Abbey Gilman, Yifei S Lin, Junyue He, Zhizhong Zheng, J Yuyang Lu, Jina Lee, Zhonghe Ke, Julia Ablaeva, Matthew J Sweet, Steve Horvath, Zhengdong Zhang, Eviatar Nevo, Andrei Seluanov, Vera Gorbunova.

Blind mole rats (BMRs) are small rodents, characterized by an exceptionally long lifespan (>21 years) and resistance to both spontaneous and induced tumorigenesis. Here we report that cancer resistance in the BMR is mediated by retrotransposable elements (RTEs). Cells and tissues of BMRs express very low levels of DNA methyltransferase 1. Following cell hyperplasia, the BMR genome DNA loses methylation, resulting in the activation of RTEs. Upregulated RTEs form cytoplasmic RNA-DNA hybrids, which activate the cGAS-STING pathway to induce cell death. Although this mechanism is enhanced in the BMR, we show that it functions in mice and humans. We propose that RTEs were co-opted to serve as tumor suppressors that monitor cell proliferation and are activated in premalignant cells to trigger cell death via activation of the innate immune response. Activation of RTEs is a double-edged sword, serving as a tumor suppressor but contributing to aging in late life via the induction of sterile inflammation.

DOI: https://doi.org/10.1038/s41590-021-01027-8
Front Physiol. 2021 ;12 729452

Mitochondrial tRNA-Derived Fragments and Their Contribution to Gene Expression Regulation.

Athanasios-Nasir Shaukat, Eleni G Kaliatsi, Vassiliki Stamatopoulou, Constantinos Stathopoulos.

  Mutations in human mitochondrial tRNAs (mt-tRNAs) are responsible for several and sometimes severe clinical phenotypes, classified among mitochondrial diseases. In addition, post-transcriptional modifications of mt-tRNAs in correlation with several stress signals can affect their stability similarly to what has been described for their nuclear-encoded counterparts. Many of the perturbations related to either point mutations or aberrant modifications of mt-tRNAs can lead to specific cleavage and the production of mitochondrial tRNA-derived fragments (mt-tRFs). Although mt-tRFs have been detected in several studies, the exact biogenesis steps and biological role remain, to a great extent, unexplored. Several mt-tRFs are produced because of the excessive oxidative stress which predominantly affects mitochondrial DNA integrity. In addition, mt-tRFs have been detected in various diseases with possible detrimental consequences, but also their production may represent a response mechanism to external stimuli, including infections from pathogens. Finally, specific point mutations on mt-tRNAs have been reported to impact the pool of the produced mt-tRFs and there is growing evidence suggesting that mt-tRFs can be exported and act in the cytoplasm. In this review, we summarize current knowledge on mitochondrial tRNA-deriving fragments and their possible contribution to gene expression regulation.

Keywords:  mitochondria; mitochondrial tRNA-derived fragments; mitochondrial tRNAs; ncRNAs; tRNA-derived fragments

DOI:  https://doi.org/10.3389/fphys.2021.729452
FASEB J. 2021 Oct;35(10): e21933

Skeletal muscle type-specific mitochondrial adaptation to high-fat diet relies on differential autophagy modulation.

Pablo E Morales, Matías Monsalves-Álvarez, Satya Murthy Tadinada, Matthew P Harris, Andrea Ramírez-Sagredo, Jafet Ortiz-Quintero, Mayarling Francisca Troncoso, Nicole De Gregorio, Ximena Calle, Renata O Pereira, Vitor A Lira, Alejandra Espinosa, E Dale Abel, Sergio Lavandero.

  In obesity, skeletal muscle mitochondrial activity changes to cope with increased nutrient availability. Autophagy has been proposed as an essential mechanism involved in the regulation of mitochondrial metabolism. Still, the contribution of autophagy to mitochondrial adaptations in skeletal muscle during obesity is unknown. Here, we show that in response to high-fat diet (HFD) feeding, distinct skeletal muscles in mice exhibit differentially regulated autophagy that may modulate mitochondrial activity. We observed that after 4 and 40 weeks of high-fat diet feeding, OXPHOS subunits and mitochondrial DNA content increased in the oxidative soleus muscle. However, in gastrocnemius muscle, which has a mixed fiber-type composition, the mitochondrial mass increased only after 40 weeks of HFD feeding. Interestingly, fatty acid-supported mitochondrial respiration was enhanced in gastrocnemius, but not in soleus muscle after a 4-week HFD feeding. This increased metabolic profile in gastrocnemius was paralleled by preserving autophagy flux, while autophagy flux in soleus was reduced. To determine the role of autophagy in this differential response, we used an autophagy-deficient mouse model with partial deletion of Atg7 specifically in skeletal muscle (SkM-Atg7+/- mice). We observed that Atg7 reduction resulted in diminished autophagic flux in skeletal muscle, alongside blunting the HFD-induced increase in fatty acid-supported mitochondrial respiration observed in gastrocnemius. Remarkably, SkM-Atg7+/- mice did not present increased mitochondria accumulation. Altogether, our results show that HFD triggers specific mitochondrial adaptations in skeletal muscles with different fiber type compositions, and that Atg7-mediated autophagy modulates mitochondrial respiratory capacity but not its content in response to an obesogenic diet.

Keywords:  Atg7; fatty acids; obesity; skeletal muscle fiber

DOI:  https://doi.org/10.1096/fj.202001593RR
Nat Metab. 2021 Sep;3(9): 1259-1274

Heritable shifts in redox metabolites during mitochondrial quiescence reprogramme progeny metabolism.

Helin Hocaoglu, Lei Wang, Mengye Yang, Sibiao Yue, Matthew Sieber.

Changes in maternal diet and metabolic defects in mothers can profoundly affect health and disease in their progeny. However, the biochemical mechanisms that induce the initial reprogramming events at the cellular level have remained largely unknown owing to limitations in obtaining pure populations of quiescent oocytes. Here, we show that the precocious onset of mitochondrial respiratory quiescence causes a reprogramming of progeny metabolic state. The premature onset of mitochondrial respiratory quiescence drives the lowering of Drosophila oocyte NAD+ levels. NAD+ depletion in the oocyte leads to reduced methionine cycle production of the methyl donor S-adenosylmethionine in embryos and lower levels of histone H3 lysine 27 trimethylation, resulting in enhanced intestinal lipid metabolism in progeny. In addition, we show that triggering cellular quiescence in mammalian cells and chemotherapy-resistant human cancer cell models induces cellular reprogramming events identical to those seen in Drosophila, suggesting a conserved metabolic mechanism in systems reliant on quiescent cells.

DOI: https://doi.org/10.1038/s42255-021-00450-3
EMBO J. 2021 Sep 20. e109529

A killer metamorphosis: catching BAK in action at the membrane.

Adedolapo M Ojoawo, Tudor Moldoveanu.

Permeabilization of the outer mitochondrial membrane initiates apoptotic cell death. B-cell lymphoma 2 (BCL-2) antagonist killer (BAK) and BCL-2-associated X (BAX) mediate mitochondrial poration, but how this process unfolds remains poorly defined. Two studies in this issue investigate the transition of dormant, inactive BAK monomer to a highly dynamic membrane-associated, pore-forming oligomer.

DOI: https://doi.org/10.15252/embj.2021109529
Nat Rev Nephrol. 2021 Sep 20.

RBC mitochondria trigger interferon release.

Monica Wang.

DOI: https://doi.org/10.1038/s41581-021-00494-4
J Biol Chem. 2021 Sep 21. pii: S0021-9258(21)01027-9. [Epub ahead of print] 101224

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage.

Kristina Bubb, Tatjana Holzer, Janica L Nolte, Marcus Krüger, Richard Wilson, Ursula Schlötzer-Schrehardt, Jürgen Brinckmann, Janine Altmüller, Attila Aszodi, Lutz Fleischhauer, Hauke Clausen-Schaumann, Kristina Probst, Bent Brachvogel.

  Energy metabolism and extracellular matrix function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single cell RNA-seq (scRNA-seq) analysis to uncover the importance of the mitochondrial respiratory chain for extracellular matrix homeostasis in mature cartilage. This tissue produces large amounts of a specialized extracellular matrix to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocytes. These changes were associated with alterations in extracellular matrix composition, a shift in collagen/non-collagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.

Keywords:  Extracellular matrix; MMP10; THBS1; atomic force microscopy; matrisome; matrix metalloproteinase (MMP); mitochondria; mitochondrial respiratory chain; single cell RNA sequencing; transcriptomics

DOI:  https://doi.org/10.1016/j.jbc.2021.101224
iScience. 2021 Sep 24. 24(9): 103055

STING nuclear partners contribute to innate immune signaling responses.

Charles R Dixon, Poonam Malik, Jose I de Las Heras, Natalia Saiz-Ros, Flavia de Lima Alves, Mark Tingey, Eleanor Gaunt, A Christine Richardson, David A Kelly, Martin W Goldberg, Greg J Towers, Weidong Yang, Juri Rappsilber, Paul Digard, Eric C Schirmer.

  STimulator of INterferon Genes (STING) is an adaptor for cytoplasmic DNA sensing by cGAMP/cGAS that helps trigger innate immune responses (IIRs). Although STING is mostly localized in the ER, we find a separate inner nuclear membrane pool of STING that increases mobility and redistributes to the outer nuclear membrane upon IIR stimulation by transfected dsDNA or dsRNA mimic poly(I:C). Immunoprecipitation of STING from isolated nuclear envelopes coupled with mass spectrometry revealed a distinct nuclear envelope-STING proteome consisting of known nuclear membrane proteins and enriched in DNA- and RNA-binding proteins. Seventeen of these nuclear envelope STING partners are known to bind direct interactors of IRF3/7 transcription factors, and testing a subset of these revealed STING partners SYNCRIP, MEN1, DDX5, snRNP70, RPS27a, and AATF as novel modulators of dsDNA-triggered IIRs. Moreover, we find that SYNCRIP is a novel antagonist of the RNA virus, influenza A, potentially shedding light on reports of STING inhibition of RNA viruses.

Keywords:  Cell biology; Immunology; Molecular physiology; Virology

DOI:  https://doi.org/10.1016/j.isci.2021.103055
Elife. 2021 Sep 21. pii: e62635. [Epub ahead of print]10

Metformin alleviates stress-induced cellular senescence of aging human adipose stromal cells and the ensuing adipocyte dysfunction.

Laura Le Pelletier, Matthieu Mantecon, Jennifer Gorwood, Martine Auclair, Roberta Foresti, Roberto Motterlini, Mireille Laforge, Michael Atlan, Bruno Fève, Jacqueline Capeau, Claire Lagathu, Veronique Bereziat.

  Aging is associated with central fat redistribution and insulin resistance. To identify age-related adipose features, we evaluated the senescence and adipogenic potential of adipose-derived-stromal cells (ASCs) from abdominal subcutaneous fat obtained from healthy normal-weight young (<25y) or older women (>60y). Increased cell passages of young-donor ASCs (in vitro aging), resulted in senescence but not oxidative stress. ASC-derived adipocytes presented impaired adipogenesis but no early mitochondrial dysfunction. Conversely, aged-donor ASCs at early passages displayed oxidative stress and mild senescence. ASC-derived adipocytes exhibited oxidative stress, and early mitochondrial dysfunction but adipogenesis was preserved. In vitro aging of aged-donor ASCs resulted in further increased senescence, mitochondrial dysfunction, oxidative stress and severe adipocyte dysfunction. When in vitro aged young-donor ASCs were treated with metformin, no alteration was alleviated. Conversely, metformin treatment of aged-donor ASCs decreased oxidative stress and mitochondrial dysfunction resulting in decreased senescence. Metformin's prevention of oxidative stress and of the resulting senescence improved the cells' adipogenic capacity and insulin sensitivity. This effect was mediated by the activation of AMP-activated-protein-kinase as revealed by its specific inhibition and activation. Overall, aging ASC-derived adipocytes presented impaired adipogenesis and insulin sensitivity. Targeting stress-induced senescence of ASCs with metformin may improve age-related adipose tissue dysfunction.

Keywords:  cell biology; human

DOI:  https://doi.org/10.7554/eLife.62635
Sci Rep. 2021 Sep 21. 11(1): 18733

Biomarkers of cellular aging during a controlled human malaria infection.

Aurelie Miglar, Isaie J Reuling, Xi Zen Yap, Anna Färnert, Robert W Sauerwein, Muhammad Asghar.

Cellular aging is difficult to study in individuals with natural infection, given the diversity of symptom duration and clinical presentation, and the high interference of aging-related processes with host and environmental factors. To address this challenge, we took advantage of the controlled human malaria infection (CHMI) model. This approach allowed us to characterize the relationship among cellular aging markers prior, during and post malaria pathophysiology in humans, controlling for infection dose, individual heterogeneity, previous exposure and co-infections. We demonstrate that already low levels of Plasmodium falciparum impact cellular aging by inducing high levels of inflammation and redox-imbalance; and that cellular senescence reversed after treatment and parasite clearance. This study provides insights into the complex relationship of telomere length, cellular senescence, telomerase expression and aging-related processes during a single malaria infection.

DOI: https://doi.org/10.1038/s41598-021-97985-y
Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01180-3. [Epub ahead of print]36(12): 109731

The protein arginine methyltransferase PRMT1 promotes TBK1 activation through asymmetric arginine methylation.

Zhenzhen Yan, Haifeng Wu, Hansen Liu, Guimin Zhao, Honghai Zhang, Wanxin Zhuang, Feng Liu, Yi Zheng, Bingyu Liu, Lei Zhang, Chengjiang Gao.

  TBK1 is an essential kinase for the innate immune response against viral infection. However, the key molecular mechanisms regulating the TBK1 activation remain elusive. Here, we identify PRMT1, a type I protein arginine methyltransferase, as an essential regulator of TBK1 activation. PRMT1 directly interacts with TBK1 and catalyzes asymmetric methylation of R54, R134, and R228 on TBK1. This modification enhances TBK1 oligomerization after viral infection, which subsequently promotes TBK1 phosphorylation and downstream type I interferon production. More important, myeloid-specific Prmt1 knockout mice are more susceptible to infection with DNA and RNA viruses than Prmt1fl/fl mice. Our findings reveal insights into the molecular regulation of TBK1 activation and demonstrate the essential function of protein arginine methylation in innate antiviral immunity.

Keywords:  PRMT1; TBK1; innate antiviral immunity; protein arginine methylation

DOI:  https://doi.org/10.1016/j.celrep.2021.109731
Nat Rev Methods Primers. 2021 ;pii: 32. [Epub ahead of print]1

Subcellular proteomics.

Josie A Christopher, Charlotte Stadler, Claire E Martin, Marcel Morgenstern, Yanbo Pan, Cora N Betsinger, David G Rattray, Diana Mahdessian, Anne-Claude Gingras, Bettina Warscheid, Janne Lehtiö, Ileana M Cristea, Leonard J Foster, Andrew Emili, Kathryn S Lilley.

The eukaryotic cell is compartmentalized into subcellular niches, including membrane-bound and membrane-less organelles. Proteins localize to these niches to fulfil their function, enabling discreet biological processes to occur in synchrony. Dynamic movement of proteins between niches is essential for cellular processes such as signalling, growth, proliferation, motility and programmed cell death, and mutations causing aberrant protein localization are associated with a wide range of diseases. Determining the location of proteins in different cell states and cell types and how proteins relocalize following perturbation is important for understanding their functions, related cellular processes and pathologies associated with their mislocalization. In this Primer, we cover the major spatial proteomics methods for determining the location, distribution and abundance of proteins within subcellular structures. These technologies include fluorescent imaging, protein proximity labelling, organelle purification and cell-wide biochemical fractionation. We describe their workflows, data outputs and applications in exploring different cell biological scenarios, and discuss their main limitations. Finally, we describe emerging technologies and identify areas that require technological innovation to allow better characterization of the spatial proteome.

DOI: https://doi.org/10.1038/s43586-021-00029-y
Mitochondrion. 2021 Sep 15. pii: S1567-7249(21)00122-7. [Epub ahead of print]

Blood cell respiration rates and mtDNA copy number: a promising tool for the diagnosis of mitochondrial disease.

Martina Alonso, Cristina Zabala, Santiago Mansilla, Laureana De Brun, Jennyfer Martínez, Mariela Garau, Gabriela Rivas, Cecilia Acosta, Daniela Lens, Alfredo Cerisola, Martín Graña, Hugo Naya, Rodrigo Puentes, Lucía Spangenberg, Víctor Raggio, Aída Lemes, Laura Castro, Celia Quijano.

  Human mitochondrial diseases are a group of heterogeneous diseases caused by defects in oxidative phosphorylation, due to mutations in mitochondrial (mtDNA) or nuclear DNA. The diagnosis of mitochondrial disease is challenging since mutations in multiple genes can affect mitochondrial function, there is considerable clinical variability and a poor correlation between genotype and phenotype. Herein we assessed mitochondrial function in peripheral blood mononuclear cells (PBMCs) and platelets from volunteers without known metabolic pathology and patients with mitochondrial disease. Oxygen consumption rates were evaluated and respiratory parameters indicative of mitochondrial function were obtained. A negative correlation between age and respiratory parameters of PBMCs from control individuals was observed. Surprisingly, respiratory parameters of PBMCs normalized by cell number were similar in patients and young controls. Considering possible compensatory mechanisms, mtDNA copy number in PBMCs was quantified and an increase was found in patients with respect to controls. Hence, respiratory parameters normalized by mtDNA copy number were determined, and in these conditions a decrease in maximum respiration rate and spare respiratory capacity was observed in patients relative to control individuals. In platelets no decay was seen in mitochondrial function with age, while a reduction in basal, ATP-independent and ATP-dependent respiration normalized by cell number was detected in patients compared to control subjects. In summary, our results offer promising perspectives regarding the assessment of mitochondrial function in blood cells for the diagnosis mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsies, and for following disease progression and response to treatments.

Keywords:  PBMC; aging; bioenergetics; mitochondrial disease; mtDNA; platelets

DOI:  https://doi.org/10.1016/j.mito.2021.09.004
Methods Mol Biol. 2022 ;2363 321-334

Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria.

Beatriz Ferrando, Ian Max Møller, Tinna Stevnsner.

  Nuclear, mitochondrial and plastidic DNA is constantly exposed to conditions, such as ultraviolet radiation or reactive oxygen species, which will induce chemical modifications to the nucleotides. Unless repaired, these modifications can lead to mutations, so the nucleus, mitochondria and plastids each contains a number of DNA repair systems. We here describe assays for measuring the enzyme activities associated with the base-excision repair pathway in potato tuber mitochondria. As the name implies, this pathway involves removing a modified base and replacing it with an undamaged base. Activity of each of the enzymes involved, DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase and DNA ligase can be measured by incubating a mitochondrial extract with a specifically designed oligonucleotide. After incubation, the reaction mixture is separated on a polyacrylamide gel, and the amounts of specific products formed is estimated by autoradiography, which makes it possible to calculate the enzymatic activity.

Keywords:  Base excision repair; DNA repair; Plant mitochondria; mtDNA

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_21
Faraday Discuss. 2021 Sep 22.

Controllable membrane remodeling by a modified fragment of the apoptotic protein Bax.

Katherine G Schaefer, Brayan Grau, Nicolas Moore, Ismael Mingarro, Gavin M King, Francisco N Barrera.

Intrinsic apoptosis is orchestrated by a group of proteins that mediate the coordinated disruption of mitochondrial membranes. Bax is a multi-domain protein that, upon activation, disrupts the integrity of the mitochondrial outer membrane by forming pores. We strategically introduced glutamic acids into a short sequence of the Bax protein that constitutively creates membrane pores. The resulting BaxE5 peptide efficiently permeabilizes membranes at acidic pH, showing low permeabilization at neutral pH. Atomic force microscopy (AFM) imaging showed that at acidic pH BaxE5 established several membrane remodeling modalities that progressively disturbed the integrity of the lipid bilayer. The AFM data offers vistas on the membrane disruption process, which starts with pore formation and progresses through localized exposure of membrane monolayers leading to stable and small (height ∼ 16 Å) lipid-peptide complexes. The different types of membrane morphology observed in the presence of BaxE5 suggest that the peptide can establish different types of membrane interactions. BaxE5 adopts a rare unstructured conformation when bound to membranes, which might facilitate the dynamic transition between those different states, and then promote membrane digestion.

DOI: https://doi.org/10.1039/d0fd00070a
iScience. 2021 Sep 24. 24(9): 103038

In situ observation of mitochondrial biogenesis as the early event of apoptosis.

Chang-Sheng Shao, Xiu-Hong Zhou, Yu-Hui Miao, Peng Wang, Qian-Qian Zhang, Qing Huang.

  Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed.

Keywords:  Biochemistry methods; Biomolecular engineering; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2021.103038
Sci Rep. 2021 Sep 22. 11(1): 18822

Metabolic drift in the aging nervous system is reflected in human cerebrospinal fluid.

Kristian Peters, Stephanie Herman, Payam Emami Khoonsari, Joachim Burman, Steffen Neumann, Kim Kultima.

Chronic diseases affecting the central nervous system (CNS) like Alzheimer's or Parkinson's disease typically develop with advanced chronological age. Yet, aging at the metabolic level has been explored only sporadically in humans using biofluids in close proximity to the CNS such as the cerebrospinal fluid (CSF). We have used an untargeted liquid chromatography high-resolution mass spectrometry (LC-HRMS) based metabolomics approach to measure the levels of metabolites in the CSF of non-neurological control subjects in the age of 20 up to 74. Using a random forest-based feature selection strategy, we extracted 69 features that were strongly related to age (page < 0.001, rage = 0.762, R2Boruta age = 0.764). Combining an in-house library of known substances with in silico chemical classification and functional semantic annotation we successfully assigned putative annotations to 59 out of the 69 CSF metabolites. We found alterations in metabolites related to the Cytochrome P450 system, perturbations in the tryptophan and kynurenine pathways, metabolites associated with cellular energy (NAD+, ADP), mitochondrial and ribosomal metabolisms, neurological dysfunction, and an increase of adverse microbial metabolites. Taken together our results point at a key role for metabolites found in CSF related to the Cytochrome P450 system as most often associated with metabolic aging.

DOI: https://doi.org/10.1038/s41598-021-97491-1
Front Genet. 2021 ;12 721864

Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma.

Grzegorz L Ciesielski, Shalom Kim, Carolina de Bovi Pontes, Laurie S Kaguni.

  The maintenance of the mitochondrial genome depends on a suite of nucleus-encoded proteins, among which the catalytic subunit of the mitochondrial replicative DNA polymerase, Pol γα, plays a pivotal role. Mutations in the Pol γα-encoding gene, POLG, are a major cause of human mitochondrial disorders. Here we present a study of direct and functional interactions of Pol γα with the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB coordinates the activity of the enzymes at the DNA replication fork. However, the mechanism of this functional relationship is elusive, and no direct interactions between the replicative factors have been identified to date. This contrasts strikingly with the extensive interactomes of SSB proteins identified in other homologous replication systems. Here we show for the first time that mtSSB binds Pol γα directly, in a DNA-independent manner. This interaction is strengthened in the absence of the loop 2.3 structure in mtSSB, and is abolished upon preincubation with Pol γβ. Together, our findings suggest that the interaction between mtSSB and polymerase gamma holoenzyme (Pol γ) involves a balance between attractive and repulsive affinities, which have distinct effects on DNA synthesis and exonucleolysis.

Keywords:  DNA polymerase gamma; intermolecular interactions; mitochondrial DNA replication; mitochondrial biogenesis; mitochondrial single-stranded DNA-binding protein

DOI:  https://doi.org/10.3389/fgene.2021.721864
Cell. 2021 Sep 15. pii: S0092-8674(21)01046-1. [Epub ahead of print]

Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.

Guilherme Pedreira de Freitas Nader, Sonia Agüera-Gonzalez, Fiona Routet, Matthieu Gratia, Mathieu Maurin, Valeria Cancila, Clotilde Cadart, Andrea Palamidessi, Rodrigo Nalio Ramos, Mabel San Roman, Matteo Gentili, Ayako Yamada, Alice Williart, Catalina Lodillinsky, Emilie Lagoutte, Catherine Villard, Jean-Louis Viovy, Claudio Tripodo, Jérôme Galon, Giorgio Scita, Nicolas Manel, Philippe Chavrier, Matthieu Piel.

  Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Keywords:  TREX1, nuclear envelope rupture, DNA damage, mammary duct carcinoma, tumor invasion, senescence, breast cancer, cGAS, confinement, epithelial to mesenchymal transition

DOI:  https://doi.org/10.1016/j.cell.2021.08.035
Sci Adv. 2021 Sep 24. 7(39): eabi7514

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance.

Giulia Rossetti, Judith A Ermer, Maike Stentenbach, Stefan J Siira, Tara R Richman, Dusanka Milenkovic, Kara L Perks, Laetitia A Hughes, Emma Jamieson, Gulibaikelamu Xiafukaiti, Natalie C Ward, Satoru Takahashi, Nicola Gray, Helena M Viola, Livia C Hool, Oliver Rackham, Aleksandra Filipovska.

[Figure: see text].

DOI: https://doi.org/10.1126/sciadv.abi7514
Methods Mol Biol. 2022 ;2363 111-119

High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes.

Lilian Vincis Pereira Sanglard, Catherine Colas des Francs-Small.

  Blue native electrophoresis (BN-PAGE) is a highly resolutive method suited to the study of high molecular weight protein complexes between 100 and >3000 kDa. One of the drawbacks of this method is that it is very time-consuming and requires high quantities of purified organelles. Here we describe a high throughput BN-PAGE method allowing to screen libraries of plants potentially altered in respiratory metabolism.

Keywords:  Blue Native PAGE; Immunoblots; Mitochondria; Respiratory complexes

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_10
Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01195-5. [Epub ahead of print]36(12): 109742

Brown adipocyte ATF4 activation improves thermoregulation and systemic metabolism.

Esther Paulo, Yun Zhang, Ruchi Masand, Tony L Huynh, Youngho Seo, Danielle L Swaney, Margaret Soucheray, Erica Stevenson, David Jimenez-Morales, Nevan J Krogan, Biao Wang.

  Cold-induced thermogenesis in endotherms demands adaptive thermogenesis fueled by mitochondrial respiration and Ucp1-mediated uncoupling in multilocular brown adipocytes (BAs). However, dietary regulation of thermogenesis in BAs isn't fully understood. Here, we describe that the deficiency of Leucine-rich pentatricopeptide repeat containing-protein (Lrpprc) in BAs reduces mtDNA-encoded ETC gene expression, causes ETC proteome imbalance, and abolishes the mitochondria-fueled thermogenesis. BA-specific Lrpprc knockout mice are cold resistant in a 4°C cold-tolerance test in the presence of food, which is accompanied by the activation of transcription factor 4 (ATF4) and proteome turnover in BAs. ATF4 activation genetically by BA-specific ATF4 overexpression or physiologically by a low-protein diet feeding can improve cold tolerance in wild-type and Ucp1 knockout mice. Furthermore, ATF4 activation in BAs improves systemic metabolism in obesogenic environment regardless of Ucp1's action. Therefore, our study reveals a diet-dependent but Ucp1-independent thermogenic mechanism in BAs that is relevant to systemic thermoregulation and energy homeostasis.

Keywords:  ATF4; brown adipocyte; thermogenesis

DOI:  https://doi.org/10.1016/j.celrep.2021.109742
Phys Chem Chem Phys. 2021 Sep 21.

Topological principles of protein folding.

Barbara Scalvini, Vahid Sheikhhassani, Alireza Mashaghi.

What is the topology of a protein and what governs protein folding to a specific topology? This is a fundamental question in biology. The protein folding reaction is a critically important cellular process, which is failing in many prevalent diseases. Understanding protein folding is also key to the design of new proteins for applications. However, our ability to predict the folding of a protein chain is quite limited and much is still unknown about the topological principles of folding. Current predictors of folding kinetics, including the contact order and size, present a limited predictive power, suggesting that these models are fundamentally incomplete. Here, we use a newly developed mathematical framework to define and extract the topology of a native protein conformation beyond knot theory, and investigate the relationship between native topology and folding kinetics in experimentally characterized proteins. We show that not only the folding rate, but also the mechanistic insight into folding mechanisms can be inferred from topological parameters. We identify basic topological features that speed up or slow down the folding process. The approach enabled the decomposition of protein 3D conformation into topologically independent elementary folding units, called circuits. The number of circuits correlates significantly with the folding rate, offering not only an efficient kinetic predictor, but also a tool for a deeper understanding of theoretical folding models. This study contributes to recent work that reveals the critical relevance of topology to protein folding with a new, contact-based, mathematically rigorous perspective. We show that topology can predict folding kinetics when geometry-based predictors like contact order and size fail.

DOI: https://doi.org/10.1039/d1cp03390e