bims-tunefa Biomed News
on Tumor necrosis factor superfamily and post-translational modifications
Issue of 2020‒03‒22
33 papers selected by
John Silke
Walter and Eliza Hall Institute of Medical Research

  1. Bioorg Med Chem Lett. 2020 Mar 10. pii: S0960-894X(20)30195-5. [Epub ahead of print] 127106
    Anderson NA, Cryan J, Ahmed A, Dai H, McGonagle GA, Rozier C, Benowitz AB.
      Inhibitors of CDK4 and CDK6 have emerged as important FDA-approved treatment options for breast cancer patients. The properties and pharmacology of CDK4/6 inhibitor medicines have been extensively profiled, and investigations into the degradation of these targets via a PROTAC strategy have also been reported. PROTACs are a novel class of small-molecules that offer the potential for differentiated pharmacology compared to traditional inhibitors by redirecting the cellular ubiquitin-proteasome system to degrade target proteins of interest. We report here the preparation of palbociclib-based PROTACs that incorporate binders for three different E3 ligases, including a novel IAP-binder, which effectively degrade CDK4 and CDK6 in cells. In addition, we show that the palbociclib-based PROTACs in this study that recruit different E3 ligases all exhibit preferential CDK6 vs. CDK4 degradation selectivity despite employing a selection of linkers between the target binder and the E3 ligase binder.
    Keywords:  CDK4; CDK6; PROTAC; Palbociclib
  2. Commun Biol. 2020 Mar 20. 3(1): 140
    Mares A, Miah AH, Smith IED, Rackham M, Thawani AR, Cryan J, Haile PA, Votta BJ, Beal AM, Capriotti C, Reilly MA, Fisher DT, Zinn N, Bantscheff M, MacDonald TT, Vossenkamper A, Dace P, Churcher I, Benowitz AB, Watt G, Denyer J, Scott-Stevens P, Harling JD.
      Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.
  3. Nature. 2020 Mar;579(7799): 415-420
    Zhang Z, Zhang Y, Xia S, Kong Q, Li S, Liu X, Junqueira C, Meza-Sosa KF, Mok TMY, Ansara J, Sengupta S, Yao Y, Wu H, Lieberman J.
      Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis)1. Gasdermin E (GSDME, also known as DFNA5)-mutated in familial ageing-related hearing loss2-can be cleaved by caspase 3, thereby converting noninflammatory apoptosis to pyroptosis in GSDME-expressing cells3-5. GSDME expression is suppressed in many cancers, and reduced GSDME levels are associated with decreased survival as a result of breast cancer2,6, suggesting that GSDME might be a tumour suppressor. Here we show that 20 of 22 tested cancer-associated GSDME mutations reduce GSDME function. In mice, knocking out Gsdme in GSDME-expressing tumours enhances, whereas ectopic expression in Gsdme-repressed tumours inhibits, tumour growth. This tumour suppression is mediated by killer cytotoxic lymphocytes: it is abrogated in perforin-deficient mice or mice depleted of killer lymphocytes. GSDME expression enhances the phagocytosis of tumour cells by tumour-associated macrophages, as well as the number and functions of tumour-infiltrating natural-killer and CD8+ T lymphocytes. Killer-cell granzyme B also activates caspase-independent pyroptosis in target cells by directly cleaving GSDME at the same site as caspase 3. Uncleavable or pore-defective GSDME proteins are not tumour suppressive. Thus, tumour GSDME acts as a tumour suppressor by activating pyroptosis, enhancing anti-tumour immunity.
  4. Exp Cell Res. 2020 Mar 12. pii: S0014-4827(20)30166-X. [Epub ahead of print] 111958
    Tu J, Zhang B, Fang G, Chang W, Zhao Y.
      The sodium-coupled bicarbonate cotransporter 1 (NBCe1) plays an essential role in the maintenance of acid-base homeostasis in the human body. However, little research has been done regarding the modification of NBCe1. Nedd4-2 is one of the most important ubiquitin E3 ligases in the kidney where it is responsible for mediating the ubiquitylation level of many important ion channel proteins; therefore, influencing their expression and membrane localization. In this study, we performed experiments based on a prediction from bioinformatics analysis that NBCe1 might be a Nedd4-2 target protein. The results of co-immunoprecipitation and glutathione S-transferase pull-down assays showed that Nedd4-2 interacted with NBCe1. An in vitro ubiquitination assay further demonstrated that Nedd4-2 is indeed the NBCe1 ubiquitin E3 ligase. The overexpression of Nedd4-2 decreased NBCe1 expression, while MG132 rescued the changes. Nedd4-2 overexpression also altered the subcellular distribution of NBCe1. Furthermore, the kidney specific Nedd4-2-knockout mice certified the alteration of NBCe1. In addition, we speculate that neddylation activates Nedd4-2. A co-immunoprecipitation analysis indicated that Nedd4-2 interacted with Nedd8. In vitro neddylation experiments further demonstrated that Nedd4-2 underwent neddylation modification. The overexpression of Nedd8 led to decreased NBCe1 expression, while Nedd4-2 inhibition rescued the changes. These findings demonstrate that Nedd4-2 acts as the ubiquitin E3 ligase of NBCe1, mediating the degradation and altering the subcellular distribution of NBCe1, and that the neddylation modification downregulated NBCe1 expression by upregulating Nedd4-2 activity.
    Keywords:  NBCe1; Nedd4-2; Nedd8; Neddylation; Ubiquitin E3 ligase; Ubiquitination
  5. J Immunol. 2020 Mar 18. pii: ji1901142. [Epub ahead of print]
    Lou Y, Han M, Song Y, Zhong J, Zhang W, Chen YH, Wang H.
      TNFAIP8-like 2 (TIPE2) is a negative regulator of immune receptor signaling that maintains immune homeostasis. Dysregulated TIPE2 expression has been observed in several types of human immunological disorders. However, how TIPE2 expression is regulated remains to be determined. We report in this study that the SCFβ-TrCP E3 ubiquitin ligase regulates TIPE2 protein abundance by targeting it for ubiquitination and subsequent degradation via the 26S proteasome. Silencing of either cullin-1 or β-TrCP1 resulted in increased levels of TIPE2 in immune cells. TAK1 phosphorylated the Ser3 in the noncanonical degron motif of TIPE2 to trigger its interaction with β-TrCP for subsequent ubiquitination and degradation. Importantly, the amount of TIPE2 protein in immune cells determined the strength of TLR 4-induced signaling and downstream gene expression. Thus, our study has uncovered a mechanism by which SCFβ-TrCP E3 ubiquitin ligase regulates TLR responses.
  6. Cells. 2020 Mar 09. pii: E663. [Epub ahead of print]9(3):
    Abbas R, Larisch S.
      Inhibitors of apoptosis (IAPs) are a family of proteins that regulate cell death and inflammation. XIAP (X-linked IAP) is the only family member that suppresses apoptosis by directly binding to and inhibiting caspases. On the other hand, cIAPs suppress the activation of the extrinsic apoptotic pathway by preventing the formation of pro-apoptotic signaling complexes. IAPs are negatively regulated by IAP-antagonist proteins such as Smac/Diablo and ARTS. ARTS can promote apoptosis by binding and degrading XIAP via the ubiquitin proteasome-system (UPS). Smac can induce the degradation of cIAPs but not XIAP. Many types of cancer overexpress IAPs, thus enabling tumor cells to evade apoptosis. Therefore, IAPs, and in particular XIAP, have become attractive targets for cancer therapy. In this review, we describe the differences in the mechanisms of action between Smac and ARTS, and we summarize efforts to develop cancer therapies based on mimicking Smac and ARTS. Several Smac-mimetic small molecules are currently under evaluation in clinical trials. Initial efforts to develop ARTS-mimetics resulted in a novel class of compounds, which bind and degrade XIAP but not cIAPs. Smac-mimetics can target tumors with high levels of cIAPs, whereas ARTS-mimetics are expected to be effective for cancers with high levels of XIAP.
    Keywords:  ARTS; IAP antagonist; Smac; XIAP; apoptosis; cIAPs; cancer therapy; small molecules
  7. Cell Death Discov. 2020 ;6 14
    von Karstedt S, Walczak H.
      Twenty-one percent of all human cancers bear constitutively activating mutations in the proto-oncogene KRAS. This incidence is substantially higher in some of the most inherently therapy-resistant cancers including 30% of non-small cell lung cancers (NSCLC), 50% of colorectal cancers, and 95% of pancreatic ductal adenocarcinomas (PDAC). Importantly, survival of patients with KRAS-mutated PDAC and NSCLC has not significantly improved since the 1970s highlighting an urgent need to re-examine how oncogenic KRAS influences cell death signaling outputs. Interestingly, cancers expressing oncogenic KRAS manage to escape antitumor immunity via upregulation of programmed cell death 1 ligand 1 (PD-L1). Recently, the development of next-generation KRASG12C-selective inhibitors has shown therapeutic efficacy by triggering antitumor immunity. Yet, clinical trials testing immune checkpoint blockade in KRAS-mutated cancers have yielded disappointing results suggesting other, additional means endow these tumors with the capacity to escape immune recognition. Intriguingly, oncogenic KRAS reprograms regulated cell death pathways triggered by death receptors of the tumor necrosis factor (TNF) receptor superfamily. Perverting the course of their intended function, KRAS-mutated cancers use endogenous TNF-related apoptosis-inducing ligand (TRAIL) and its receptor(s) to promote tumor growth and metastases. Yet, endogenous TRAIL-TRAIL-receptor signaling can be therapeutically targeted and, excitingly, this may not only counteract oncogenic KRAS-driven cancer cell migration, invasion, and metastasis, but also the immunosuppressive reprogramming of the tumor microenvironment it causes. Here, we provide a concise summary of the current literature on oncogenic KRAS-mediated reprogramming of cell death signaling and antitumor immunity with the aim to open novel perspectives on combinatorial treatment strategies involving death receptor targeting.
    Keywords:  Cell biology; Cell death
  8. Adv Exp Med Biol. 2020 ;1248 295-324
    Yao H, Xu J.
      The antagonism, stalemate and compromise between the immune system and tumor cells is closely associated with tumor development and progression. In recent years, tumor immunotherapy has made continuous breakthroughs. It has become an important approach for cancer treatment, improving the survival and prognosis of more and more tumor patients. Further investigating the mechanism of tumor immune regulation, and exploring tumor immunotherapy targets with high specificity and wide applicability will provide researchers and clinicians with favorable weapons towards cancer. Ubiquitination affects protein fate through influencing the activity, stability and location of target protein. The regulation of substrate protein fate by ubiquitination is involved in cell cycle, apoptosis, transcriptional regulation, DNA repair, immune response, protein degradation and quality control. E3 ubiquitin ligase selectively recruits specific protein substrates through specific protein-protein interactions to determine the specificity of the overall ubiquitin modification reaction. Immune-checkpoint inhibitory pathway is an important mechanism for tumor cells to evade immune killing, which can inhibit T cell activity. Blocking the immune checkpoints and activating T cells through targeting the negative regulatory factors of T cell activation and removing the "brake" of T lymphocytes can enhance T cells immune response against tumors. Therefore, blocking the immune checkpoint is one of the methods to enhance the activity of T cells, and it is also a hot target for the development of anti-tumor drugs in recent years, whose inhibitors have shown good effect in specific tumor treatment. Ubiquitination, as one of the most important posttranslational modification of proteins, also modulates the expression, intracellular trafficking, subcellular and membranous location of immune checkpoints, regulating the immune surveillance of T cells to tumors.
    Keywords:  Cancer therapy; Immune-checkpoint; Lysosome; Proteasomal degradation; Ubiquitination
  9. Semin Cancer Biol. 2020 Mar 11. pii: S1044-579X(20)30064-X. [Epub ahead of print]
    Wang ZW, Hu X, Ye M, Lin M, Chu M, Shen X.
      A growing amount of evidence indicates that the neuronally expressed developmentally downregulated 4 (NEDD4, also known as NEDD4-1) E3 ligase plays a critical role in a variety of cellular processes via the ubiquitination-mediated degradation of multiple substrates. The abnormal regulation of NEDD4 protein has been implicated in cancer development and progression. In this review article, we briefly delineate the downstream substrates and upstream regulators of NEDD4, which are involved in carcinogenesis. Moreover, we succinctly elucidate the functions of NEDD4 protein in tumorigenesis and progression, including cell proliferation, apoptosis, cell cycle, migration, invasion, epithelial mesenchymal transition (EMT), cancer stem cells, and drug resistance. The findings regarding NEDD4 functions are further supported by knockout mouse models and human tumor tissue studies. This review could provide a promising and optimum anticancer therapeutic strategy via targeting the NEDD4 protein.
    Keywords:  Cancer; E3 ligases; NEDD4; Therapy; Ubiquitination
  10. Front Mol Biosci. 2020 ;7 21
    Kliza K, Husnjak K.
      Ubiquitination regulates nearly all cellular processes by coordinated activity of ubiquitin writers (E1, E2, and E3 enzymes), erasers (deubiquitinating enzymes) and readers (proteins that recognize ubiquitinated proteins by their ubiquitin-binding domains). By differentially modifying cellular proteome and by recognizing these ubiquitin modifications, ubiquitination machinery tightly regulates execution of specific cellular events in space and time. Dynamic and complex ubiquitin architecture, ranging from monoubiquitination, multiple monoubiquitination, eight different modes of homotypic and numerous types of heterogeneous polyubiquitin linkages, enables highly dynamic and complex regulation of cellular processes. We discuss available tools and approaches to study ubiquitin networks, including methods for the identification and quantification of ubiquitin-modified substrates, as well as approaches to quantify the length, abundance, linkage type and architecture of different ubiquitin chains. Furthermore, we also summarize the available approaches for the discovery of novel ubiquitin readers and ubiquitin-binding domains, as well as approaches to monitor and visualize activity of ubiquitin conjugation and deconjugation machineries. We also discuss benefits, drawbacks and limitations of available techniques, as well as what is still needed for detailed spatiotemporal dissection of cellular ubiquitination networks.
    Keywords:  E3 ligase; affinity purification; deubiquitinating enzyme; mass spectrometry; ubiquitin; ubiquitin receptor
  11. Adv Exp Med Biol. 2020 ;1248 7-32
    Van Coillie S, Wiernicki B, Xu J.
      Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is an inhibitory receptor belonging to the CD28 immunoglobulin subfamily, expressed primarily by T-cells. Its ligands, CD80 and CD86, are typically found on the surface of antigen-presenting cells and can either bind CD28 or CTLA-4, resulting in a costimulatory or a co-inhibitory response, respectively. Because of its dampening effect, CTLA-4 is a crucial regulator of T-cell homeostasis and self-tolerance. The mechanisms by which CTLA-4 exerts its inhibitory function can be categorized as either cell-intrinsic (affects the CTLA-4 expressing T-cell) or cell-extrinsic (affects secondary cells). Research from the last decade has shown that CTLA-4 mainly acts in a cell-extrinsic manner via its competition with CD28, CTLA-4-mediated trans-endocytosis of CD80 and CD86, and its direct tolerogenic effects on the interacting cell. Nonetheless, intrinsic CTLA-4 signaling has been implicated in T-cell motility and the regulation of CTLA-4 its subcellular localization amongst others. CTLA-4 is well recognized as a key immune checkpoint and has gained significant momentum as a therapeutic target in the field of autoimmunity and cancer. In this chapter, we describe the role of costimulation in immune response induction as well as the main mechanisms by which CTLA-4 can inhibit this process.
    Keywords:  CD28; CD80; CD86; CTLA-4; Immune tolerance
  12. Expert Opin Ther Targets. 2020 Mar 17.
    Ding L, Billadeau DD.
      IntroductionPancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and is the third leading cause of adult cancer death in the United States with a single-digit 5-year survival rate despite significant advances in understanding the genetics and biology of the disease. Glycogen synthase kinase-3α (GSK-3α) and GSK-3β are serine/threonine kinases encoded by distinct genes that have been shown to localize to the cytoplasm, mitochondria and nucleus. Although they are highly homologous within their kinase domains and known to phosphorylate an overlapping set of target proteins, genetic studies have shown that GSK-3β phosphorylates and regulates the activity of several proteins that participate in pathways that promote neoplastic transformation. Significantly, GSK-3β is progressively overexpressed during PDAC development and has been shown to play an important role in tumor development, progression and resistance to chemotherapy. Thus, novel therapeutic approaches designed to target GSK-3β or the signaling cascades that regulate its expression have become attractive targets for treating PDAC.Areas coveredThis review describes and summarizes the expanding cellular mechanisms regulating GSK-3β activity, including upstream translational and post-translational regulation, as well as the downstream cellular targets and their functions in PDAC cell growth, cell fate, metastasis and chemotherapeutic resistance.Expert opinionWith approximately 100 identified substrates impacting a large number of signaling pathways and transcriptional regulation, the role of GSK-3 kinases are generally considered to be cell- and context-specific. Mutation of the KRas gene is found in over 95% of PDAC patients, where it plays an essential role in PDAC initiation. In addition, oncogenic KRas drives the transcriptional expression of the GSK-3β gene which has been shown to regulate the proliferation and survival of PDAC cells, as well as resistance to various chemotherapies. Thus, the combination of GSK-3 inhibitors with chemotherapeutic drugs could be a promising therapeutic strategy for PDAC.
    Keywords:  GSK-3; GSK-3 inhibitors; KRas; pancreatic cancer; pancreatic ductal adenocarcinoma; targeted therapy
  13. Gastroenterology. 2020 Mar 13. pii: S0016-5085(20)30339-5. [Epub ahead of print]
    Ma Y, Li J, Wang H, Chiu Y, Kingsley CV, Fry D, Delaney SN, Wei SC, Zhang J, Maitra A, Yee C.
      BACKGROUND AIMS: Advanced pancreatic ductal adenocarcinoma (PDAC) is resistant to therapy, including immune checkpoint inhibitors. We evaluated the effects of a neutralizing antibody against programmed cell death 1 (PDCD1, also called PD1) and an agonist of OX40 (provides a survival signal to activated T cells) in mice with pancreatic tumors.METHODS: We performed studies in C57BL/6 mice (controls), KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, and mice with orthotopic tumors grown from Panc02 cells, KrasG12D;P53flox/flox;PDX-1-Cre;Luciferase (KPC-Luc) cells, or mT4 cells. After tumors developed, mice were given injections of control antibody or anti-OX40 and/or anti-PD1 antibody. Some mice were then given injections of antibodies against CD8, CD4, or NK1.1 to deplete immune cells, and IL4 or IL7RA to block cytokine signaling. Bioluminescence imaging was used to monitor tumor growth. Tumor tissues collected and single-cell suspensions were analyzed by time of flight mass spectrometry analysis. Mice that were tumor-free 100 days after implantation of orthotopic tumors were rechallenged with PDAC cells (KPC-Luc or mT4) and survival was measured. Median levels of PD1 and OX40 mRNAs in PDACs were determined from the Cancer Genome Atlas and compared with patient survival times.
    RESULTS: In mice with orthotopic tumors, all those given control antibody or anti-PD1 died within 50 days, whereas 43% of mice given anti-OX40 survived for 225 days; almost 100% of mice given the combination of anti-PD1 and anti-OX40 survived for 225 days, and tumors were no longer detected. KPC mice given control antibody, anti-PD1, or anti-OX40 had median survival times of 50 days or less, whereas mice given the combination of anti-PD1 and anti-OX40 survived for a median 88 days. Mice with orthotopic tumors that were given the combination of anti-PD1 and anti-OX40 and survived 100 days were rechallenged with a second tumor; those re-challenged with mT4 cells survived an additional median 70 days and those re-challenged with KPC-Luc cells survived long term, tumor free. The combination of anti-PD1 and anti-OX40 did not slow tumor growth in mice with antibody-mediated depletion of CD4+ T cells. Mice with orthotopic tumors given the combination of anti-PD1 and anti-OX40 that survived after complete tumor rejection were re-challenged with KPC-Luc cells; those with depletion of CD4+ T cells before the re-challenge had uncontrolled tumor growth. Furthermore, KPC orthotopic tumors from mice given the combination contained an increased number of CD4+ T cells that expressed CD127, compared with mice given control antibody. The combination of agents reduced the proportion of T-regulatory and exhausted T cells and decreased T-cell expression of GATA3; tumor size was negatively associated with numbers of infiltrating CD4+ T cells, CD4+CD127+ T cells, and CD8+CD127+ T cells and positively associated with numbers of CD4+PD1+ T cells, CD4+CD25+ T cells, and CD8+PD1+ T cells. PDACs with high levels of OX40 and low levels of PD1 were associated with longer survival times of patients.
    CONCLUSIONS: Pancreatic tumors appear to evade the immune response by inducing development of immune-suppressive T cells. In mice, the combination of anti-PD1 inhibitory and anti-OX40 agonist antibodies reduces the proportion of T-regulatory and exhausted T cells in pancreatic tumors and increases numbers of memory CD4+ and CD8+ T cells, eradicating all detectable tumor. This information might be used in development of immune-based combination therapies for PDAC.
    Keywords:  CyTOF; immune checkpoint inhibitor; immune-based therapy; mouse model
  14. Biochem Soc Trans. 2020 Mar 20. pii: BST20190535. [Epub ahead of print]
    Wu HQ, Baker D, Ovaa H.
      Eukaryotic life depends upon the interplay between vast networks of signaling pathways composed of upwards of 109-1010 proteins per cell. The integrity and normal operation of the cell requires that these proteins act in a precise spatial and temporal manner. The ubiquitin system is absolutely central to this process and perturbation of its function contributes directly to the onset and progression of a wide variety of diseases, including cancer, metabolic syndromes, neurodegenerative diseases, autoimmunity, inflammatory disorders, infectious diseases, and muscle dystrophies. Whilst the individual components and the overall architecture of the ubiquitin system have been delineated in some detail, how ubiquitination might be successfully targeted, or harnessed, to develop novel therapeutic approaches to the treatment of disease, currently remains relatively poorly understood. In this review, we will provide an overview of the current status of selected small molecule ubiquitin system inhibitors. We will further discuss the unique challenges of targeting this ubiquitous and highly complex machinery, and explore and highlight potential ways in which these challenges might be met.
    Keywords:  PROTAC; chemical probe; drug development; inhibitor; post-translational modification; ubiquitin
  15. Front Immunol. 2020 ;11 323
    Wang S, Zhou L, Ling L, Meng X, Chu F, Zhang S, Zhou F.
      Recognition of pathogen-associated molecular patterns (PAMPs) triggers expression of antiviral interferons and proinflammatory cytokines, which functions as the frontier of host defense against microbial pathogen invasion. Hippo-YAP pathway regulates cell proliferation, survival, differentiation and is involved in diverse life processes, including tissue homeostasis and tumor suppression. Emerging discoveries elucidated that the components of Hippo-YAP pathway, such as MST1/2, NDR1/2, and YAP/TAZ played crucial regulatory roles in innate immunity. Meanwhile the innate immune signaling also exhibited regulatory effect on Hippo-YAP pathway. As for the importance of these two pathways, it would be interesting to figure out the deeper biological implications of their interplays. This review focuses on the regulation between Hippo-YAP pathway and innate immune signaling. We also propose the possible contribution of these interplays to tumor development.
    Keywords:  Hippo-YAP pathway; NF-kB signaling; innate immunity; tumor development; type I interferon
  16. Biochem Biophys Res Commun. 2020 Mar 17. pii: S0006-291X(20)30549-0. [Epub ahead of print]
    Nishida T, Yamada Y.
      The transcriptional repressor PARIS, which is a substrate of the ubiquitin E3 ligase parkin, represses the expression of the transcriptional co-activator, PGC-1α. However, little is known about how its repression activity is regulated. We have previously shown that PARIS is SUMOylated, and this SUMOylation plays an important role in regulating its transcriptional repression activity. In this study, we demonstrated that PARIS SUMOylation induced its ubiquitination and subsequent proteasomal degradation, which was mediated by the SUMO-targeted ubiquitin ligase RNF4. Reporter gene assays revealed that co-expression of SUMO3 and RNF4 relieved PARIS-mediated transcriptional repression. Conversely, the SUMO E3 ligase PIASy inhibited the RNF4-mediated ubiquitination of PARIS and blocked the RNF4-mediated relief of PARIS-mediated transcriptional repression. These results suggest that RNF4 regulates PARIS ubiquitination to control its transcriptional repression activity.
    Keywords:  Parkin interacting substrate (PARIS); SUMO-Targeted ubiquitin ligases; SUMOylation; Transcriptional repression; Ubiquitination
  17. J Clin Invest. 2020 Mar 19. pii: 124382. [Epub ahead of print]
    Kober-Hasslacher M, Oh-Strauß H, Kumar D, Soberón V, Diehl C, Lech M, Engleitner T, Katab E, Fernandez Saiz V, Piontek G, Li H, Menze B, Ziegenhain C, Enard W, Rad R, Böttcher JP, Anders HJ, Rudelius M, Schmidt-Supprian M.
      Single nucleotide polymorphisms and locus amplification link the NF-κB transcription factor c-Rel to human autoimmune diseases and B cell lymphomas, respectively. However, the functional consequences of enhanced c-Rel levels remain enigmatic. Here, we overexpressed c-Rel specifically in mouse B cells from BAC-transgenic gene loci and demonstrate that c-Rel protein levels linearly dictated expansion of germinal center (GC) B cells and isotype-switched plasma cells. c-Rel expression in B cells of otherwise c-Rel-deficient mice fully rescued terminal B cell differentiation, underscoring its critical B cell-intrinsic roles. Unexpectedly, in GCB cells transcription-independent regulation produced the highest c-Rel protein levels amongst B cell subsets. In c-Rel overexpressing GCB cells this caused enhanced nuclear translocation, a profoundly altered transcriptional program and increased proliferation. Finally, we provide a link between c-Rel gain and autoimmunity by showing that c-Rel overexpression in B cells caused autoantibody production and renal immune complex deposition.
    Keywords:  Autoimmune diseases; Autoimmunity; B cells; Immunology; NF-kappaB
  18. Nat Catal. 2020 Mar;3(3): 295-306
    Rosier BJHM, Markvoort AJ, Gumí Audenis B, Roodhuizen JAL, den Hamer A, Brunsveld L, de Greef TFA.
      Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximity-induced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein-protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity, and additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami-based protein assembly platforms have the potential to inform the function of other multi-enzyme complexes involved in inflammation, innate immunity and cell death.
  19. Immunity. 2020 Mar 17. pii: S1074-7613(20)30075-3. [Epub ahead of print]52(3): 499-512.e5
    Goepfert A, Lehmann S, Blank J, Kolbinger F, Rondeau JM.
      Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases.
    Keywords:  IL-17; IL-17 signaling; IL-17F; IL-17RC; X-ray structure; cytokine receptors
  20. Nat Commun. 2020 Mar 16. 11(1): 1407
    Basilico S, Wang X, Kennedy A, Tzelepis K, Giotopoulos G, Kinston SJ, Quiros PM, Wong K, Adams DJ, Carnevalli LS, Huntly BJP, Vassiliou GS, Calero-Nieto FJ, Göttgens B.
      Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets.
  21. Sci Rep. 2020 Mar 19. 10(1): 5028
    Kook S, Zhan X, Thibeault K, Ahmed MR, Gurevich VV, Gurevich EV.
      Loss-of-function mutations in the E3 ubiquitin ligase parkin have been implicated in the death of dopaminergic neurons in the substantia nigra, which is the root cause of dopamine deficit in the striatum in Parkinson's disease. Parkin ubiquitinates proteins on mitochondria that lost membrane potential, promoting the elimination of damaged mitochondria. Neuroprotective activity of parkin has been linked to its critical role in the mitochondria maintenance. Here we report a novel regulatory mechanism: another E3 ubiquitin ligase Mdm2 directly binds parkin and enhances its enzymatic activity in vitro and in intact cells. Mdm2 translocates to damaged mitochondria independently of parkin, enhances parkin-dependent ubiquitination of the outer mitochondria membrane protein mitofusin1. Mdm2 facilitates and its knockdown reduces parkin-dependent mitophagy. Thus, ubiquitously expressed Mdm2 might enhance cytoprotective parkin activity. The data suggest that parkin activation by Mdm2 could be targeted to increase its neuroprotective functions, which has implications for anti-parkinsonian therapy.
  22. Expert Opin Drug Saf. 2020 Mar 17.
    Campanati A, Diotallevi F, Martina E, Paolinelli M, Radi G, Offidani A.
      Introduction: Conventional topical therapies and disease-modifying anti-rheumatic drugs (DMARDs) for patients with psoriasis are often linked to inadequate outcomes and risk of multiple adverse effects. Biologic agents such as etanercept (ETN) have revolutionized the therapeutic management of psoriasis, allowing the treatment of most difficult cases, and fragile patients.Areas covered: The authors searched PubMed using the term "psoriasis", "etanercept" and "safety". Articles considered by the authors to be most relevant, such as randomized controlled studies, cohort studies, and review articles placing emphasis on studies of efficacy and safety, were selected. Case reports and letters relating to safety were also included. The main sources of data referenced by these articles were also included in the review. Besides, to get the relevant studies, the reference lists were examined to identify the potentially available studies. The aim of this review is to describe the safety profile of ETN, used for psoriasis treatment, focusing on related clinical implications.Expert opinion: ETN has a favourable safety profile, and its use should be largely considered in psoriatic patients. Caution should be recommended in case of chronic heart failure, autoimmune disease, previous malignancies, familial history of demyelinating diseases, latent TBC infection, chronic HBV and HCV infection or HIV.
    Keywords:  TNF-alpha inhibitors; adverse events; biologic therapy; etanercept; plaque psoriasis; safety
  23. Cancer Cell. 2020 Mar 16. pii: S1535-6108(20)30100-8. [Epub ahead of print]37(3): 267-269
    Gundry MC, Goodell MA, Brunetti L.
      Several acute myeloid leukemia genetic sub-types converge on high expression of HOX genes, critical for their self-renewal. A new orally bioavailable Menin-MLL inhibitor (VTP-50469) appears to promote their differentiation through direct effects on the HOX cofactor MEIS1, paving the way for clinical trials.
  24. Int J Mol Sci. 2020 Mar 13. pii: E1957. [Epub ahead of print]21(6):
    Murakoshi M, Gohda T, Suzuki Y.
      Despite considerable advancements in medicine, the optimal treatment for chronic kidney disease (CKD), especially diabetic kidney disease (DKD), remains a major challenge. More patients with DKD succumb to death due to cardiovascular events than due to progression to end-stage renal disease (ESRD). Moreover, patients with DKD and ESRD have remarkably poor prognosis. Current studies have appreciated the contribution of inflammation and inflammatory mediators, such as tumor necrosis factor (TNF)-related biomarkers, on the development/progression of DKD. The present review focuses on molecular roles, serum concentrations of TNF receptors (TNFRs), and their association with increased albuminuria, eGFR decline, and all-cause mortality in diabetes. Experimental studies have suggested that DKD progression occurs through the TNFα-TNFR2 inflammatory pathway. Moreover, serum TNFR levels were positively associated with albuminuria and negatively associated with estimated glomerular filtration rate (eGFR), while circulating levels of TNFRs exhibited an independent effect on all-cause mortality and eGFR decline, including ESRD, even after adjusting for existing risk factors. However, their precise function has yet to be elucidated and requires further studies.
    Keywords:  TNF receptor; TNFα; biomarker; diabetic kidney disease
  25. J Cancer. 2020 ;11(7): 1657-1667
    Hollomon MG, Patterson L, Santiago-O'Farrill J, Kleinerman ES, Gordon N.
      Fas-associated protein with death domain (FADD) was first identified for its role in linking death receptors to the apoptotic signaling pathway with subsequent cell death. Later studies reported non-apoptotic functions for FADD in normal cells and cancer cells. Non-apoptotic functions for FADD in osteosarcoma (OS) have not been reported. In this study, FADD protein expression was knocked down in human CCHOSD, LM7, and SaOS2 OS cell lines followed by assessment of sensitivity to TNFα- or TRAIL-induced cell death. Knock down of FADD significantly increased TNFα-induced cell death in LM7 and SaOS2 cell lines. The mode of TNFα-induced cell death was apoptosis and not necroptosis. Inhibition of nuclear factor kappa B (NFκB) in wildtype cells increased TNFα-induced cell death to similar levels observed in FADD knockdown cells, suggesting a role for FADD in NFκB pro-survival cell signaling. In addition, knock down of FADD increased SMAC mimetic-mediated TNFα-induced cell death in all cell lines studied. The results of this study indicate that FADD has a pro-survival function in OS following TNFα treatment that involves NFκB signaling. The results also indicate that the pro-survival function of FADD is associated with XIAP activity.
    Keywords:  FADD; NFκB; TNFα; XIAP; osteosarcoma
  26. ACS Med Chem Lett. 2020 Mar 12. 11(3): 237-240
    Gao H, Sun X, Rao Y.
      PROTACs-induced targeted protein degradation has emerged as a novel therapeutic strategy in drug development and attracted the favor of academic institutions, large pharmaceutical enterprises (e.g., AstraZeneca, Bayer, Novartis, Amgen, Pfizer, GlaxoSmithKline, Merck, and Boehringer Ingelheim, etc.), and biotechnology companies. PROTACs opened a new chapter for novel drug development. However, any new technology will face many new problems and challenges. Perspectives on the potential opportunities and challenges of PROTACs will contribute to the research and development of new protein degradation drugs and degrader tools.
  27. Nat Chem Biol. 2020 Apr;16(4): 369-378
    Gerry CJ, Schreiber SL.
      Nature uses a variety of tools to mediate the flow of information in cells, many of which control distances between key biomacromolecules. Researchers have thus generated compounds whose activities stem from interactions with two (or more) proteins simultaneously. In this Perspective, we describe how these 'bifunctional' small molecules facilitate the study of an increasingly wide range of complex biological phenomena and enable the drugging of otherwise challenging therapeutic targets and processes. Despite their structural and functional differences, all bifunctional molecules employ Nature's strategy of altering interactomes and inducing proximity to modulate biology. They therefore exhibit a shared set of chemical and biophysical principles that have not yet been appreciated fully. By highlighting these commonalities-and their wide-ranging consequences-we hope to chip away at the artificial barriers that threaten to constrain this interdisciplinary field. Doing so promises to yield remarkable benefits for biological research and therapeutics discovery.
  28. Virus Res. 2020 Mar 17. pii: S0168-1702(20)30079-4. [Epub ahead of print] 197938
    Le-Trilling VTK, Trilling M.
      Human cytomegalovirus (HCMV) is a ubiquitous member of the Betaherpesvirinae subfamily, causing life-threatening diseases in individuals with impaired, immature, or senescent immunity. Accordingly, HIV-infected AIDS patients, transplant recipients, and congenitally infected neonates frequently suffer from symptomatic episodes of HCMV replication. Like all viruses, HCMV has a split relationship with the host proteome. Efficient virus replication can only be achieved if proteins involved in intrinsic, innate, and adaptive immune responses are sufficiently antagonized. Simultaneously, the abundance and function of proteins involved in the synthesis of chemical building blocks required for virus production, such as nucleotides, amino acids, and fatty acids, must be preserved or even enriched. The ubiquitin (Ub) proteasome system (UPS) constitutes one of the most relevant protein decay systems of eukaryotic cells. In addition to the regulation of the turn-over and abundance of thousands of proteins, the UPS also generates the majority of peptides presented by major histocompatibility complex (MHC) molecules to allow surveillance by T lymphocytes. Cytomegaloviruses exploit the UPS to regulate the abundance of viral proteins and to manipulate the host proteome in favour of viral replication and immune evasion. After summarizing the current knowledge of CMV-mediated misuse of the UPS, we discuss the evolution of viral proteins utilizing the UPS for the degradation of defined target proteins. We propose two alternative routes of adapter protein development and their mechanistic consequences.
    Keywords:  cytomegalovirus; evolution; immune evasion; proteasome; protein degradation; ubiquitin
  29. Proc Natl Acad Sci U S A. 2020 Mar 19. pii: 201902298. [Epub ahead of print]
    Yip MCJ, Bodnar NO, Rapoport TA.
      The Saccharomyces cerevisiae protein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.
    Keywords:  Ddi1; Nrf1; protease; proteasome; ubiquitin
  30. Front Pharmacol. 2020 ;11 166
    Gurgone D, McShane L, McSharry C, Guzik TJ, Maffia P.
      Cardiovascular disease (CVD) is an important comorbidity in a number of chronic inflammatory diseases. However, evidence in highly prevalent respiratory disease such as asthma are still limited. Epidemiological and clinical data are not univocal in supporting the hypothesis that asthma and CVD are linked and the mechanisms of this relationship remain poorly defined. In this review, we explore the relationship between asthma and cardiovascular disease, with a specific focus on cytokine contribution to vascular dysfunction and atherosclerosis. This is important in the context of recent evidence linking broad inflammatory signaling to cardiovascular events. However inflammatory regulation in asthma is different to the one typically observed in atherosclerosis. We focus on the contribution of cytokine networks encompassing IL-4, IL-6, IL-9, IL-17A, IL-33 but also IFN-γ and TNF-α to vascular dysfunction in atherosclerosis. In doing so we highlight areas of unmet need and possible therapeutic implications.
    Keywords:  asthma; atherosclerosis; cardiovascular disease; cytokines; interleukin
  31. Nature. 2020 Mar;579(7799): 452-455
    Han Y, Reyes AA, Malik S, He Y.
      The chromatin-remodelling complex SWI/SNF is highly conserved and has critical roles in various cellular processes, including transcription and DNA-damage repair1,2. It hydrolyses ATP to remodel chromatin structure by sliding and evicting histone octamers3-8, creating DNA regions that become accessible to other essential factors. However, our mechanistic understanding of the remodelling activity is hindered by the lack of a high-resolution structure of complexes from this family. Here we report the cryo-electron microscopy structure of Saccharomyces cerevisiae SWI/SNF bound to a nucleosome, at near-atomic resolution. In the structure, the actin-related protein (Arp) module is sandwiched between the ATPase and the rest of the complex, with the Snf2 helicase-SANT associated (HSA) domain connecting all modules. The body contains an assembly scaffold composed of conserved subunits Snf12 (also known as SMARCD or BAF60), Snf5 (also known as SMARCB1, BAF47 or INI1) and an asymmetric dimer of Swi3 (also known as SMARCC, BAF155 or BAF170). Another conserved subunit, Swi1 (also known as ARID1 or BAF250), resides in the core of SWI/SNF, acting as a molecular hub. We also observed interactions between Snf5 and the histones at the acidic patch, which could serve as an anchor during active DNA translocation. Our structure enables us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and to propose a model for how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation9.
  32. J Med Chem. 2020 Mar 17.
    Johnson BM, Shu YZ, Zhuo X, Meanwell NA.
      The applications of fluorine in drug design continue to expand, facilitated by an improved understanding of its effects on physicochemical properties and the development of synthetic methodologies that are providing access to new fluorinated motifs. In turn, studies of fluorinated molecules are providing deeper insights into the effects of fluorine on metabolic pathways, distribution, and disposition. Despite the high strength of the C-F bond, the departure of fluoride from metabolic intermediates can be facile. This reactivity has been leveraged in the design of mechanism-based enzyme inhibitors and has influenced the metabolic fate of fluorinated compounds. In this Perspective, we summarize the literature associated with the metabolism of fluorinated molecules, focusing on examples where the presence of fluorine influences the metabolic profile. These studies have revealed potentially problematic outcomes with some fluorinated motifs and are enhancing our understanding of how fluorine should be deployed.
  33. Cell Rep. 2020 Mar 17. pii: S2211-1247(20)30275-8. [Epub ahead of print]30(11): 3671-3681.e5
    Adomati T, Cham LB, Hamdan TA, Bhat H, Duhan V, Li F, Ali M, Lang E, Huang A, Naser E, Khairnar V, Friedrich SK, Lang J, Friebus-Kardash J, Bergerhausen M, Schiller M, Machlah YM, Lang F, Häussinger D, Ferencik S, Hardt C, Lang PA, Lang KS.
      Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factor β (TGF-β). Lack of Mertk in Mertk-/- mice prevents induction of IL-10 and TGF-β, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV.