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Jiang, H., Xue, X., Panda, S., Kawale, A., Hooy, R. M., Liang, F., . . . Gekara, N. O. (2019). Chromatin-bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death. EMBO Journal, 38(21), Article ID e102718.
Open this publication in new window or tab >>Chromatin-bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death
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2019 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 38, no 21, article id e102718Article in journal (Refereed) Published
Abstract [en]

DNA repair via homologous recombination (HR) is indispensable for genome integrity and cell survival but if unrestrained can result in undesired chromosomal rearrangements. The regulatory mechanisms of HR are not fully understood. Cyclic GMP‐AMP synthase (cGAS) is best known as a cytosolic innate immune sensor critical for the outcome of infections, inflammatory diseases, and cancer. Here, we report that cGAS is primarily a chromatin‐bound protein that inhibits DNA repair by HR, thereby accelerating genome destabilization, micronucleus generation, and cell death under conditions of genomic stress. This function is independent of the canonical STING‐dependent innate immune activation and is physiologically relevant for irradiation‐induced depletion of bone marrow cells in mice. Mechanistically, we demonstrate that inhibition of HR repair by cGAS is linked to its ability to self‐oligomerize, causing compaction of bound template dsDNA into a higher‐ordered state less amenable to strand invasion by RAD51‐coated ssDNA filaments. This previously unknown role of cGAS has implications for understanding its involvement in genome instability‐associated disorders including cancer.

Place, publisher, year, edition, pages
EMBOpress, 2019
Keywords
cancer, cell death, cGAS, chromatin compaction, DNA repair
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164894 (URN)10.15252/embj.2019102718 (DOI)000487392000001 ()31544964 (PubMedID)
Funder
Swedish Research Council, 2015-02857Swedish Research Council, 2016-00890Swedish Cancer Society, CAN 2017/421NIH (National Institute of Health), R01 CA220123NIH (National Institute of Health), P30 CA054174NIH (National Institute of Health), R01 GM GM 129342-01-A1
Available from: 2019-11-06 Created: 2019-11-06 Last updated: 2019-11-26Bibliographically approved
Jiang, H., Panda, S. & Gekara, N. O. (2019). Comet and micronucleus assays for analyzing DNA damage and genome integrity. In: Sohn, J (Ed.), DNA SENSORS AND INFLAMMASOMES: (pp. 299-307). ELSEVIER ACADEMIC PRESS INC
Open this publication in new window or tab >>Comet and micronucleus assays for analyzing DNA damage and genome integrity
2019 (English)In: DNA SENSORS AND INFLAMMASOMES / [ed] Sohn, J, ELSEVIER ACADEMIC PRESS INC , 2019, p. 299-307Chapter in book (Refereed)
Abstract [en]

Detection of DNA damage in cells is fundamental for the study of DNA repair and genome-instability associated processes including carcinogenesis. Many studies often rely on cytotoxicity assays to estimate genotoxicity. However, measurements of cytotoxicity, a delayed outcome requiring high threshold genotoxicity to induce, does not provide information about the subtle, early genotoxic effects relevant for mechanistic understanding of DNA repair processes. Here describe how to combine two simple procedures for monitoring the presence of DNA damage in individual eukaryotic cells using: (1) the Comet assay for measuring initial DNA breaks and (2) the Micronucleus assay for detecting delayed outcome DNA breaks in dividing cells. We discuss the principles, experimental design considerations and troubleshooting tips for optimizing these methods. They require standard molecular biology instruments and a fluorescent microscope.

Place, publisher, year, edition, pages
ELSEVIER ACADEMIC PRESS INC, 2019
Series
Methods in Enzymology, ISSN 0076-6879 ; 625
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164490 (URN)10.1016/bs.mie.2019.05.015 (DOI)000488782900019 ()31455533 (PubMedID)978-0-12-818360-1 (ISBN)978-0-12-818359-5 (ISBN)
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-10-25Bibliographically approved
Panda, S., Jiang, H. & Gekara, N. O. (2019). TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes. In: Sohn, J (Ed.), DNA SENSORS AND INFLAMMASOMES: (pp. 339-350). ELSEVIER ACADEMIC PRESS INC
Open this publication in new window or tab >>TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes
2019 (English)In: DNA SENSORS AND INFLAMMASOMES / [ed] Sohn, J, ELSEVIER ACADEMIC PRESS INC , 2019, p. 339-350Chapter in book (Refereed)
Abstract [en]

Ubiquitination is a reversible posttranslational modification that regulates nearly all cellular processes. The ubiquitin polypeptide is conjugated via its C-terminus to amine groups of lysine residues on target protein. Additionally, ubiquitins moieties can be conjugated in tandem to the initial ubiquitin via any of its internal lysine residues or N terminal methionine residue, resulting in the formation of polyubiquitin chains with distinct biophysical properties and biological functions. Elucidating the types of polyubiquitin chains present in proteins is essential for understanding their function and mechanism of regulation. Traditionally, ubiqutin modifications have been elucidated by exogenously co-expressing proteins of interest with epitope-tagged ubiquitins mutated in specific lysine residues. However, this strategy is prone experimental artifacts. In this protocol, we describe how to elucidate endogenous ubiquitin modifications. This procedure combines TUBE (Tandem Ubiquitin Binding Entity)-based isolation of ubiquitin conjugates, digestion with linkage specific deubiquitinases and immunoblotting. This procedure is very robust can be applied to profile types and architectural organization polyubiquitin chains present on the any proteins of interest and has been instrumental in elucidating ubiquitin modifications in NOD2 signaling in our recent study (Panda & Gekara, 2018).

Place, publisher, year, edition, pages
ELSEVIER ACADEMIC PRESS INC, 2019
Series
Methods in Enzymology, ISSN 0076-6879 ; 625
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164489 (URN)10.1016/bs.mie.2019.05.006 (DOI)000488782900021 ()31455535 (PubMedID)978-0-12-818360-1 (ISBN)978-0-12-818359-5 (ISBN)
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-10-25Bibliographically approved
Panda, S. & Gekara, N. O. (2018). The deubiquitinase MYSM1 dampens NOD2-mediated inflammation and tissue damage by inactivating the RIP2 complex. Nature Communications, 9, Article ID 4654.
Open this publication in new window or tab >>The deubiquitinase MYSM1 dampens NOD2-mediated inflammation and tissue damage by inactivating the RIP2 complex
2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 4654Article in journal (Refereed) Published
Abstract [en]

NOD2 is essential for antimicrobial innate immunity and tissue homeostasis, but require tight regulation to avert pathology. A focal point of NOD2 signaling is RIP2, which upon polyubiquitination nucleates the NOD2:RIP2 complex, enabling signaling events leading to inflammation, yet the precise nature and the regulation of the polyubiquitins coordinating this process remain unclear. Here we show that NOD2 signaling involves conjugation of RIP2 with lysine 63 (K63), K48 and M1 polyubiquitin chains, as well as with non-canonical K27 chains. In addition, we identify MYSM1 as a proximal deubiquitinase that attenuates NOD2:RIP2 complex assembly by selectively removing the K63, K27 and M1 chains, but sparing the K48 chains. Consequently, MYSM1 deficient mice have unrestrained NOD2-mediated peritonitis, systemic inflammation and liver injury. This study provides a complete overview of the polyubiquitins in NOD2:RIP2 signaling and reveal MYSM1 as a central negative regulator restricting these polyubiquitins to prevent excessive inflammation.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Basic Medicine Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-153237 (URN)10.1038/s41467-018-07016-0 (DOI)000449363900003 ()30405132 (PubMedID)
Funder
Swedish Research Council, 2015-02857Swedish Research Council, 2016-00890Swedish Cancer Society, CAN 2017/421
Available from: 2018-11-12 Created: 2018-11-12 Last updated: 2018-12-11Bibliographically approved
Panda, S. (2018). The role and mechanism of ubiquitin system in innate immune regulation. (Doctoral dissertation). Umeå: Umeå University
Open this publication in new window or tab >>The role and mechanism of ubiquitin system in innate immune regulation
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pattern-recognition receptors (PRRs) include the cell surface or endosomal membrane localized Toll-like receptors (TLRs) and the cytoplasmic PRRs such as RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and cytoplasmic DNA receptors (CDRs). Triggering of PRRs culminates in the transcriptional induction of pro-inflammatory cytokines and type I interferons (IFNs) that coordinate protection against pathogens but require tight control to avert inflammatory diseases. The mechanisms underlying this strict regulation are unclear.

Ubiquitiation is a reversible post-translational modification that controls nearly all cellular processes including the immune system. We identified, H2A deubiquitinase myb-like SWIM and MPN domains 1 (MYSM1) a previously described as a key component of epigenetic signaling machinery as a key negative regulator of the innate immune system that guards against an overzealous self-destructive immune response. In response to microbial stimuli, MYSM1 accumulated in the cytoplasm where it interacted with and inactivated TRAF3 and TRAF6 complexes to terminate TLR, RLR and CDR pathways for pro-inflammatory and type I interferone responses. Consequently, MYSM1 deficiency in mice resulted in hyper-inflammation and enhanced viral clearance but also susceptibility to septic shock.

NOD2, belonging to the intracellular NLR family. A focal point of NOD2 signalling is RIP2, which upon polyubiquitination nucleates the NOD2:RIP2 complex, enabling signaling events leading to inflammation, yet the precise nature and the regulation of the polyubiquitins coordinating this process remains unclear. We show that NOD2 signaling involves conjugation of RIP2 with K63, K48 and M1 polyubiquitin chains as well as with non-canonical K27 chains. Furthermore, we identify MYSM1 as the proximal deubiquitinase that attenuates NOD2- RIP2 complex assembly by selectively removing the K63, M1 and K27 chains. Consequently, MYSM1 deficient mice have unrestrained NOD2-mediated peritonitis and liver injury. Henceforth, this study provide a complete description of the polyubiquitin modifications in the NOD2:RIP2 signalling and reveal MYSM1 as a central negative regulator that prevents excessive inflammation.

In order to overcome the host barrier to infection, some pathogens elude the immune defense by hijacking the ubiquitin system. Francisella tularensis is one of the most infectious bacteria. It employs several mechanisms to evade detection by the innate immune system, but how remains obscure. Here, we showed that Francisella triggers but concomitantly inhibits the TLRs, RLRs and CDRs pathway by inhibiting K63-linked polyubiquitination and assembly of TRAF6 and TRAF3 complexes that control the transcriptional responses of PRRs.

In summary, my work identify MYSM1 as a key negative regulator of the innate immune system. Although, mainly located in the nucleus MYSM1 rapidly amass in the cytoplasm, where it interacts with and inactivates the key PRR signalling complexes. Afterward, MYSM1 undergoes proteaosomal degradation to avert sustained immune suppression. Thus, MYSM1 is part of a highly versatile negative feedback regulatory mechanism, which in response to biological danger is swiftly activated in “on-and-off” manner to restore immune homeostasis. Furthermore, Francisella targets the ubiquitin system to inhibt multiple PRRs hence allowing this bacterium to invade and proliferate in the host without evoking a self-limiting innate immune response.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 54
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1998
Keywords
The ubiquitin system, Deubiquitinase, MYSM1, Innate immune regulation Toll-like receptor, RIG-l like receptors, Cytoplasmic DNA receptors, NOD-like receptors, Francisella tularensis, Immune subversion
National Category
Immunology in the medical area Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-153987 (URN)978-91-7601-988-7 (ISBN)
Public defence
2019-01-25, Föreläsningssal A unod T9, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-12-17 Created: 2018-12-11 Last updated: 2018-12-11Bibliographically approved
Putzova, D., Panda, S., Härtlova, A., Stulík, J. & Gekara, N. O. (2017). Subversion of innate immune responses by Francisella involves the disruption of TRAF3 and TRAF6 signalling complexes. Cellular Microbiology, 19(11), Article ID e12769.
Open this publication in new window or tab >>Subversion of innate immune responses by Francisella involves the disruption of TRAF3 and TRAF6 signalling complexes
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2017 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 19, no 11, article id e12769Article in journal (Refereed) Published
Abstract [en]

The success of pathogens depends on their ability to circumvent immune defences. Francisella tularensis is one of the most infectious bacteria known. The remarkable virulence of Francisella is believed to be due to its capacity to evade or subvert the immune system, but how remains obscure. Here, we show that Francisella triggers but concomitantly inhibits the Toll-like receptor, RIG-I-like receptor, and cytoplasmic DNA pathways. Francisella subverts these pathways at least in part by inhibiting K63-linked polyubiquitination and assembly of TRAF6 and TRAF3 complexes that control the transcriptional responses of pattern recognition receptors. We show that this mode of inhibition requires a functional type VI secretion system and/or the presence of live bacteria in the cytoplasm. The ability of Francisella to enter the cytosol while simultaneously inhibiting multiple pattern recognition receptor pathways may account for the notable capacity of this bacterium to invade and proliferate in the host without evoking a self-limiting innate immune response.

Place, publisher, year, edition, pages
Hoboken: Wiley-Blackwell, 2017
Keywords
inflammatory responses, listeria monocytogenes, murine macrophages, tularensis lvs, cytosolic dna, in vitro, system, infection, activation, trif
National Category
Cell and Molecular Biology
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-139636 (URN)10.1111/cmi.12769 (DOI)000412834200008 ()28745813 (PubMedID)
Funder
Swedish Research Council, 2013-8621Swedish Research Council, 2015-02857
Available from: 2017-09-19 Created: 2017-09-19 Last updated: 2019-03-15Bibliographically approved
Panda, S., Nilsson, J. A. & Gekara, N. O. (2015). Deubiquitinase MYSM1 Regulates Innate Immunity through Inactivation of TRAF3 and TRAF6 Complexes. Immunity, 43(4), 647-659
Open this publication in new window or tab >>Deubiquitinase MYSM1 Regulates Innate Immunity through Inactivation of TRAF3 and TRAF6 Complexes
2015 (English)In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 43, no 4, p. 647-659Article in journal (Refereed) Published
Abstract [en]

Pattern-recognition receptors (PRRs) including Toll-like receptors, RIG-I-like receptors, and cytoplasmic DNA receptors are essential for protection against pathogens but require tight control to avert inflammatory diseases. The mechanisms underlying this strict regulation are unclear. MYSM1 was previously described as a key component of epigenetic signaling machinery. We found that in response to microbial stimuli, MYSM1 accumulated in the cytoplasm where it interacted with and inactivated TRAF3 and TRAF6 complexes to terminate PRR pathways for pro-inflammatory and type I interferon responses. Consequently, Mysm1 deficiency in mice resulted in hyper-inflammation and enhanced viral clearance but also susceptibility to septic shock. We identified two motifs in MYSM1 that were essential for innate immune suppression: the SWIRM domain that interacted with TRAF3 and TRAF6 and the metalloproteinase domain that removed K63 polyubiquitins. This study identifies MYSM1 as a key negative regulator of the innate immune system that guards against an overzealous self-destructive immune response.

Place, publisher, year, edition, pages
Elsevier, 2015
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-111482 (URN)10.1016/j.immuni.2015.09.010 (DOI)000363478700009 ()26474655 (PubMedID)
Available from: 2015-12-08 Created: 2015-11-13 Last updated: 2018-12-11Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-7622-5116

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