bims-tunefa 2020-04-26 papers

bims-tunefa

Biomed News

on Tumor necrosis factor superfamily and post-translational modifications

Issue of 2020–04–26
thirty-two papers selected by
John Silke, Walter and Eliza Hall Institute of Medical Research

Modes of allosteric regulation of the ubiquitination machinery.
Coupling Conjugation and Deconjugation Activities to Achieve Cellular Ubiquitin Dynamics.
Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity.
Selective Degradation of Target Proteins by Chimeric Small-Molecular Drugs, PROTACs and SNIPERs.
Selective inhibition of Tumor necrosis factor receptor-1 (TNFR1) for the treatment of autoimmune diseases.
The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L).
CARD6 protects against collagen-induced rheumatoid arthritis in mice through attenuating the inflammatory response and joint destruction via suppression of TNFR1/TRAF2 signaling.
The structure of the extracellular domains of human interleukin 11 α-receptor reveals mechanisms of cytokine engagement.
The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.
LUBAC accelerates B-cell lymphomagenesis by conferring B cells resistance to genotoxic stress.
The CRL3BTBD9 E3 ubiquitin ligase complex targets TNFAIP1 for degradation to suppress cancer cell migration.
Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination.
Molecular imaging of inflammation - Current and emerging technologies for diagnosis and treatment.
Inhibition of RIPK1/RIPK3 ameliorates osteoclastogenesis through regulating NLRP3-dependent NF-κB and MAPKs signaling pathways.
Monovalent protein-degraders - Insights and future perspectives.
Separase-triggered apoptosis enforces minimal length of mitosis.
Toll-like receptors: Triggers of regulated cell death and promising targets for cancer therapy.
Learning from mouse models of MLL fusion gene-driven acute leukemia.
Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats.
RIPK3 and Caspase-1/11 Are Necessary for Optimal Antigen-Specific CD8 T Cell Response Elicited by Genetically Modified Listeria monocytogenes.
Discovery and biological evaluation of proteolysis targeting chimeras (PROTACs) as an EGFR degraders based on osimertinib and lenalidomide.
Suppressing PARylation by 2',5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death.
Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs.
The necroptosis pathway and its role in age-related neurodegenerative diseases: will it open up new therapeutic avenues in the next decade?
Tnfaip3 expression in pulmonary conventional type 1 Langerin-expressing dendritic cells regulates T helper 2-mediated airway inflammation in mice.
Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats.
TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response.
TNF-Signaling Modulates Neutrophil-Mediated Immunity at the Feto-Maternal Interface During LPS-Induced Intrauterine Inflammation.
Comparison of Target Recognition by TRAF1 and TRAF2.
Selective Janus Kinase 2 (JAK2) Pseudokinase Ligands with a Diaminotriazole Core.
Novel Apoptotic Mediators Identified by Conservation of Vertebrate Caspase Targets.
A necroptotic-independent function of MLKL in regulating endothelial cell adhesion molecule expression.

Curr Opin Struct Biol. 2020 Apr 15. pii: S0959-440X(20)30020-8. [Epub ahead of print]62 189-196

Modes of allosteric regulation of the ubiquitination machinery.

Martin L Rennie, Viduth K Chaugule, Helen Walden.

Ubiquitination is a post-translational modification crucial for cellular signaling. A diverse range of enzymes constitute the machinery that mediates attachment of ubiquitin onto target proteins. This diversity allows the targeting of various proteins in a highly regulated fashion. Many of the enzymes have multiple domains or subunits that bind allosteric effectors and exhibit large conformational rearrangements to facilitate regulation. Here we consider recent examples of ubiquitin itself as an allosteric effector of RING and RBR E3 ligases, as well as advances in the understanding of allosteric regulatory elements within HECT E3 ligases.

DOI: https://doi.org/10.1016/j.sbi.2020.02.003
Trends Biochem Sci. 2020 May;pii: S0968-0004(20)30030-X. [Epub ahead of print]45(5): 427-439

Coupling Conjugation and Deconjugation Activities to Achieve Cellular Ubiquitin Dynamics.

Casey P Nielsen, Jason A MacGurn.

  In eukaryotic cells, proteome remodeling is mediated by the ubiquitin-proteasome system, which regulates protein degradation, trafficking, and signaling events in the cell. Interplay between the cellular proteome and ubiquitin is complex and dynamic and many regulatory features that support this system have only recently come into focus. An unexpected recurring feature in this system is the physical interaction between E3 ubiquitin ligases and deubiquitylases (DUBs). Recent studies have reported on the regulatory significance of DUB-E3 interactions and it is becoming clear that they play important but complicated roles in the regulation of diverse cellular processes. Here, we summarize the current understanding of interactions between ubiquitin conjugation and deconjugation machineries and we examine the regulatory logic of these enigmatic complexes.

Keywords:  E3 ubiquitin ligase; deubiquitylase; polyubiquitin chain editing; ubiquitin

DOI:  https://doi.org/10.1016/j.tibs.2020.01.008
Nat Commun. 2020 Apr 24. 11(1): 1996

Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity.

Yonghan He, Xuan Zhang, Jianhui Chang, Ha-Neui Kim, Peiyi Zhang, Yingying Wang, Sajid Khan, Xingui Liu, Xin Zhang, Dongwen Lv, Lin Song, Wen Li, Dinesh Thummuri, Yaxia Yuan, Janet S Wiegand, Yuma T Ortiz, Vivekananda Budamagunta, Jennifer H Elisseeff, Judith Campisi, Maria Almeida, Guangrong Zheng, Daohong Zhou.

Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors.

DOI: https://doi.org/10.1038/s41467-020-15838-0
Pharmaceuticals (Basel). 2020 Apr 21. pii: E74. [Epub ahead of print]13(4):

Selective Degradation of Target Proteins by Chimeric Small-Molecular Drugs, PROTACs and SNIPERs.

Minoru Ishikawa, Shusuke Tomoshige, Yosuke Demizu, Mikihiko Naito.

  New therapeutic modalities are needed to address the problem of pathological but undruggable proteins. One possible approach is the induction of protein degradation by chimeric drugs composed of a ubiquitin ligase (E3) ligand coupled to a ligand for the target protein. This article reviews chimeric drugs that decrease the level of specific proteins such as proteolysis targeting chimeric molecules (PROTACs) and specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs), which target proteins for proteasome-mediated degradation. We cover strategies for increasing the degradation activity induced by small molecules, and their scope for application to undruggable proteins.

Keywords:  PROTACs; SNIPERs; chemical protein degradation

DOI:  https://doi.org/10.3390/ph13040074
Cytokine Growth Factor Rev. 2020 Apr 12. pii: S1359-6101(20)30031-9. [Epub ahead of print]

Selective inhibition of Tumor necrosis factor receptor-1 (TNFR1) for the treatment of autoimmune diseases.

Nan Zhang, Ziyi Wang, Yongxing Zhao.

  Anti-TNF biologics have achieved great success in the treatment of autoimmune diseases and have been the most selling biologics on market. However, the anti-TNF biologics have shown some disadvantages such as poor efficacy to some patients and high risk of infection and malignancies during clinical application. Current anti-TNF biologics are antibodies or antibody fragments that bind to TNF-α and subsequently block both TNF-TNFR1 and TNF-TNFR2 signaling. Transgenic animal studies indicate that TNFR1 signaling is responsible for chronic inflammation and cell apoptosis whereas TNFR2 signaling regulates tissue regeneration and inflammation. Recent studies propose to selectively inhibit TNFR1 to enhance efficacy and avoid side effects. In this review, we introduce the biology of TNF-TNFR1 and TNF-TNFR2 signaling, the advantages of selective inhibition of TNF-TNFR1 signaling and research updates on the development of selective inhibitors for TNF-TNFR1 signaling. Antibodies, small molecules and aptamers that selectively inhibit TNFR1 have showed therapeutic potential and less side effects in preclinical studies. Development of selective inhibitors for TNFR1 is a good strategy to enhance the efficacy and reduce the side effects of anti-TNF inhibitors and will be a trend for next-generation of anti-TNF inhibitors.

Keywords:  Inflammation; NF-κB; TNF inhibitors; TNF receptors; TNF-α

DOI:  https://doi.org/10.1016/j.cytogfr.2020.03.002
Cell Death Differ. 2020 Apr 20.

The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L).

Jamie Z Roberts, Caitriona Holohan, Tamas Sessler, Jennifer Fox, Nyree Crawford, Joel S Riley, Hajrah Khawaja, Joanna Majkut, Emma Evergren, Luke M Humphreys, Jennifer Ferris, Catherine Higgins, Margarita Espona-Fiedler, Paul Moynagh, Simon S McDade, Daniel B Longley.

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2's pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1's DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1's recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)'s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

DOI: https://doi.org/10.1038/s41418-020-0539-7
Biochem Biophys Res Commun. 2020 Apr 18. pii: S0006-291X(20)30703-8. [Epub ahead of print]

CARD6 protects against collagen-induced rheumatoid arthritis in mice through attenuating the inflammatory response and joint destruction via suppression of TNFR1/TRAF2 signaling.

Min Zhao, Hongying He, Jianbao Yin.

  Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, characterized by chronic inflammation and bone destruction. However, the pathogenesis that contributes to RA is still unclear. Caspase recruitment domain protein 6 (CARD6) is a typical member of CARD domain-containing proteins, and shows regulatory effects on nuclear factor-κB (NF-κB) activation to meditate inflammation. In the present study, the role of CARD6 in the progression of inflammatory bone erosion in RA was investigated using the in vitro and in vivo experiments. In vitro results indicated that CARD expression was markedly down-regulated in the activated macrophages induced by lipopolysaccharide (LPS), accompanied with time-dependently increased expression of pro-inflammatory cytokines. Notably, over-expressing CARD6 in macrophages by adenoviral (Ad) vector significantly abolished the expression levels of pro-inflammatory cytokines and chemokines. We found that CARD6 over-expression-suppressed inflammatory response was associated with the blockage of tumor necrosis factor receptor-1/tumor necrosis factor receptor-associated factor-2 (TNFR1/TRAF2) signaling, inhibiting NF-κB pathway subsequently. In addition, LPS-induced apoptosis in macrophages was also blunted due to AdCARD6 infection. CARD6-alleviated inflammatory response and apoptotic cell death were further confirmed in TNF-α-stimulated macrophages. Then, the in vivo studies showed that promoting CARD6 expression using adeno-associated virus (AAV) effectively attenuated the severity of arthritis, improved histopathological damage, and hindered the bone erosion in collagen-induced arthritis (CIA) mice. Moreover, pro-inflammatory factors in the joint samples were also markedly decreased in CIA mice with CARD6 over-expression, which was related to the down-regulation of TNFR1/TRAF2/NF-κB signaling pathway. Meanwhile, apoptosis in joint of CIA mice was also ameliorated by AAV-CARD6, as evidenced by the obviously reduced expression of cleaved Caspase-3. These results clearly suggested that CARD6 might have anti-inflammatory and anti-apoptotic effects during RA progression, and thus could be defined as a novel therapeutic target for RA treatment in future.

Keywords:  Bone erosion; CARD6; Inflammation and apoptosis; Rheumatoid arthritis; TNFR1/TRAF2

DOI:  https://doi.org/10.1016/j.bbrc.2020.04.006
J Biol Chem. 2020 Apr 24. pii: jbc.RA119.012351. [Epub ahead of print]

The structure of the extracellular domains of human interleukin 11 α-receptor reveals mechanisms of cytokine engagement.

Riley D Metcalfe, Kaheina Aizel, Courtney O Zlatic, Paul M Nguyen, Craig J Morton, Daisy Sio-Seng Lio, Heung-Chin Cheng, Renwick C J Dobson, Michael W Parker, Paul R Gooley, Tracy L Putoczki, Michael D W Griffin.

  Interleukin 11 (IL-11) activates multiple intracellular signalling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the β-subunit receptor, gp130. Dysregulated IL-11 signalling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signalling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα, and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand binding sites. Molecular dynamics simulations showed that specific mutations destabilise IL-11Rα and may have indirect effects on the cytokine binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11/IL-11Rα complex.

Keywords:  IL6 family cytokine; cancer; cytokine; fibrosis; gp130; inflammation; interleukin; receptor structure-function; signaling; structural biology

DOI:  https://doi.org/10.1074/jbc.RA119.012351
Cell Rep. 2020 Apr 21. pii: S2211-1247(20)30457-5. [Epub ahead of print]31(3): 107547

The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.

Wenxian Wu, Xiaojing Wang, Niklas Berleth, Jana Deitersen, Nora Wallot-Hieke, Philip Böhler, David Schlütermann, Fabian Stuhldreier, Jan Cox, Katharina Schmitz, Sabine Seggewiß, Christoph Peter, Gary Kasof, Anja Stefanski, Kai Stühler, Astrid Tschapek, Axel Gödecke, Björn Stork.

  Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.

Keywords:  MLKL; RIPK1; RIPK3; TNF; ULK1; autophagy; complex I; complex II; necroptosis; necrosome

DOI:  https://doi.org/10.1016/j.celrep.2020.107547
Blood. 2020 Apr 23. pii: blood.2019002654. [Epub ahead of print]

LUBAC accelerates B-cell lymphomagenesis by conferring B cells resistance to genotoxic stress.

Tomoyasu Jo, Momoko Nishikori, Yasunori Kogure, Hiroshi Arima, Katsuhiro Sasaki, Yoshiteru Sasaki, Tomoko Nakagawa, Fumie Iwai, Shuji Momose, Aki Shiraishi, Hiroshi Kiyonari, Noritaka Kagaya, Tetsuo Onuki, Kazuo Shin-Ya, Minoru Yoshida, Keisuke Kataoka, Seishi Ogawa, Kazuhiro Iwai, Akifumi Takaori-Kondo.

Linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-kB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL), and expression of HOIP which parallels LUBAC activity is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-kB activation, and the analysis of human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-kB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-kB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicates that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death, and is a suitable therapeutic target for B-cell lymphomas.

DOI: https://doi.org/10.1182/blood.2019002654
Signal Transduct Target Ther. 2020 Apr 24. 5(1): 42

The CRL3BTBD9 E3 ubiquitin ligase complex targets TNFAIP1 for degradation to suppress cancer cell migration.

Lihui Li, Wenjuan Zhang, Yue Liu, Xiaojun Liu, Lili Cai, Jihui Kang, Yunjing Zhang, Wenlian Chen, Changsheng Dong, Yanmei Zhang, Mingsong Wang, Wenyi Wei, Lijun Jia.

Tumor necrosis factor alpha-induced protein 1 (TNFAIP1) modulates a plethora of important biological processes, including tumorigenesis and cancer cell migration. However, the regulatory mechanism of TNFAIP1 degradation remains largely elusive. In the present study, with a label-free quantitative proteomic approach, TNFAIP1 was identified as a novel ubiquitin target of the Cullin-RING E3 ubiquitin ligase (CRL) complex. More importantly, Cul3-ROC1 (CRL3), a subfamily of CRLs, was identified to specifically interact with TNFAIP1 and promote its polyubiquitination and degradation. Mechanistically, BTBD9, a specific adaptor component of CRL3 complex, was further defined to bind and promote the ubiquitination and degradation of TNFAIP1 in cells. As such, downregulation of BTBD9 promoted lung cancer cell migration by upregulating the expression of TNFAIP1, whereas TNFAIP1 deletion abrogated this effect. Finally, bioinformatics and clinical sample analyses revealed that BTBD9 was downregulated while TNFAIP1 was overexpressed in human lung cancer, which was associated with poor overall survival of patients. Taken together, these findings reveal a previously unrecognized mechanism by which the CRL3BTBD9 ubiquitin ligase controls TNFAIP1 degradation to regulate cancer cell migration.

DOI: https://doi.org/10.1038/s41392-020-0140-z
Mol Cell. 2020 Apr 02. pii: S1097-2765(20)30164-7. [Epub ahead of print]

Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination.

Fujie Yan, Chunfeng Huang, Xiaofei Wang, Jiaxing Tan, Sen Cheng, Muyang Wan, Zhao Wang, Shuangyu Wang, Shuhui Luo, Arong Li, Xing Guo, Mingguang Feng, Xiaoyun Liu, Yongqun Zhu, Yan Zhou.

  Ubiquitination is essential for numerous eukaryotic cellular processes. Here, we show that the type III effector CteC from Chromobacterium violaceum functions as an adenosine diphosphate (ADP)-ribosyltransferase that specifically modifies ubiquitin via threonine ADP-ribosylation on residue T66. The covalent modification prevents the transfer of ubiquitin from ubiquitin-activating enzyme E1 to ubiquitin-conjugating enzyme E2, which inhibits subsequent ubiquitin activation by E2 and E3 enzymes in the ubiquitination cascade and leads to the shutdown of polyubiquitin synthesis in host cells. This unique modification also causes dysfunction of polyubiquitin chains in cells, thereby blocking host ubiquitin signaling. The disruption of host ubiquitination by CteC plays a crucial role in C. violaceum colonization in mice during infection. CteC represents a family of effector proteins in pathogens of hosts from different kingdoms. All the members of this family specifically ADP-ribosylate ubiquitin. The action of CteC reveals a new mechanism for interfering with host ubiquitination by pathogens.

Keywords:  ADP-ribosylation; Chromobacterium violaceum; NF-κB; bacterial pathogens; effector protein; polyubiquitin chain; posttranslational modification; type III secretion system; ubiquitin; ubiquitination

DOI:  https://doi.org/10.1016/j.molcel.2020.03.016
Pharmacol Ther. 2020 Apr 20. pii: S0163-7258(20)30078-4. [Epub ahead of print] 107550

Molecular imaging of inflammation - Current and emerging technologies for diagnosis and treatment.

Neil MacRitchie, Marina Frleta-Gilchrist, Atsuko Sugiyama, Tristan Lawton, Iain B McInnes, Pasquale Maffia.

  Inflammation is a key factor in multiple diseases including primary immune-mediated inflammatory diseases e.g. rheumatoid arthritis but also, less obviously, in many other common conditions, e.g. cardiovascular disease and diabetes. Together, chronic inflammatory diseases contribute to the majority of global morbidity and mortality. However, our understanding of the underlying processes by which the immune response is activated and sustained is limited by a lack of cellular and molecular information obtained in situ. Molecular imaging is the visualization, detection and quantification of molecules in the body. The ability to reveal information on inflammatory biomarkers, pathways and cells can improve disease diagnosis, guide and monitor therapeutic intervention and identify new targets for research. The optimum molecular imaging modality will possess high sensitivity and high resolution and be capable of non-invasive quantitative imaging of multiple disease biomarkers while maintaining an acceptable safety profile. The mainstays of current clinical imaging are computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US) and nuclear imaging such as positron emission tomography (PET). However, none of these have yet progressed to routine clinical use in the molecular imaging of inflammation, therefore new approaches are required to meet this goal. This review sets out the respective merits and limitations of both established and emerging imaging modalities as clinically useful molecular imaging tools in addition to potential theranostic applications.

Keywords:  Inflammation; Magnetic resonance imaging (MRI); Molecular imaging; Photoacoustic imaging (PAI); Positron emission tomography (PET); Surface-enhanced Raman spectroscopy (SERS)

DOI:  https://doi.org/10.1016/j.pharmthera.2020.107550
Biochem Biophys Res Commun. 2020 Apr 19. pii: S0006-291X(20)30675-6. [Epub ahead of print]

Inhibition of RIPK1/RIPK3 ameliorates osteoclastogenesis through regulating NLRP3-dependent NF-κB and MAPKs signaling pathways.

Shuang Liang, Zhenghao Nian, Kun Shi.

  Osteoblast-induced bone formation and osteoclast-regulated bone resorption are the essential events contributing to bone homeostasis. It is critical to investigate the underlying molecular mechanisms. In this study, we explored the effects of receptor-interacting serine-threonine kinases (RIPKs) on osteoclastogenesis and bone loss in vitro and in vivo. We found that both RIPK1 and RIPK3 expression levels were highly up-regulated during osteoclastogenesis. Inhibiting RIPK1 and RIPK3 by their inhibitors Necrostatin-1 (Nec-1) and GSK-872, respectively, showed effective activities against osteoclast differentiation and bone resorption induced by receptor activator of nuclear factor-κB ligand (Rankl). Osteoclast-specific gene expression levels were also impeded by RIPK1/RIPK3 blockage in a time-dependent manner. Subsequently, we found that the pyrin domain-containing protein 3 (NLRP3) inflammasome stimulated by Rankl during osteoclastogenesis was greatly inhibited by Nec-1 and GSK-872. Additionally, reducing RIPK1/RIPK3 overtly reduced the activation of NF-κB (p65) and mitogen-activated protein kinases (MAPKs) signaling during Rankl-induced osteoclast formation. Notably, adenovirus-regulated NLRP3 over-expression significantly abrogated the inhibitory effects of Nec-1 and GSK-872 on NF-κB and MAPKs signaling pathways, as well as the osteoclastogenesis. Finally, the in vivo studies indicated that suppressing RIPK1/RIPK3 could effectively ameliorate ovariectomy (OVX)-induced bone loss in mice through repressing osteoclastogenesis, as proved by the clearly down-regulated number of osteoclasts via histological staining. In conclusion, our study elucidated that restraining RIPK1/RIPK3 could hinder osteoclastogenesis and attenuate bone loss through suppressing NLRP3-dependent NF-κB and MAPKs signaling pathways. Therefore, targeting RIPK1/RIPK3 signaling might be a potential therapeutic strategy to develop effective treatments against osteoclast-related bone lytic diseases.

Keywords:  MAPKs; NF-κB; NLRP3; Osteoclastogenesis; RIPK1/RIPK3

DOI:  https://doi.org/10.1016/j.bbrc.2020.03.177
Bioorg Med Chem Lett. 2020 Apr 18. pii: S0960-894X(20)30302-4. [Epub ahead of print] 127202

Monovalent protein-degraders - Insights and future perspectives.

Ivan Cornella-Taracido, Carlos Garcia-Echeverria.

  The therapeutic potential of interfering with dysregulated proteins by inducing its selective degradation has been pursued using different mechanisms. In the present article, we review representative examples of monovalent protein-degraders that, contrary to the proteolysis targeting chimeras, achieve target degradation without displaying recognition motifs for the recruitment of E3 ubiquitin ligases. We also highlight new technologies and assays that may brought to bear on the discovery of common elements that could predict and enable the selective degradation of pathogenic targets by monovalent protein-degraders. The successful application of these methods would pave the way to the advancement of new drugs with unique efficacy and tolerability properties.

Keywords:  Degradation; Monovalent; PROTAC

DOI:  https://doi.org/10.1016/j.bmcl.2020.127202
Nature. 2020 Apr;580(7804): 542-547

Separase-triggered apoptosis enforces minimal length of mitosis.

Susanne Hellmuth, Olaf Stemmann.

Prolonged mitosis often results in apoptosis1. Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery2. Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death3,4. Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-XL by separase. Cleavage not only prevents MCL1 and BCL-XL from sequestering pro-apoptotic BAK, but also converts them into active promoters of death in mitosis. Our data strongly suggest that the deadliest cleavage fragment, the C-terminal half of MCL1, forms BAK/BAX-like pores in the mitochondrial outer membrane. MCL1 and BCL-XL are turned into separase substrates only upon phosphorylation by NEK2A. Early mitotic degradation of this kinase is therefore crucial for preventing apoptosis upon scheduled activation of separase in metaphase. Speeding up mitosis by abrogation of the spindle assembly checkpoint results in a temporal overlap of the enzymatic activities of NEK2A and separase and consequently in cell death. We propose that NEK2A and separase jointly check on spindle assembly checkpoint integrity and eliminate cells that are prone to chromosome missegregation owing to accelerated progression through early mitosis.

DOI: https://doi.org/10.1038/s41586-020-2187-y
Immunol Lett. 2020 Apr 18. pii: S0165-2478(20)30005-5. [Epub ahead of print]223 1-9

Toll-like receptors: Triggers of regulated cell death and promising targets for cancer therapy.

Yun Yang, Rang Feng, Yuan-Zhong Wang, Hong-Wu Sun, Quan-Ming Zou, Hai-Bo Li.

  Toll-like receptors (TLRs) belong to a family of pattern recognition receptors (PRRs). It is well known that TLRs play an essential role in activating innate and adaptive immune responses. TLRs are involved in mediating inflammatory responses and maintaining epithelial barrier homeostasis, and they are highly likely to activate various signalling pathways during cancer chemotherapy. For a long time, much research focused on the immune modulating function of TLRs in cancer genesis, pathology and therapeutic strategies. However, recent reports have suggested that except for the innate and adaptive immune responses that they initiate, TLRs can signal to induce regulated cell death (RCD), which also plays an important role in the antitumor process. TLR agonists also have been investigated as cancer therapeutic agents under clinical evaluation. In this review, we focused on the mechanism of RCD induced by TLR signals and the important role that they play in anticancer therapy combined with recent experimental and clinical trial data to discuss the possibility of TLRs as promising targets for cancer therapy. TLRs represent triggers of regulated cell death and targets for cancer therapy. The molecular mechanisms of TLR-induced RCD and relationship between TLR-signalling pathways and cancer remain to be investigated by further studies.

Keywords:  Toll-like receptors; cancer therapy; regulated cell death; review

DOI:  https://doi.org/10.1016/j.imlet.2020.04.002
Biochim Biophys Acta Gene Regul Mech. 2020 Apr 19. pii: S1874-9399(19)30348-7. [Epub ahead of print] 194550

Learning from mouse models of MLL fusion gene-driven acute leukemia.

Juerg Schwaller.

5-10% of human acute leukemias carry chromosomal translocations involving the mixed lineage leukemia (MLL) gene that result in the expression of chimeric protein fusing MLL to >80 different partners of which AF4, ENL and AF9 are the most prevalent. In contrast to many other leukemia-associated mutations, several MLL-fusions are powerful oncogenes that transform hematopoietic stem cells but also more committed progenitor cells. Here, I review different approaches that were used to express MLL fusions in the murine hematopoietic system which often, but not always, resulted in highly penetrant and transplantable leukemias that closely phenocopied the human disease. Due to its simple and reliable nature, reconstitution of irradiated mice with bone marrow cells retrovirally expressing the MLL-AF9 fusion became the most frequently in vivo model to study the biology of acute myeloid leukemia (AML). I review some of the most influential studies that used this model to dissect critical protein interactions, the impact of epigenetic regulators, microRNAs and microenvironment-dependent signals for MLL fusion-driven leukemia. In addition, I highlight studies that used this model for shRNA- or genome editing-based screens for cellular vulnerabilities that allowed to identify novel therapeutic targets of which some entered clinical trials. Finally, I discuss some inherent characteristics of the widely used mouse model based on retroviral expression of the MLL-AF9 fusion that can limit general conclusions for the biology of AML. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.

DOI: https://doi.org/10.1016/j.bbagrm.2020.194550
Sci Transl Med. 2020 Apr 22. pii: eaaw3172. [Epub ahead of print]12(540):

Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats.

Yaohui Wang, Hailong Zhang, Zhizeng Wang, Yinxiang Wei, Mingli Wang, Meichen Liu, Xuance Wang, Yinan Jiang, Gongning Shi, Dongmei Zhao, Zhengyan Yang, Zhiguang Ren, Jing Li, Zhenkai Zhang, Zhenfeng Wang, Bei Zhang, Beibei Zong, Xueke Lou, Chengguo Liu, Zihui Wang, Hao Zhang, Ningya Tao, Xuefang Li, Xingkun Zhang, Yafei Guo, Yang Ye, Yu Qi, Hui Li, Man Wang, Rongxin Guo, Guanchang Cheng, Shulian Li, Jun Zhang, Guangchao Liu, Lihui Chai, Qiang Lou, Xia Li, Xiukun Cui, Erhe Gao, Zheng Dong, Yanzhong Hu, Youhai H Chen, Yuanfang Ma.

Myocardial infarction (MI) is a leading cause of death worldwide for which there is no cure. Although cardiac cell death is a well-recognized pathological mechanism of MI, therapeutic blockade of cell death to treat MI is not straightforward. Death receptor 5 (DR5) and its ligand TRAIL [tumor necrosis factor (TNF)-related apoptosis-inducing ligand] are up-regulated in MI, but their roles in pathological remodeling are unknown. Here, we report that blocking TRAIL with a soluble DR5 immunoglobulin fusion protein diminished MI by preventing cardiac cell death and inflammation in rats, pigs, and monkeys. Mechanistically, TRAIL induced the death of cardiomyocytes and recruited and activated leukocytes, directly and indirectly causing cardiac injury. Transcriptome profiling revealed increased expression of inflammatory cytokines in infarcted heart tissue, which was markedly reduced by TRAIL blockade. Together, our findings indicate that TRAIL mediates MI directly by targeting cardiomyocytes and indirectly by affecting myeloid cells, supporting TRAIL blockade as a potential therapeutic strategy for treating MI.

DOI: https://doi.org/10.1126/scitranslmed.aaw3172
Front Immunol. 2020 ;11 536

RIPK3 and Caspase-1/11 Are Necessary for Optimal Antigen-Specific CD8 T Cell Response Elicited by Genetically Modified Listeria monocytogenes.

Aamir Rana, Felipe Campos de Almeida, Henry A Paico Montero, Maryanne M Gonzales Carazas, Karina R Bortoluci, Subash Sad, Gustavo P Amarante-Mendes.

  Efficient induction of effector and long-term protective antigen-specific CD8+ T memory response by vaccination is essential to eliminate malignant and pathogen-infected cells. Intracellular infectious bacteria, including Listeria monocytogenes, have been considered potent vectors to carry multiple therapeutic proteins and generate antigen-specific CD8+ T cell responses. Although the role of molecules involved in inflammatory cell death pathways, such as necroptosis (RIPK3-mediated) and pyroptosis (Caspase-1/11-mediated), as effectors of immune response against intracellular bacteria are relatively well understood, their contribution to the adjuvant effect of recombinant bacterial vectors in the context of antigen-specific CD8+ T cell response remained obscure. Therefore, we evaluated the impact of RIPK3 and Caspase-1/11 (Casp-1/11) individual and combined deficiencies on the modulation of antigen-specific CD8+ T cell response during vaccination of mice with ovalbumin-expressing L. monocytogenes (LM-OVA). We observed that Casp-1/11 but not RIPK3 deficiency negatively impacts the capacity of mice to clear LM-OVA. Importantly, both RIPK3 and Casp-1/11 are necessary for optimal LM-OVA-mediated antigen-specific CD8+ T cell response, as measured by in vivo antigen-specific CD8+ T cell proliferation, target cell elimination, and cytokine production. Furthermore, Casp-1/11 and Casp-1/11/RIPK3 combined deficiencies restrict the early initiation of antigen-specific CD8+ T cell memory response. Taken together, our findings demonstrate that RIPK3 and Casp-1/11 influence the quality of CD8+ T cell responses induced by recombinant L. monocytogenes vectors.

Keywords:  Casp-1/11; Effector CD8+ T cells; Listeria monocytogenes; RIPK3; ovalbumin

DOI:  https://doi.org/10.3389/fimmu.2020.00536
Bioorg Med Chem Lett. 2020 Apr 04. pii: S0960-894X(20)30264-X. [Epub ahead of print] 127167

Discovery and biological evaluation of proteolysis targeting chimeras (PROTACs) as an EGFR degraders based on osimertinib and lenalidomide.

Kailun He, Zhuo Zhang, Wenbing Wang, Xiaoliang Zheng, Xiaoju Wang, Xingxian Zhang.

  Epidermal growth factor receptor (EGFR) is one of the important and valuable drug targets. Overexpression of EGFR is associated with the development of many types of cancer. In this study, three PROTACs small molecules (16a-16c) were designed, synthesized and evaluated for their cytotoxicity against the growth in different NSCLC cell line and the degradation effect. The bioassay results indicated that 16c has a good inhibition in PC9 cells and H1975 cells, and the corresponding IC50 value was 0.413 μM and 0.657 μM, respectively. Western blotting results demonstrated that compound 16c could serve as an effective EGFRdel19-targeting degrader in PC9 cells.

Keywords:  CRBN; EGFR-TK degrader; Lenalidomide; Osimertinib; Proteolysis targeting chimera

DOI:  https://doi.org/10.1016/j.bmcl.2020.127167
EMBO J. 2020 Apr 23. e101573

Suppressing PARylation by 2',5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death.

Anna A Kondratova, HyeonJoo Cheon, Beihua Dong, Elise G Holvey-Bates, Metis Hasipek, Irina Taran, Christina Gaughan, Babal K Jha, Robert H Silverman, George R Stark.

  High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis in vitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.

Keywords:  PARP1; PARylation; oligoadenylate synthetase; parthanatos

DOI:  https://doi.org/10.15252/embj.2019101573
Nat Immunol. 2020 May;21(5): 535-545

Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs.

Tomoya Muto, Callum S Walker, Kwangmin Choi, Kathleen Hueneman, Molly A Smith, Zartash Gul, Guillermo Garcia-Manero, Averil Ma, Yi Zheng, Daniel T Starczynowski.

Despite evidence of chronic inflammation in myelodysplastic syndrome (MDS) and cell-intrinsic dysregulation of Toll-like receptor (TLR) signaling in MDS hematopoietic stem and progenitor cells (HSPCs), the mechanisms responsible for the competitive advantage of MDS HSPCs in an inflammatory milieu over normal HSPCs remain poorly defined. Here, we found that chronic inflammation was a determinant for the competitive advantage of MDS HSPCs and for disease progression. The cell-intrinsic response of MDS HSPCs, which involves signaling through the noncanonical NF-κB pathway, protected these cells from chronic inflammation as compared to normal HSPCs. In response to inflammation, MDS HSPCs switched from canonical to noncanonical NF-κB signaling, a process that was dependent on TLR-TRAF6-mediated activation of A20. The competitive advantage of TLR-TRAF6-primed HSPCs could be restored by deletion of A20 or inhibition of the noncanonical NF-κB pathway. These findings uncover the mechanistic basis for the clonal dominance of MDS HSPCs and indicate that interfering with noncanonical NF-κB signaling could prevent MDS progression.

DOI: https://doi.org/10.1038/s41590-020-0663-z
Expert Opin Ther Targets. 2020 Apr 20.

The necroptosis pathway and its role in age-related neurodegenerative diseases: will it open up new therapeutic avenues in the next decade?

Natalia Salvadores, Felipe A Court.

  Introduction: Necroptosis is a programmed form of necrotic cell death. Growing evidence demonstrates that necroptosis contributes to cell demise in different pathological conditions including age-dependent neurodegenerative diseases (NDs). These findings opened new avenues for understanding the mechanisms of neuronal loss in these diseases, which might eventually translate into novel therapeutic interventions.Areas covered: We reviewed key aspects of necroptosis, in health and disease, focusing on evidence demonstrating its involvement in the pathogenesis of age-related NDs. We then highlight the engagement of this pathway in the mechanism of axonal degeneration. We searched on PubMed the literature regarding necroptosis published between 2008-2020 and reviewed all publications were necroptosis was studied in the context of age-related NDs.Expert opinion: Axonal loss and neuronal death are the ultimate consequences of NDs that translate into disease phenotypes. Targeting degenerative mechanisms of the neuron appears as a strategy that might cover a wide range of diseases. Thus, the participation of necroptosis as a common mediator of neuronal demise emerges as a promising target for therapeutic intervention. Considering evidence demonstrating that necroptosis mediates axonal degeneration, we propose and discuss the potential of targeting necroptosis-mediated axonal destruction as a strategy to tackle NDs before neuronal loss occurs.

Keywords:  aging; axonal degeneration; necroaxoptosis; necroptosis; neurodegenerative diseases; neuroprotection

DOI:  https://doi.org/10.1080/14728222.2020.1758668
Allergy. 2020 Apr 23.

Tnfaip3 expression in pulmonary conventional type 1 Langerin-expressing dendritic cells regulates T helper 2-mediated airway inflammation in mice.

Heleen Vroman, Denise van Uden, Ingrid M Bergen, Jennifer A C van Hulst, Melanie Lukkes, Geert van Loo, Björn E Clausen, Louis Boon, Bart N Lambrecht, Hamida Hammad, Rudi W Hendriks, Mirjam Kool.

   BACKGROUND: Conventional type 1 dendritic cells (cDC1s) control antiviral and antitumor immunity by inducing antigen-specific cytotoxic CD8+ T-cell responses. Controversy exists whether cDC1s also control CD4+ T helper 2 (Th2) cell responses, since suppressive and activating roles have been reported. DC activation status, controlled by the transcription factor NF-κB, might determine the precise outcome of Th-cell differentiation upon encounter with cDC1s. To investigate the role of activated cDC1s in Th2-driven immune responses, pulmonary cDC1s were activated by targeted deletion of A20/Tnfaip3, a negative regulator of NF-κB signaling METHODS: To target pulmonary cDC1s, Cd207 (Langerin)-mediated excision of A20/Tnfaip3 was used, generating Tnfaip3fl/fl xCd207+/cre (Tnfaip3Lg-KO ) mice. Mice were exposed to house dust mite (HDM) to provoke Th2-mediated immune responses.
RESULTS: Mice harboring Tnfaip3-deficient cDC1s did not develop Th2-driven eosinophilic airway inflammation upon HDM exposure, but rather showed elevated numbers of IFNγ-expressing CD8+ T-cells. In addition, Tnfaip3Lg-KO mice harbored increased numbers of IL-12-expressing cDC1s and elevated PD-L1 expression in all pulmonary DC subsets. Blocking either IL-12 or IFNγ in Tnfaip3Lg-KO mice restored Th2-responses, whereas administration of recombinant IFNγ during HDM sensitization in C57Bl/6 mice blocked Th2-development.
CONCLUSIONS: These findings indicate that the activation status of cDC1s, shown by their specific expression of co-inhibitory molecules and cytokines, critically contributes to the development of Th2-cell-mediated disorders, most likely by influencing IFNγ production in CD8+ T-cells.

Keywords:  A20/Tnfaip3; CD8+ T-cells; Th2-driven airway inflammation; Type 1 conventional dendritic cells; interferon gamma

DOI:  https://doi.org/10.1111/all.14334
MAbs. 2020 Jan-Dec;12(1):12(1): 1752529

Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats.

Ami Miller, Stephen Carr, Terry Rabbitts, Hanif Ali.

  The success of therapeutic antibodies is largely attributed for their exquisite specificity, homogeneity, and functionality. There is, however, a need to engineer antibodies to extend and enhance their potency. One parameter is functional affinity augmentation, since antibodies matured in vivo have a natural affinity threshold. Generation of multivalent antibodies is one option capable of surpassing this affinity threshold through increased avidity. In this study, we present a novel platform consisting of an array of multivalent antibody formats, termed Quads, generated using the self-assembling tetramerization domain from p53. We demonstrate the versatility of this tetramerization domain by engineering anti-tumor necrosis factor (TNF) Quads that exhibit major increases in binding potency and in neutralizing TNF-mediated cytotoxicity compared to parental anti-TNF molecules. Further, Quads are amenable to fusion with different binding domains, allowing generation of novel multivalent monospecific and bispecific formats. Quads are thus a novel group of molecules that can be engineered to yield potential therapeutics with novel modalities and potencies.

Keywords:  Multivalent; antibody; avidity; bispecific; octavalent; tetravalent

DOI:  https://doi.org/10.1080/19420862.2020.1752529
Nat Commun. 2020 Apr 20. 11(1): 1913

TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response.

Jianwen Chen, Ruirui He, Wanwei Sun, Ru Gao, Qianwen Peng, Liwen Zhu, Yanyun Du, Xiaojian Ma, Xiaoli Guo, Huazhi Zhang, Chengcheng Tan, Junhan Wang, Wei Zhang, Xiufang Weng, Jianghong Man, Hermann Bauer, Qing K Wang, Bradley N Martin, Cun-Jin Zhang, Xiaoxia Li, Chenhui Wang.

The TAGAP gene locus has been linked to several infectious diseases or autoimmune diseases, including candidemia and multiple sclerosis. While previous studies have described a role of TAGAP in T cells, much less is known about its function in other cell types. Here we report that TAGAP is required for Dectin-induced anti-fungal signaling and proinflammatory cytokine production in myeloid cells. Following stimulation with Dectin ligands, TAGAP is phosphorylated by EPHB2 at tyrosine 310, which bridges proximal Dectin-induced EPHB2 activity to downstream CARD9-mediated signaling pathways. During Candida albicans infection, mice lacking TAGAP mount defective immune responses, impaired Th17 cell differentiation, and higher fungal burden. Similarly, in experimental autoimmune encephalomyelitis model of multiple sclerosis, TAGAP deficient mice develop significantly attenuated disease. In summary, we report that TAGAP plays an important role in linking Dectin-induced signaling to the promotion of effective T helper cell immune responses, during both anti-fungal host defense and autoimmunity.

DOI: https://doi.org/10.1038/s41467-020-15564-7
Front Immunol. 2020 ;11 558

TNF-Signaling Modulates Neutrophil-Mediated Immunity at the Feto-Maternal Interface During LPS-Induced Intrauterine Inflammation.

Pietro Presicce, Monica Cappelletti, Paranthaman Senthamaraikannan, Feiyang Ma, Marco Morselli, Courtney M Jackson, Shibabrata Mukherjee, Lisa A Miller, Matteo Pellegrini, Alan H Jobe, Claire A Chougnet, Suhas G Kallapur.

  Accumulation of activated neutrophils at the feto-maternal interface is a defining hallmark of intrauterine inflammation (IUI) that might trigger an excessive immune response during pregnancy. Mechanisms responsible of this massive neutrophil recruitment are poorly investigated. We have previously showed that intraamniotic injection of LPS in rhesus macaques induced a neutrophil predominant inflammatory response similar to that seen in human IUI. Here, we demonstrate that anti-TNF antibody (Adalimumab) inhibited ~80% of genes induced by LPS involved in inflammatory signaling and innate immunity in chorio-decidua neutrophils. Consistent with the gene expression data, TNF-blockade decreased LPS-induced neutrophil accumulation and activation at the feto-maternal interface. We also observed a reduction in IL-6 and other pro-inflammatory cytokines but not prostaglandins concentrations in the amniotic fluid. Moreover, TNF-blockade decreased mRNA expression of inflammatory cytokines in the chorio-decidua but not in the uterus, suggesting that inhibition of TNF-signaling decreased the inflammation in a tissue-specific manner within the uterine compartment. Taken together, our results demonstrate a predominant role for TNF-signaling in modulating the neutrophilic infiltration at the feto-maternal interface during IUI and suggest that blockade of TNF-signaling could be considered as a therapeutic approach for IUI, the major leading cause of preterm birth.

Keywords:  chorioamnionitis; inflammation; innate immunity; neutrophils; reproductive immunology

DOI:  https://doi.org/10.3389/fimmu.2020.00558
Int J Mol Sci. 2020 Apr 21. pii: E2895. [Epub ahead of print]21(8):

Comparison of Target Recognition by TRAF1 and TRAF2.

Chang Min Kim, Hyun Ho Park.

  Although TRAF1 and TRAF2 share common receptors and have extremely conserved amino acid residues, recent studies have shown that key differences in receptor binding preferences with different affinities exist, which might be important for their different functions in TRAF-mediated signal transduction. To better understand TRAF1 and TRAF2 signaling, we analyzed and compared their receptor binding-affinities. Our study revealed that TRADD, TANK, and caspase-2 bind to both TRAF1 and TRAF2 with different affinities in vitro. Sequence and structural analyses revealed that S454 on TRAF2 (corresponding to A369 of TRAF1) is critical for the binding of TRADD, and F347 on TRAF1 (corresponding to L432 of TRAF2) is a critical determinant for high affinity binding of TANK and caspase-2.

Keywords:  TRADD; TRAF; apoptosis; inflammation; protein interaction

DOI:  https://doi.org/10.3390/ijms21082895
J Med Chem. 2020 Apr 24.

Selective Janus Kinase 2 (JAK2) Pseudokinase Ligands with a Diaminotriazole Core.

Maria-Elena Liosi, Stefan G Krimmer, Ana S Newton, Thomas Dawson, David E Puleo, Kara J Cutrona, Yoshihisa Suzuki, Joseph Schlessinger, William L Jorgensen.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules which preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.

DOI: https://doi.org/10.1021/acs.jmedchem.0c00192
Biomolecules. 2020 Apr 15. pii: E612. [Epub ahead of print]10(4):

Novel Apoptotic Mediators Identified by Conservation of Vertebrate Caspase Targets.

Nina Gubina, Dominique Leboeuf, Konstantin Piatkov, Maxim Pyatkov.

  Caspases are proteases conserved throughout Metazoans and responsible for initiating and executing the apoptotic program. Currently, there are over 1800 known apoptotic caspase substrates, many of them known regulators of cell proliferation and death, which makes them attractive therapeutic targets. However, most caspase substrates are by-standers, and identifying novel apoptotic mediators amongst all caspase substrates remains an unmet need. Here, we conducted an in silico search for significant apoptotic caspase targets across different species within the Vertebrata subphylum, using different criteria of conservation combined with structural features of cleavage sites. We observed that P1 aspartate is highly conserved while the cleavage sites are extensively variable and found that cleavage sites are located primarily in coiled regions composed of hydrophilic amino acids. Using the combination of these criteria, we determined the final list of the 107 most relevant caspase substrates including 30 novel targets previously unknown for their role in apoptosis and cancer. These newly identified substrates can be potential regulators of apoptosis and candidates for anti-tumor therapy.

Keywords:  N-degron pathway; apoptosis; caspases; cleavage site; conservation; evolution; regulation

DOI:  https://doi.org/10.3390/biom10040612
Cell Death Dis. 2020 Apr 24. 11(4): 282

A necroptotic-independent function of MLKL in regulating endothelial cell adhesion molecule expression.

Jialin Dai, Chonghe Zhang, Lin Guo, Hao He, Kai Jiang, Yingying Huang, Xixi Zhang, Haibing Zhang, Wu Wei, Yaoyang Zhang, Lihua Lu, Junhao Hu.

Mixed-lineage kinase domain-like protein (MLKL) is known as the terminal executor of necroptosis. However, its function outside of necroptosis is still not clear. Herein, we demonstrate that MLKL promotes vascular inflammation by regulating the expression of adhesion molecules ICAM1, VCAM1, and E-selectin in endothelial cells (EC). MLKL deficiency suppresses the expression of these adhesion molecules, thereby reducing EC-leukocyte interaction in vitro and in vivo. Mechanistically, we show that MLKL interacts with RBM6 to promote the mRNA stability of adhesion molecules. In conclusion, this study identified a novel role of MLKL in regulating endothelial adhesion molecule expression and local EC-leukocyte interaction during acute inflammation.

DOI: https://doi.org/10.1038/s41419-020-2483-3