bims-tunefa Biomed News
on Tumor necrosis factor superfamily and post-translational modifications
Issue of 2020‒09‒27
fifteen papers selected by
John Silke
Walter and Eliza Hall Institute of Medical Research

  1. Nature. 2020 Sep 24.
    Gitlin AD, Heger K, Schubert AF, Reja R, Yan D, Pham VC, Suto E, Zhang J, Kwon YC, Freund EC, Kang J, Pham A, Caothien R, Bacarro N, Hinkle T, Xu M, McKenzie BS, Haley B, Lee WP, Lill JR, Roose-Girma M, Dohse M, Webster JD, Newton K, Dixit VM.
      Mutations in the death receptor Fas1,2 or its ligand FasL3 cause autoimmune lymphoproliferative syndrome (ALPS), whereas mutations in caspase-8 or its adaptor FADD - which mediate cell death downstream of Fas/FasL - cause severe immunodeficiency in addition to ALPS4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms underlying immunodeficiency remain undefined. Here, we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased production of select cytokines upon Toll-like receptor (TLR) 1/2, TLR7, or TLR9 stimulation, but not upon TLR3 or TLR4 engagement. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, impaired TLR3 and TLR4 cytokine responses of caspase-8-deficient macrophages13 were largely rescued by co-deletion of N4BP1. Thus, persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumor necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by FADD-caspase-8 mutations.
  2. Crit Rev Biochem Mol Biol. 2020 Sep 23. 1-14
    Sanderson DJ, Cohen MS.
      Poly-(ADP)-ribose polymerases (PARPs) are a family of 17 enzymes in humans that have diverse roles in cell physiology including DNA damage repair, transcription, innate immunity, and regulation of signaling pathways. The modular domain architecture of PARPs gives rise to this functional diversity. PARPs catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to targets-proteins and poly-nucleic acids. This enigmatic post-translational modification comes in two varieties: the transfer of a single unit of ADP-ribose, known as mono-ADP-ribosylation (MARylation) or the transfer of multiple units of ADP-ribose, known as poly-ADP-ribosylation (PARylation). Emerging data shows that PARPs are regulated at multiple levels to control when and where PARP-mediated M/PARylation occurs in cells. In this review, we will discuss the latest knowledge regarding the regulation of PARPs in cells: from transcription and protein stability to subcellular localization and modulation of catalytic activity.
    Keywords:  ADP-ribosylation; MARylation; PARylation; allosteric activation; Poly(ADP)-ribose polymerase; nicotinamide adenine dinucleotide (NAD+)
  3. EMBO Rep. 2020 Sep 21. e50400
    Heim VJ, Dagley LF, Stafford CA, Hansen FM, Clayer E, Bankovacki A, Webb AI, Lucet IS, Silke J, Nachbur U.
      Signaling via the intracellular pathogen receptors nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2 requires receptor interacting kinase 2 (RIPK2), an adaptor kinase that can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG-tagged RIPK2 knock-in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice, we were able to generate a detailed map of post-translational modifications on RIPK2. Similar to other reports, we did not detect ubiquitination of RIPK2 lysine 209 during NOD2 signaling. However, using site-directed mutagenesis we identified a new regulatory region on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP and downstream signaling outcomes.
    Keywords:   XIAP ; NOD signaling; RIPK2; inflammation; ubiquitin
  4. Signal Transduct Target Ther. 2020 Sep 21. 5(1): 209
    Yu H, Lin L, Zhang Z, Zhang H, Hu H.
      NF-κB pathway consists of canonical and non-canonical pathways. The canonical NF-κB is activated by various stimuli, transducing a quick but transient transcriptional activity, to regulate the expression of various proinflammatory genes and also serve as the critical mediator for inflammatory response. Meanwhile, the activation of the non-canonical NF-κB pathway occurs through a handful of TNF receptor superfamily members. Since the activation of this pathway involves protein synthesis, the kinetics of non-canonical NF-κB activation is slow but persistent, in concordance with its biological functions in the development of immune cell and lymphoid organ, immune homeostasis and immune response. The activation of the canonical and non-canonical NF-κB pathway is tightly controlled, highlighting the vital roles of ubiquitination in these pathways. Emerging studies indicate that dysregulated NF-κB activity causes inflammation-related diseases as well as cancers, and NF-κB has been long proposed as the potential target for therapy of diseases. This review attempts to summarize our current knowledge and updates on the mechanisms of NF-κB pathway regulation and the potential therapeutic application of inhibition of NF-κB signaling in cancer and inflammatory diseases.
  5. Chem Sci. 2020 Jun 21. 11(23): 6058-6069
    Rut W, Zmudzinski M, Snipas SJ, Bekes M, Huang TT, Drag M.
      Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs-MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell lysate experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.
  6. J Immunol. 2020 Sep 25. pii: ji1901311. [Epub ahead of print]
    Jasenosky LD, Nambu A, Tsytsykova AV, Ranjbar S, Haridas V, Kruidenier L, Tough DF, Goldfeld AE.
      The human TNF/LT locus genes TNF, LTA, and LTB are expressed in a cell type-specific manner. In this study, we show that a highly conserved NFAT binding site within the distal noncoding element hHS-8 coordinately controls TNF and LTA gene expression in human T cells. Upon activation of primary human CD4+ T cells, hHS-8 and the TNF and LTA promoters display increased H3K27 acetylation and nuclease sensitivity and coordinate induction of TNF, LTA, and hHS-8 enhancer RNA transcription occurs. Functional analyses using CRISPR/dead(d)Cas9 targeting of the hHS-8-NFAT site in the human T cell line CEM demonstrate significant reduction of TNF and LTA mRNA synthesis and of RNA polymerase II recruitment to their promoters. These studies elucidate how a distal element regulates the inducible cell type-specific gene expression program of the human TNF/LT locus and provide an approach for modulation of TNF and LTA transcription in human disease using CRISPR/dCas9.
  7. Mol Cell Proteomics. 2020 Sep 21. pii: mcp.RA120.002290. [Epub ahead of print]
    Nie L, Wang C, Li N, Feng X, Lee N, Su D, Tang M, Yao F, Chen J.
      Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the β-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies.
    Keywords:  Cancer Biology*; E3 ubiquitin ligase; Label-free quantification; Mass Spectrometry; Protein Degradation*; RNF146; Substrate identification; TNKS; Ubiquitin; Ubiquitinases
  8. Nat Metab. 2020 Sep 21.
    de Kivit S, Mensink M, Hoekstra AT, Berlin I, Derks RJE, Both D, Aslam MA, Amsen D, Berkers CR, Borst J.
      Following activation, conventional T (Tconv) cells undergo an mTOR-driven glycolytic switch. Regulatory T (Treg) cells reportedly repress the mTOR pathway and avoid glycolysis. However, here we demonstrate that human thymus-derived Treg (tTreg) cells can become glycolytic in response to tumour necrosis factor receptor 2 (TNFR2) costimulation. This costimulus increases proliferation and induces a glycolytic switch in CD3-activated tTreg cells, but not in Tconv cells. Glycolysis in CD3-TNFR2-activated tTreg cells is driven by PI3-kinase-mTOR signalling and supports tTreg cell identity and suppressive function. In contrast to glycolytic Tconv cells, glycolytic tTreg cells do not show net lactate secretion and shuttle glucose-derived carbon into the tricarboxylic acid cycle. Ex vivo characterization of blood-derived TNFR2hiCD4+CD25hiCD127lo effector T cells, which were FOXP3+IKZF2+, revealed an increase in glucose consumption and intracellular lactate levels, thus identifying them as glycolytic tTreg cells. Our study links TNFR2 costimulation in human tTreg cells to metabolic remodelling, providing an additional avenue for drug targeting.
  9. Nat Commun. 2020 Sep 25. 11(1): 4869
    Janisiw E, Raices M, Balmir F, Paulin LF, Baudrimont A, von Haeseler A, Yanowitz JL, Jantsch V, Silva N.
      Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.
  10. Open Biol. 2020 09;10(9): 200160
    Fara A, Mitrev Z, Rosalia RA, Assas BM.
      Coronavirus disease 2019 (COVID-19) has swept the world, unlike any other pandemic in the last 50 years. Our understanding of the disease has evolved rapidly since the outbreak; disease prognosis is influenced mainly by multi-organ involvement. Acute respiratory distress syndrome, heart failure, renal failure, liver damage, shock and multi-organ failure are strongly associated with morbidity and mortality. The COVID-19 disease pathology is plausibly linked to the hyperinflammatory response of the body characterized by pathological cytokine levels. The term 'cytokine storm syndrome' is perhaps one of the critical hallmarks of COVID-19 disease severity. In this review, we highlight prominent cytokine families and their potential role in COVID-19, the type I and II interferons, tumour necrosis factor and members of the Interleukin family. We address various changes in cellular components of the immune response corroborating with changes in cytokine levels while discussing cytokine sources and biological functions. Finally, we discuss in brief potential therapies attempting to modulate the cytokine storm.
    Keywords:  COVID-19; IFN-γ; IL-6; SARS; TNF-α; coronavirus
  11. Nature. 2020 Sep 23.
    Xu D, Zhao H, Jin M, Zhu H, Shan B, Geng J, Dziedzic SA, Amin P, Mifflin L, Naito MG, Najafov A, Xing J, Yan L, Liu J, Qin Y, Hu X, Wang H, Zhang M, Manuel VJ, Tan L, He Z, Sun ZJ, Lee VMY, Wagner G, Yuan J.
      Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.
  12. J Clin Invest. 2020 Sep 22. pii: 129374. [Epub ahead of print]
    Song K, Cai X, Dong Y, Wu H, Wei Y, Shankavaram U, Cui K, Lee Y, Zhu B, Bhattacharjee S, Wang B, Zhang K, Wen A, Wong S, Yu L, Xia L, Welm AL, Bielenberg DR, Camphausen K, Kang Y, Chen H.
      Estrogen receptor (ER)-negative breast cancer is thought to be more malignant and devastating than ER-positive breast cancer and exhibit elevated NF-κB activity. How abnormally high NF-κB activity is maintained in ER-negative breast cancer is poorly understood. The importance of linear ubiquitination, which is generated by the linear ubiquitin chain assembly complex (LUBAC), is increasingly appreciated in NF-κB signaling, which regulates cell activation and death. Here, we showed that epsin proteins, a family of ubiquitin-binding endocytic adaptors, interacted with LUBAC via its Ubiquitin-Interacting Motif (UIM) and bound LUBAC's bona fide substrate NEMO via its N-terminal homolog (ENTH) domain. Furthermore, epsins promoted NF-κB essential modulator (NEMO) linear ubiquitination and served as scaffolds for recruiting other components of the IκB kinase (IKK) complex; thereby, resulting in the heightened IKK activation and sustained NF-κB signaling essential for the development of ER-negative breast cancer. Heightened epsin levels in ER-negative human breast cancer are associated with poor, relapse-free survival. We showed that transgenic and pharmacological approaches eliminating epsins potently impeded breast cancer development in both spontaneous and patient-derived xenograft breast cancer mouse models. Our findings established the pivotal role epsins played in promoting breast cancer. Thus, targeting epsins may represent a strategy to restrain NF-κB signaling, and provide an important perspective into ER-negative breast cancer treatment.
    Keywords:  Adaptor proteins; Breast cancer; Cell Biology; NF-kappaB; Oncology
  13. Nature. 2020 Sep;585(7826): 530-537
    Josephson B, Fehl C, Isenegger PG, Nadal S, Wright TH, Poh AWJ, Bower BJ, Giltrap AM, Chen L, Batchelor-McAuley C, Roper G, Arisa O, Sap JBI, Kawamura A, Baldwin AJ, Mohammed S, Compton RG, Gouverneur V, Davis BG.
      Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (β-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent β-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.
  14. Nature. 2020 Sep;585(7826): S7-S9
    Makin S.
    Keywords:  Cancer; Personalized medicine; Proteomics; Transcriptomics
  15. Cell Rep. 2020 Sep 22. pii: S2211-1247(20)31165-7. [Epub ahead of print]32(12): 108176
    Buch-Larsen SC, Hendriks IA, Lodge JM, Rykær M, Furtwängler B, Shishkova E, Westphall MS, Coon JJ, Nielsen ML.
      ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of activated ion electron transfer dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profile 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress and identify 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and electron-transfer higher-energy collisional dissociation (EThcD), respectively. Under physiological conditions, AI-ETD identifies 450 ADPr sites on low-abundant proteins, including in vivo cysteine modifications on poly(ADP-ribosyl)polymerase (PARP) 8 and tyrosine modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provide insights into the physiological regulation of ADPr.
    Keywords:  ADP-ribosylation; AI-ETD; Golgi apparatus; PARP14; PARP8; endoplasmic reticulum; glycosylation; mass spectrometry; physiological ADPr; proteomics