bims-minimp 2021-11-21 papers

on Mitochondria, innate immunity, proteostasis

Issue of 2021‒11‒21
nineteen papers selected by
Hanna Salmonowicz
International Institute of Molecular Mechanisms and Machines of the Polish Academy of Sciences

Mitochondrial chaperone in human health and diseases.
Beyond mitophagy: mitochondrial-derived vesicles can get the job done!
Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen.
Nuclear pore complex maintenance and implications for age-related diseases.
Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells.
Carrying Excess Baggage Can Slowdown Life: Protein Clearance Machineries That Go Awry During Aging and the Relevance of Maintaining Them.
NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix.
Widespread use of unconventional targeting signals in mitochondrial ribosome proteins.
Mapping protein carboxymethylation sites provides insights into their role in proteostasis and cell proliferation.
A novel HSF1 activator ameliorates nonalcoholic steatohepatitis by stimulating mitochondrial adaptive oxidation.
Putative role of uncoupling proteins in mitochondria-nucleus communications and DNA damage response.
Accumulation of cholesterol suppresses oxidative phosphorylation and altered responses to inflammatory stimuli of macrophages.
Repair or Degrade: the Thermodynamic Dilemma of Cellular Protein Quality-Control.
CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.
Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease.
Constitutive IFNα Protein Production in Bats.
Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells.
MicroRNAs and the DNA damage response: How is cell fate determined?
The cryo-EM structure of the chloroplast ClpP complex.

Free Radic Biol Med. 2021 Nov 12. pii: S0891-5849(21)00812-1. [Epub ahead of print]

Mitochondrial chaperone in human health and diseases.

Tyler Bahr, Joshua Katuri, Ting Liang, Yidong Bai.

  Molecular chaperones are a family of proteins that maintain cellular protein homeostasis through non-covalent peptide folding and quality control mechanisms. The chaperone proteins found within mitochondria play significant protective roles in mitochondrial biogenesis, quality control, and stress response mechanisms. Defective mitochondrial chaperones have been implicated in aging, neurodegeneration, and cancer. In this review, we focus on the two most prominent mitochondrial chaperones: mtHsp60 and mtHsp70. These proteins demonstrate different cellular localization patterns, interact with different targets, and have different functional activities. We discuss the structure and function of these prominent mitochondrial chaperone proteins and give an update on newly discovered regulatory mechanisms and disease implications.

Keywords:  Mitochondrial chaperone; Mitochondrial homeostasis; Stressresponse; mtHsp60; mtHsp70

DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.11.015
Autophagy. 2021 Nov 15. 1-3

Beyond mitophagy: mitochondrial-derived vesicles can get the job done!

Payel Mondal, Christina Towers.

  Mitochondria are critical organelles that maintain cellular metabolism and overall function. The catabolic pathway of autophagy plays a central role in recycling damaged mitochondria. Although the autophagy pathway is indispensable for some cancer cell survival, our latest study shows that rare autophagy-dependent cancer cells can adapt to loss of this core pathway. In the process, the autophagy-deficient cells acquire unique dependencies on alternate forms of mitochondrial homeostasis. These rare autophagy-deficient clones circumvent the lack of canonical autophagy by increasing mitochondrial dynamics and by recycling damaged mitochondria via mitochondrial-derived vesicles (MDVs). These studies are the first to implicate MDVs in cancer cell metabolism although many unanswered questions remain about this non-canonical pathway.

Keywords:  Cancer; mitochondrial fusion; mitochondrial-derived vesicles; mitophagy; non-canonical autophagy

DOI:  https://doi.org/10.1080/15548627.2021.1999562
EMBO Rep. 2021 Nov 15. e53054

Metabolic resistance to the inhibition of mitochondrial transcription revealed by CRISPR-Cas9 screen.

Mara Mennuni, Roberta Filograna, Andrea Felser, Nina A Bonekamp, Patrick Giavalisco, Oleksandr Lytovchenko, Nils-Göran Larsson.

  Cancer cells depend on mitochondria to sustain their increased metabolic need and mitochondria therefore constitute possible targets for cancer treatment. We recently developed small-molecule inhibitors of mitochondrial transcription (IMTs) that selectively impair mitochondrial gene expression. IMTs have potent antitumor properties in vitro and in vivo, without affecting normal tissues. Because therapy-induced resistance is a major constraint to successful cancer therapy, we investigated mechanisms conferring resistance to IMTs. We employed a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats)-(CRISP-associated protein 9) whole-genome screen to determine pathways conferring resistance to acute IMT1 treatment. Loss of genes belonging to von Hippel-Lindau (VHL) and mammalian target of rapamycin complex 1 (mTORC1) pathways caused resistance to acute IMT1 treatment and the relevance of these pathways was confirmed by chemical modulation. We also generated cells resistant to chronic IMT treatment to understand responses to persistent mitochondrial gene expression impairment. We report that IMT1-acquired resistance occurs through a compensatory increase of mitochondrial DNA (mtDNA) expression and cellular metabolites. We found that mitochondrial transcription factor A (TFAM) downregulation and inhibition of mitochondrial translation impaired survival of resistant cells. The identified susceptibility and resistance mechanisms to IMTs may be relevant for different types of mitochondria-targeted therapies.

Keywords:  CRISPR-Cas9 screen; cancer; chemoresistance; inhibitor of mitochondrial transcription; mtDNA

DOI:  https://doi.org/10.15252/embr.202153054
Trends Cell Biol. 2021 Nov 12. pii: S0962-8924(21)00202-6. [Epub ahead of print]

Nuclear pore complex maintenance and implications for age-related diseases.

Jinqiang Liu, Martin W Hetzer.

  Nuclear pore complexes (NPCs) bridge the nucleus and the cytoplasm and are indispensable for crucial cellular activities, such as bidirectional molecular trafficking and gene transcription regulation. The discovery of long-lived proteins (LLPs) in NPCs from postmitotic cells raises the exciting possibility that the maintenance of NPC integrity might play an inherent role in lifelong cell function. Age-dependent deterioration of NPCs and loss of nuclear integrity have been linked to age-related decline in postmitotic cell function and degenerative diseases. In this review, we discuss our current understanding of NPC maintenance in proliferating and postmitotic cells, and how malfunction of nucleoporins (Nups) might contribute to the pathogenesis of various neurodegenerative and cardiovascular diseases.

Keywords:  aging; neurodegenerative diseases; nuclear pore complex; protein turnover

DOI:  https://doi.org/10.1016/j.tcb.2021.10.001
Nat Nanotechnol. 2021 Nov 18.

Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells.

Tanmoy Saha, Chinmayee Dash, Ruparoshni Jayabalan, Sachin Khiste, Arpita Kulkarni, Kiran Kurmi, Jayanta Mondal, Pradip K Majumder, Aditya Bardia, Hae Lin Jang, Shiladitya Sengupta.

Cancer progresses by evading the immune system. Elucidating diverse immune evasion strategies is a critical step in the search for next-generation immunotherapies for cancer. Here we report that cancer cells can hijack the mitochondria from immune cells via physical nanotubes. Mitochondria are essential for metabolism and activation of immune cells. By using field-emission scanning electron microscopy, fluorophore-tagged mitochondrial transfer tracing and metabolic quantification, we demonstrate that the nanotube-mediated transfer of mitochondria from immune cells to cancer cells metabolically empowers the cancer cells and depletes the immune cells. Inhibiting the nanotube assembly machinery significantly reduced mitochondrial transfer and prevented the depletion of immune cells. Combining a farnesyltransferase and geranylgeranyltransferase 1 inhibitor, namely, L-778123, which partially inhibited nanotube formation and mitochondrial transfer, with a programmed cell death protein 1 immune checkpoint inhibitor improved the antitumour outcomes in an aggressive immunocompetent breast cancer model. Nanotube-mediated mitochondrial hijacking can emerge as a novel target for developing next-generation immunotherapy agents for cancer.

DOI: https://doi.org/10.1038/s41565-021-01000-4
Mol Neurobiol. 2021 Nov 18.

Carrying Excess Baggage Can Slowdown Life: Protein Clearance Machineries That Go Awry During Aging and the Relevance of Maintaining Them.

Arunabh Sarkar, Aamir Nazir.

  Cellular homeostasis is maintained by rapid and systematic cleansing of aberrant and aggregated proteins within cells. Neurodegenerative diseases (NDs) especially Parkinson's and Alzheimer's disease are known to be associated with multiple factors, most important being impaired clearance of aggregates, resulting in the accumulation of specific aggregated protein in the brain. Protein quality control (PQC) of proteostasis network comprises proteolytic machineries and chaperones along with their regulators to ensure precise operation and maintenance of proteostasis. Such regulatory factors coordinate among each other multiple functional aspects related to proteins, including their synthesis, folding, transport, and degradation. During aging due to inevitable endogenous and external stresses, sustaining a proteome balance is a challenging task. Such stresses decline the capacity of the proteostasis network compromising the proteome integrity, affecting the fundamental physiological processes including reproductive fitness of the organism. This review focuses on highlighting proteome-wide changes during aging and the strategies for proteostasis improvements. The possibility of augmenting the proteostasis network either via genetic or pharmacological interventions may be a promising strategy towards delaying age-associated pathological consequences due to proteome disbalance, thus promoting healthy aging and prolonged longevity.

Keywords:  Aging; Neurodegenerative diseases; Protein quality control; Proteostasis

DOI:  https://doi.org/10.1007/s12035-021-02640-2
Cell Biosci. 2021 Nov 17. 11(1): 195

NME6 is a phosphotransfer-inactive, monomeric NME/NDPK family member and functions in complexes at the interface of mitochondrial inner membrane and matrix.

Bastien Proust, Martina Radić, Nikolina Škrobot Vidaček, Cécile Cottet, Stéphane Attia, Frédéric Lamarche, Lucija Ačkar, Vlatka Godinić Mikulčić, Malgorzata Tokarska-Schlattner, Helena Ćetković, Uwe Schlattner, Maja Herak Bosnar.

  BACKGROUND: NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6.RESULTS: We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation.
CONCLUSIONS: NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.

Keywords:  Mitochondria; NDP kinase; NME; RCC1L; WBSCR16; nm23

DOI:  https://doi.org/10.1186/s13578-021-00707-0
EMBO J. 2021 Nov 17. e109519

Widespread use of unconventional targeting signals in mitochondrial ribosome proteins.

Yury S Bykov, Tamara Flohr, Felix Boos, Naama Zung, Johannes M Herrmann, Maya Schuldiner.

  Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Proteomic and structural data as well as computational predictions indicate that up to 25% of yeast MRPs do not have a conventional N-terminal mitochondrial targeting signal (MTS). We experimentally characterized a set of 15 yeast MRPs in vivo and found that five use internal MTSs. Further analysis of a conserved model MRP, Mrp17/bS6m, revealed the identity of the internal targeting signal. Similar to conventional MTS-containing proteins, the internal sequence mediates binding to TOM complexes. The entire sequence of Mrp17 contains positive charges mediating translocation. The fact that these sequence properties could not be reliably predicted by standard methods shows that mitochondrial protein targeting is more versatile than expected. We hypothesize that structural constraints imposed by ribosome assembly interfaces may have disfavored N-terminal presequences and driven the evolution of internal targeting signals in MRPs.

Keywords:  mitochondria; mitochondrial ribosome; mitochondrial targeting signal; targeting; translocation

DOI:  https://doi.org/10.15252/embj.2021109519
Nat Commun. 2021 Nov 18. 12(1): 6743

Mapping protein carboxymethylation sites provides insights into their role in proteostasis and cell proliferation.

Simone Di Sanzo, Katrin Spengler, Anja Leheis, Joanna M Kirkpatrick, Theresa L Rändler, Tim Baldensperger, Therese Dau, Christian Henning, Luca Parca, Christian Marx, Zhao-Qi Wang, Marcus A Glomb, Alessandro Ori, Regine Heller.

Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devise a proteomic strategy to identify sites of carboxymethyllysine modification, one of the most abundant advanced glycation end products. We identify over 1000 sites of protein carboxymethylation in mouse and primary human cells treated with the glycating agent glyoxal. By using quantitative proteomics, we find that protein glycation triggers a proteotoxic response and indirectly affects the protein degradation machinery. In primary endothelial cells, we show that glyoxal induces cell cycle perturbation and that carboxymethyllysine modification reduces acetylation of tubulins and impairs microtubule dynamics. Our data demonstrate the relevance of carboxymethyllysine modification for cellular function and pinpoint specific protein networks that might become compromised during aging.

DOI: https://doi.org/10.1038/s41467-021-26982-6
Br J Pharmacol. 2021 Nov 16.

A novel HSF1 activator ameliorates nonalcoholic steatohepatitis by stimulating mitochondrial adaptive oxidation.

Yong Rao, Chan Li, Yu-Tao Hu, Yao-Hao Xu, Bing-Bing Song, Shi-Yao Guo, Zhi Jiang, Dan-Dan Zhao, Shuo-Bin Chen, Jia-Heng Tan, Shi-Liang Huang, Qing-Jiang Li, Xiao-Jun Wang, Ying-Jun Zhang, Ji-Ming Ye, Zhi-Shu Huang.

  BACKGROUND AND PURPOSE: Nonalcoholic steatohepatitis (NASH) is the more severe form of metabolic associated fatty liver disease (MAFLD), and no pharmacologic treatment approved as yet. Identification of novel therapeutic targets and their agents are critical to overcome the current inadequacy of drug treatment for NASH.EXPERIMENTAL APPROACH: The correlation between heat shock factor 1 (HSF1) levels and the development of NASH and the target genes of HSF1 in hepatocyte were revealed by chromatin-immunoprecipitation sequencing. The effects and mechanisms of SYSU-3d in alleviating NASH were examined in relevant cell models and mouse models (the Ob/Ob mice, high-fat and high-cholesterol diet, the methionine-choline deficient diet fed mice). The drug-like properties of SYSU-3d in vivo were evaluated.
KEY RESULTS: HSF1 is progressively reduced with mitochondrial dysfunction in NASH pathogenesis and activation of this transcription factor by its newly-identified activator SYSU-3d efficiently ameliorated all manifestations of NASH in mice. When activated, the phosphorylated HSF1 (Ser326) translocated to nucleus and bound to the promoter of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) to induce mitochondrial biogenesis, thus increasing mitochondrial adaptive oxidation and inhibiting oxidative stress. The deletion of HSF1 and PGC-1α or recovery of HSF1 in HSF1-deficiency cells revealed the HSF1/PGC-1α metabolic axis mainly responsible for the anti-NASH effects of SYSU-3d independent of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK).
CONCLUSION AND IMPLICATIONS: Activation of HSF1 is a practicable therapeutic approach for NASH treatment via the HSF1/PGC-1α/mitochondrial axis, and SYSU-3d would take into consideration as a potential candidate for the treatment of NASH.

Keywords:  HSF1 activation; PGC-1α; activator; mitochondrial adaptive oxidation; non-alcoholic steatohepatitis

DOI:  https://doi.org/10.1111/bph.15727
J Biosci. 2021 ;pii: 99. [Epub ahead of print]46

Putative role of uncoupling proteins in mitochondria-nucleus communications and DNA damage response.

Yahan Niu, Chang Liu, Rui Zhang, Ying Jing, Donghai Li.

Mitochondria-nucleus communications and DNA damage response (DDR) play roles in cellular stress and closely associate with a range of diseases. Mitochondrial uncoupling proteins (UCPs) are capable of uncoupling mitochondrial oxidative phosphorylation and protecting against oxidative stress. However, the potential role of UCPs in DDR and DDR-related mitochondria-nucleus communications remains unknown. The review deduces UCPs functions in mitochondria-nucleus communications implicated in metabolite regulation (e.g., reactive oxygen species) and Ca2+ signaling, and in DDR (e.g., base excision repair, double-strand DNA break repair, mitophagy and nuclear DNA degradation). Represented are shared microRNAs that regulate UCPs and DDR. It would provide novel insight into UCPs-mediated mitochondria-nucleus communications and DDR, and potentially promote drug target identification, drug discovery and clinical therapy of DDR-related diseases.
Biochem Biophys Rep. 2021 Dec;28 101166

Accumulation of cholesterol suppresses oxidative phosphorylation and altered responses to inflammatory stimuli of macrophages.

Yi-Chou Chiu, Pei-Wen Chu, Hua-Ching Lin, Shau-Kwaun Chen.

  Hypercholesterolemia induces intracellular accumulation of cholesterol in macrophages and other immune cells, causing immunological dysfunctions. On cellular levels, cholesterol enrichment might lead to mitochondrial metabolic reprogramming and change macrophage functions. Additionally, as cholesterol is permeable to the plasma membrane and might integrate into the membranous organelles, such as endoplasmic reticulum or mitochondria, cholesterol enrichment might change the functions or properties of these organelles, and ultimately alters the cellular functions. In this study, we investigate the mitochondrial alterations and intracellular oxidative stress induced by accumulation of cholesterol in the macrophages, and the possible immunological impacts caused by these alterations. Macrophage cells RAW264.7 were treated with cholesterol to induce intracellular accumulation of cholesterol, which further triggered the reduced production of reactive oxygen/nitrogen species, as well as decrease of oxidative phosphorylation. Basal respiration rate, ATP production and non-mitochondrial oxygen consumption are all suppressed. In contrast, glycolysis remained unaltered in this cholesterol-enriched condition. Previous studies demonstrated that metabolic profiles are associated with macrophage polarization. We further verified whether this metabolic reprogramming influences the macrophage responses to pro-inflammatory or anti-inflammatory stimuli. Our results showed the changes of transcriptional regulations in both pro-inflammatory and anti-inflammatory genes, but not specific toward M1 or M2 polarization. Collectively, the accumulation of cholesterol induced mitochondrial metabolic reprogramming and suppressed the production of oxidative stress, and induced the alterations of macrophage functions.

Keywords:  Cholesterol; Inflammatory response; Macrophage; Oxidative phosphorylation; Oxidative stress

DOI:  https://doi.org/10.1016/j.bbrep.2021.101166
Front Mol Biosci. 2021 ;8 768888

Repair or Degrade: the Thermodynamic Dilemma of Cellular Protein Quality-Control.

Bruno Fauvet, Mathieu E Rebeaud, Satyam Tiwari, Paolo De Los Rios, Pierre Goloubinoff.

  Life is a non-equilibrium phenomenon. Owing to their high free energy content, the macromolecules of life tend to spontaneously react with ambient oxygen and water and turn into more stable inorganic molecules. A similar thermodynamic picture applies to the complex shapes of proteins: While a polypeptide is emerging unfolded from the ribosome, it may spontaneously acquire secondary structures and collapse into its functional native conformation. The spontaneity of this process is evidence that the free energy of the unstructured state is higher than that of the structured native state. Yet, under stress or because of mutations, complex polypeptides may fail to reach their native conformation and form instead thermodynamically stable aggregates devoid of biological activity. Cells have evolved molecular chaperones to actively counteract the misfolding of stress-labile proteins dictated by equilibrium thermodynamics. HSP60, HSP70 and HSP100 can inject energy from ATP hydrolysis into the forceful unfolding of stable misfolded structures in proteins and convert them into unstable intermediates that can collapse into the native state, even under conditions inauspicious for that state. Aggregates and misfolded proteins may also be forcefully unfolded and degraded by chaperone-gated endo-cellular proteases, and in eukaryotes also by chaperone-mediated autophagy, paving the way for their replacement by new, unaltered functional proteins. The greater energy cost of degrading and replacing a polypeptide, with respect to the cost of its chaperone-mediated repair represents a thermodynamic dilemma: some easily repairable proteins are better to be processed by chaperones, while it can be wasteful to uselessly try recover overly compromised molecules, which should instead be degraded and replaced. Evolution has solved this conundrum by creating a host of unfolding chaperones and degradation machines and by tuning their cellular amounts and activity rates.

Keywords:  chaperones; protein degradation; protein repair; proteostasis; thermodynamics

DOI:  https://doi.org/10.3389/fmolb.2021.768888
Nature. 2021 Nov 18.

CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.

Lingyun Long, Jun Wei, Seon Ah Lim, Jana L Raynor, Hao Shi, Jon P Connelly, Hong Wang, Cliff Guy, Boer Xie, Nicole M Chapman, Guotong Fu, Yanyan Wang, Hongling Huang, Wei Su, Jordy Saravia, Isabel Risch, Yong-Dong Wang, Yuxin Li, Mingming Niu, Yogesh Dhungana, Anil Kc, Peipei Zhou, Peter Vogel, Jiyang Yu, Shondra M Pruett-Miller, Junmin Peng, Hongbo Chi.

Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2-4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.

DOI: https://doi.org/10.1038/s41586-021-04109-7
J Biol Chem. 2021 Nov 12. pii: S0021-9258(21)01214-X. [Epub ahead of print] 101407

Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease.

Kristin L Zuromski, Sora Kim, Robert T Sauer, Tania A Baker.

  ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the AAA+ (ATPases associated with diverse cellular activities) superfamily and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adaptor, ClpS, that modifies ClpA's substrate profile towards N-degron substrates. Conserved tyrosines in the twelve pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adaptor-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each AAA+ ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to "tug on" and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines.

Keywords:  ATPases associated with diverse cellular activities (AAA); cysteine-mediated cross-linking; enzyme mechanism; enzyme mutation; protein degradation; structure-function

DOI:  https://doi.org/10.1016/j.jbc.2021.101407
Front Immunol. 2021 ;12 735866

Constitutive IFNα Protein Production in Bats.

Vincent Bondet, Maxime Le Baut, Sophie Le Poder, Alexis Lécu, Thierry Petit, Rudy Wedlarski, Darragh Duffy, Delphine Le Roux.

  Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this symbiotic relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this, we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFNα protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but were not associated with age, sex, or health status suggesting constitutive IFNα protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.

Keywords:  Chiroptera; Simoa digital ELISA; antiviral immunity; bats; protein levels; type I IFN

DOI:  https://doi.org/10.3389/fimmu.2021.735866
World J Stem Cells. 2021 Oct 26. 13(10): 1595-1609

Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells.

Francesco Bellanti, Giorgia di Bello, Rosanna Tamborra, Marco Amatruda, Aurelio Lo Buglio, Michał Dobrakowski, Aleksandra Kasperczyk, Sławomir Kasperczyk, Gaetano Serviddio, Gianluigi Vendemiale.

  BACKGROUND: Senescence is characterized by a decline in hepatocyte function, with impairment of metabolism and regenerative capacity. Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism, liver diseases, and organ regeneration. The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors. However, the impact of senescence on HepaRG cells is not yet known.AIM: To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells.
METHODS: We compared the transdifferentiation capacity of cells over 10 (passage 10 [P10]) vs P20. Aging was evaluated by senescence-associated (SA) beta-galactosidase activity and the comet assay. HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry (expression of cluster of differentiation 49a [CD49a], CD49f, CD184, epithelial cell adhesion molecule [EpCAM], and cytokeratin 19 [CK19]), quantitative PCR analysis (expression of albumin, cytochrome P450 3A4 [CYP3A4], γ-glutamyl transpeptidase [γ-GT], and carcinoembryonic antigen [CEA]), and functional analyses (albumin secretion, CYP3A4, and γ-GT). Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide (NAD+)/NAD with hydrogen (NADH) content were also measured.
RESULTS: SA β-galactosidase staining was higher in P20 than P10 HepaRG cells; in parallel, the comet assay showed consistent DNA damage in P20 HepaRG cells. With respect to P10, P20 HepaRG cells exhibited a reduction of CD49a, CD49f, CD184, EpCAM, and CK19 after the induction of transdifferentiation. Furthermore, lower gene expression of albumin, CYP3A4, and γ-GT, as well as reduced albumin secretion capacity, CYP3A4, and γ-GT activity were reported in transdifferentiated P20 compared to P10 cells. By contrast, the gene expression level of CEA was not reduced by transdifferentiation in P20 cells. Of note, both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells. Finally, both ATP and NAD+/NADH were depleted in P20 cells with respect to P10 cells.
CONCLUSION: SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells, with consequent impairment of metabolic and regenerative properties, which may alter applications in basic studies.

Keywords:  HepaRG cells; Mitochondria; Nicotinamide adenine dinucleotide; Regeneration; Senescence; Transdifferentiation

DOI:  https://doi.org/10.4252/wjsc.v13.i10.1595
DNA Repair (Amst). 2021 Nov 02. pii: S1568-7864(21)00201-9. [Epub ahead of print]108 103245

MicroRNAs and the DNA damage response: How is cell fate determined?

Hartwig Visser, Adam D Thomas.

  It is becoming clear that the DNA damage response orchestrates an appropriate response to a given level of DNA damage, whether that is cell cycle arrest and repair, senescence or apoptosis. It is plausible that the alternative regulation of the DNA damage response (DDR) plays a role in deciding cell fate following damage. MicroRNAs (miRNAs) are associated with the transcriptional regulation of many cellular processes. They have diverse functions, affecting, presumably, all aspects of cell biology. Many have been shown to be DNA damage inducible and it is conceivable that miRNA species play a role in deciding cell fate following DNA damage by regulating the expression and activation of key DDR proteins. From a clinical perspective, miRNAs are attractive targets to improve cancer patient outcomes to DNA-damaging chemotherapy. However, cancer tissue is known to be, or to become, well adapted to DNA damage as a means of inducing chemoresistance. This frequently results from an altered DDR, possibly owing to miRNA dysregulation. Though many studies provide an overview of miRNAs that are dysregulated within cancerous tissues, a tangible, functional association is often lacking. While miRNAs are well-documented in 'ectopic biology', the physiological significance of endogenous miRNAs in the context of the DDR requires clarification. This review discusses miRNAs of biological relevance and their role in DNA damage response by potentially 'fine-tuning' the DDR towards a particular cell fate in response to DNA damage. MiRNAs are thus potential therapeutic targets/strategies to limit chemoresistance, or improve chemotherapeutic efficacy.

Keywords:  ATM; Apoptosis; Cell fate; DNA damage response; MiRNA; P53

DOI:  https://doi.org/10.1016/j.dnarep.2021.103245
Nat Plants. 2021 Nov;7(11): 1505-1515

The cryo-EM structure of the chloroplast ClpP complex.

Ning Wang, Yifan Wang, Qian Zhao, Xiang Zhang, Chao Peng, Wenjuan Zhang, Yanan Liu, Olivier Vallon, Michael Schroda, Yao Cong, Cuimin Liu.

Protein homoeostasis in plastids is strategically regulated by the protein quality control system involving multiple chaperones and proteases, among them the Clp protease. Here, we determined the structure of the chloroplast ClpP complex from Chlamydomonas reinhardtii by cryo-electron microscopy. ClpP contains two heptameric catalytic rings without any symmetry. The top ring contains one ClpR6, three ClpP4 and three ClpP5 subunits while the bottom ring is composed of three ClpP1C subunits and one each of the ClpR1-4 subunits. ClpR3, ClpR4 and ClpT4 subunits connect the two rings and stabilize the complex. The chloroplast Cpn11/20/23 co-chaperonin, a co-factor of Cpn60, forms a cap on the top of ClpP by protruding mobile loops into hydrophobic clefts at the surface of the top ring. The co-chaperonin repressed ClpP proteolytic activity in vitro. By regulating Cpn60 chaperone and ClpP protease activity, the co-chaperonin may play a role in coordinating protein folding and degradation in the chloroplast.

DOI: https://doi.org/10.1038/s41477-021-01020-x