bims-tunefa 2020-03-15 papers

bims-tunefa

Biomed News

on Tumor necrosis factor superfamily and post-translational modifications

Issue of 2020–03–15
forty-four papers selected by
John Silke, Walter and Eliza Hall Institute of Medical Research

A Systematic Test of Receptor Binding Kinetics for Ligands in Tumor Necrosis Factor Superfamily by Computational Simulations.
Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1.
RIPK3 collaborates with GSDMD to drive tissue injury in lethal polymicrobial sepsis.
New insights into the regulation of apoptosis, necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8.
Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.
Azo-PROTAC: novel light-controlled small-molecule tool for protein knockdown.
RNA-Binding Protein HuR Promotes Th17 Cell Differentiation and Can Be Targeted to Reduce Autoimmune Neuroinflammation.
Toll-like Receptors and the Control of Immunity.
Immune Recognition of Pathogen-Derived Glycolipids Through Mincle.
The essentials of developmental apoptosis.
Proteasome inhibitors and Smac mimetics cooperate to induce cell death in Diffuse Large B-cell Lymphoma by stabilizing NOXA and triggering mitochondrial apoptosis.
UBB pseudogene 4 encodes functional ubiquitin variants.
Proteolysis targeting chimeras (PROTACs) for epigenetics research.
Monobodies as enabling tools for structural and mechanistic biology.
Combined reduction in the expression of MCL-1 and BCL-2 reduces organismal size in mice.
Pleiotropic roles of tankyrase/PARP proteins in the establishment and maintenance of human naïve pluripotency.
Regulation of Cullin-RING E3 ligase dynamics by Inositol hexakisphosphate.
Ubiquitin-Fold Modifier-1 Participates in the Diabetic Inflammatory Response by Regulating NF-κB p65 Nuclear Translocation and the Ubiquitination and Degradation of IκBα.
The many flavors of monocyte/macrophage--endothelial cell interactions.
Decorating bacteria with self-assembled synthetic receptors.
Transferrin Receptor Is a Specific Ferroptosis Marker.
Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis.
Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies.
Old Drugs, New Uses: Drug Repurposing in Hematological Malignancies.
Toward targeting inflammasomes: insights into their regulation and activation.
Treatment of immune checkpoint inhibitor-induced inflammatory arthritis.
The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis.
Recent Advances in the Design and Development of anticancer molecules based on PROTAC technology.
Caspase-8: The double-edged sword.
Ninjurin-1 upregulated by TNFα receptor 1 stimulates monocyte adhesion to human TNFα-activated endothelial cells; benefic effects of amlodipine.
A Review on Poly (ADP-ribose) Polymerase (PARP) Inhibitors and Synthetic Methodologies.
Bifunctional Molecules beyond PROTACs.
An Extended Conformation for K48 Ubiquitin Chains Revealed by the hRpn2:Rpn13:K48-Diubiquitin Structure.
BET protein inhibitor JQ1 downregulates chromatin accessibility and suppresses metastasis of gastric cancer via inactivating RUNX2/NID1 signaling.
Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.
A hack allows microscopes to capture the ultrafast and ultrasmall.
NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture.
The Trend of ripk1/ripk3 and mlkl Mediated Necroptosis Pathway in Patients with Different Stages of Prostate Cancer as Promising Progression Biomarkers.
Purinergic signalling, DAMPs and inflammation.
IFN-Stimulated Gene 15 Is an Alarmin that Boosts the CTL Response via an Innate, NK Cell-Dependent Route.
Innate Lymphocytes in Psoriasis.
Interferon inducible GBPs restrict Burkholderia thailandensis motility induced cell-cell fusion.
"Mind the gap": What patients and clinicians believe is "unknown" about psoriasis.
RUNX1: an emerging therapeutic target for cardiovascular disease.

Int J Mol Sci. 2020 Mar 05. pii: E1778. [Epub ahead of print]21(5):

A Systematic Test of Receptor Binding Kinetics for Ligands in Tumor Necrosis Factor Superfamily by Computational Simulations.

Zhaoqian Su, Yinghao Wu.

  Ligands in the tumor necrosis factor (TNF) superfamily are one major class of cytokines that bind to their corresponding receptors in the tumor necrosis factor receptor (TNFR) superfamily and initiate multiple intracellular signaling pathways during inflammation, tissue homeostasis, and cell differentiation. Mutations in the genes that encode TNF ligands or TNFR receptors result in a large variety of diseases. The development of therapeutic treatment for these diseases can be greatly benefitted from the knowledge on binding properties of these ligand-receptor interactions. In order to complement the limitations in the current experimental methods that measure the binding constants of TNF/TNFR interactions, we developed a new simulation strategy to computationally estimate the association and dissociation between a ligand and its receptor. We systematically tested this strategy to a comprehensive dataset that contained structures of diverse complexes between TNF ligands and their corresponding receptors in the TNFR superfamily. We demonstrated that the binding stabilities inferred from our simulation results were compatible with existing experimental data. We further compared the binding kinetics of different TNF/TNFR systems, and explored their potential functional implication. We suggest that the transient binding between ligands and cell surface receptors leads into a dynamic nature of cross-membrane signal transduction, whereas the slow but strong binding of these ligands to the soluble decoy receptors is naturally designed to fulfill their functions as inhibitors of signal activation. Therefore, our computational approach serves as a useful addition to current experimental techniques for the quantitatively comparison of interactions across different members in the TNF and TNFR superfamily. It also provides a mechanistic understanding to the functions of TNF-associated cell signaling pathways.

Keywords:  binding kinetics; computational simulations; tumor necrosis factor superfamily

DOI:  https://doi.org/10.3390/ijms21051778
J Interferon Cytokine Res. 2020 Mar 10.

Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1.

Fatma Zehra Hapil, Fatma Ece Çopuroğlu, Mustafa Gökhan Ertosun, Ufuk Mert, Derya Özeş, Osman Nidai Özeş.

  Tumor necrosis factor alpha (TNF-α) plays a paramount role in homeostasis by inducing tumor cytotoxicity and activating immune system. The signaling complexes formed by TNFR1 to activate JNK, p38, and nuclear factor-kappa B pathways and to subsequently induce apoptosis and necroptosis are well known. However, this "canonical TNF-α signaling" does not explain how ERK, AKT, and STAT3 can be activated by TNF-α. In addition, little to nothing is known about negative regulation of TNFR1 signaling. Because cyclic AMP-activated kinase (PKA) shows anti-TNF and anti-inflammatory activities, we postulated that PKA might affect TNF-α signaling by directly phosphorylating TNFR1. In line with this, we identified 2 putative PKA-phosphorylation motifs RRRT411 and REAT417 within the death domain of TNFR1, and investigated whether "canonical" and "noncanonical" TNFR1 signaling is regulated by modifications of T411 and T417. In this study, we demonstrate for the first time that PKA directly binds to and phosphorylates TNFR1 after TNF-α stimulation. To further support our hypothesis, we generated alanine and phosphomimetic (aspartic acid) mutants of TNFR1 at positions T411 and T417, ectopically expressed these mutants, and determined their influence on TNF-α-induced activations of ERKs, AKT, STAT3, p38α, and JNK1/2. Our results clearly showed that phosphomimetic mutants significantly suppressed and alanine mutants augmented TNF-α-induced phosphorylations of ERKs, AKT, Stat3, p38α, and JNKs. These findings strongly suggest that PKA-mediated phosphorylation of T411 and T417 of TNFR1 interferes with both "canonical" and "noncanonical" TNF-α signaling. [Figure: see text].

Keywords:  Akt; Erk; PKA; TNF-α; TNFR1; phosphorylation

DOI:  https://doi.org/10.1089/jir.2019.0128
Cell Death Differ. 2020 Mar 09.

RIPK3 collaborates with GSDMD to drive tissue injury in lethal polymicrobial sepsis.

Hui Chen, Yinshuang Li, Jianfeng Wu, Guoping Li, Xuan Tao, Kunmei Lai, Ying Yuan, Xiaohong Zhang, Zhenhuan Zou, Yanfang Xu.

Sepsis is a systemic inflammatory disease causing life-threatening multi-organ dysfunction. Accumulating evidences suggest that two forms of programmed necrosis, necroptosis and pyroptosis triggered by the pathogen component lipopolysaccharide (LPS) and inflammatory cytokines, play important roles in the development of bacterial sepsis-induced shock and tissue injury. Sepsis-induced shock and tissue injury required receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) phosphorylation, caspase11 activation and gasdermin D (GSDMD) cleavage. However, the synergistic effect of necroptosis and pyroptosis in the pathological progress of sepsis remains elusive. In this study, we found that blockage of both necroptosis and pyroptosis (double deletion of Ripk3/Gsdmd or Mlkl/Gsdmd) resulted in accumulative protection against septic shock, systemic blood clotting and multi-organ injury in mice. Bone marrow transplantation confirmed that necroptosis and pyroptosis in both myeloid and nonmyeloid cells are indispensable in the progression of sepsis-induced multi-organ injury. Both RIPK3 and GSDMD signaling collaborated to amplify necroinflammation and tissue factor release in macrophages and endothelial cells, which led to tissue injury. Furthermore, cell death induced by inflammatory cytokines and high-mobility group box 1 could be prevented by double ablation of Ripk3/Gsdmd or Mlkl/Gsdmd, suggesting that a positive feedback loop interconnecting RIPK3/MLKL and GSDMD machinery and inflammation facilitated sepsis progression. Collectively, our findings demonstrated that RIPK3-mediated necroptosis and GSDMD-mediated pyroptosis collaborated to amply inflammatory signaling and enhance tissue injury in the process of sepsis, which may shed new light on two potential targets of combined therapeutic interventions for this highly lethal disorder.

DOI: https://doi.org/10.1038/s41418-020-0524-1
Curr Opin Cell Biol. 2020 Mar 09. pii: S0955-0674(20)30027-2. [Epub ahead of print]63 186-193

New insights into the regulation of apoptosis, necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8.

Robin Schwarzer, Lucie Laurien, Manolis Pasparakis.

  Necroptosis and pyroptosis are inflammatory forms of regulated necrotic cell death as opposed to apoptosis that is generally considered immunologically silent. Recent studies revealed unexpected links in the pathways regulating and executing cell death in response to activation of signaling cascades inducing apoptosis, necroptosis, and pyroptosis. Emerging evidence suggests that receptor interacting protein kinase 1 and caspase-8 control the cross-talk between apoptosis, necroptosis, and pyroptosis and determine the type of cell death induced in response to activation of cell death signaling.

Keywords:  Apoptosis; Caspase-8; Cell death; Inflammation; Necroptosis; Pyroptosis; RIPK1

DOI:  https://doi.org/10.1016/j.ceb.2020.02.004
Science. 2020 Mar 13. 367(6483): 1264-1269

Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.

Sampath K Loganathan, Krista Schleicher, Ahmad Malik, Rene Quevedo, Ellen Langille, Katie Teng, Robin H Oh, Bhavisha Rathod, Ricky Tsai, Payman Samavarchi-Tehrani, Trevor J Pugh, Anne-Claude Gingras, Daniel Schramek.

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.

DOI: https://doi.org/10.1126/science.aax0902
J Med Chem. 2020 Mar 10.

Azo-PROTAC: novel light-controlled small-molecule tool for protein knockdown.

Yuhui Jin, Mengchen Lu, Yan Wang, Wenxin Shan, Xuanyu Wang, Qi-Dong You, Zhengyu Jiang.

Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable Azo-PROTACs by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photo-switch ability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off property.

DOI: https://doi.org/10.1021/acs.jmedchem.9b02058
J Immunol. 2020 Mar 13. pii: ji1900769. [Epub ahead of print]

RNA-Binding Protein HuR Promotes Th17 Cell Differentiation and Can Be Targeted to Reduce Autoimmune Neuroinflammation.

Jing Chen, Jennifer L Martindale, Kotb Abdelmohsen, Gaurav Kumar, Paolo M Fortina, Myriam Gorospe, Abdolmohamad Rostami, Shiguang Yu.

Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help find a novel therapeutic target for treating Th17 cell-mediated diseases. In this study, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by posttranscriptionally regulating transcription factors Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation in C57BL/6J mice. Knockout of HuR altered the transcriptome of Th17 cells characterized by reducing the levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells in the spleen during experimental autoimmune encephalomyelitis. In keeping with the fact that HuR increased the abundance of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired splenic Th17 cell migration to the CNS and abolished the disease. Accordingly, targeting HuR by its inhibitor DHTS inhibited splenic Th17 cell differentiation and reduced experimental autoimmune encephalomyelitis severity. In sum, we uncovered the molecular mechanism of HuR regulating Th17 cell functions, underscoring the therapeutic value of HuR for treatment of autoimmune neuroinflammation.

DOI: https://doi.org/10.4049/jimmunol.1900769
Cell. 2020 Mar 06. pii: S0092-8674(20)30218-X. [Epub ahead of print]

Toll-like Receptors and the Control of Immunity.

Katherine A Fitzgerald, Jonathan C Kagan.

The study of innate immunity and its link to inflammation and host defense encompasses diverse areas of biology, ranging from genetics and biophysics to signal transduction and physiology. Central to our understanding of these events are the Toll-like receptors (TLRs), an evolutionarily ancient family of pattern recognition receptors. Herein, we describe the mechanisms and consequences of TLR-mediated signal transduction with a focus on themes identified in the TLR pathways that also explain the operation of other immune signaling pathways. These themes include the detection of conserved microbial structures to identify infectious agents and the use of supramolecular organizing centers (SMOCs) as signaling organelles that ensure digital cellular responses. Further themes include mechanisms of inducible gene expression, the coordination of gene regulation and metabolism, and the influence of these activities on adaptive immunity. Studies in these areas have informed the development of next-generation therapeutics, thus ensuring a bright future for research in this area.

DOI: https://doi.org/10.1016/j.cell.2020.02.041
Adv Exp Med Biol. 2020 ;1204 31-56

Immune Recognition of Pathogen-Derived Glycolipids Through Mincle.

Yasunobu Miyake, Sho Yamasaki.

  Mincle (macrophage inducible C-type lectin, Clec4e, Clecsf9) was originally identified as a member of the C-type lectin receptor family in 1999. Then, the function of Mincle to control antifungal immunity by binding to Candida albicans was reported in 2008. Around the same time, it was reported that Mincle recognized damaged cells and induced sterile inflammation by coupling with the ITAM-adaptor molecule FcRγ. In the following year, a breakthrough discovery reported that Mincle was an essential receptor for mycobacterial cord factor (trehalose-6,6'-dimycolate, TDM). Mincle gained increasing attention immediately after this critical finding. Although our understanding of the recognition of Mycobacteria has been advanced significantly, it was also revealed that Mincle interacts with pathogens other than Mycobacteria. In addition, endogenous ligands of Mincle were identified recently. Therefore, Mincle is now considered a danger receptor both for self and non-self ligands, so-called damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). This chapter will give an overview of the accumulated knowledge of the multi-task danger receptor Mincle from its discovery to the latest findings.

Keywords:  C-type lectin receptor; Danger signal; Glycolipids

DOI:  https://doi.org/10.1007/978-981-15-1580-4_2
F1000Res. 2020 ;pii: F1000 Faculty Rev-148. [Epub ahead of print]9

The essentials of developmental apoptosis.

Anne K Voss, Andreas Strasser.

  Apoptotic cells are commonly observed in a broad range of tissues during mammalian embryonic and fetal development. Specific requirements and functions of programmed cell death were inferred from early observations. These inferences did not hold up to functional proof for a requirement of apoptosis for normal tissue development in all cases. In this review, we summarize how the appraisal of the importance of developmental apoptosis has changed over the years, in particular with detailed functional assessment, such as by using gene-targeted mice lacking essential initiators or mediators of apoptosis. In recent years, the essentials of developmental apoptosis have emerged. We hypothesize that apoptosis is predominantly required to balance cell proliferation. The two interdependent processes-cell proliferation and apoptosis-together more powerfully regulate tissue growth than does each process alone. We proposed that this ensures that tissues and cell populations attain the appropriate size that allows fusion in the body midline and retain the size of cavities once formed. In addition, a limited number of tissues, albeit not all previously proposed, rely on apoptosis for remodeling, chiefly aortic arch remodeling, elimination of supernumerary neurons, removal of vaginal septa, and removal of interdigital webs in the formation of hands and feet.

Keywords:  A1; APAF-1; BAD; BAK; BAX; BCL-2; BCL-W; BCL-XL; BID; BIK; BIM; BMF; BOK; Embryo; HRK; MCL-1; NOXA; PUMA; apoptosis; caspases; development; fetus; programmed cell death

DOI:  https://doi.org/10.12688/f1000research.21571.1
Int J Cancer. 2020 Mar 13.

Proteasome inhibitors and Smac mimetics cooperate to induce cell death in Diffuse Large B-cell Lymphoma by stabilizing NOXA and triggering mitochondrial apoptosis.

Anna Dietz, Nahide Dalda, Svenja Zielke, Jessica Dittmann, Sjoerd J L van Wijk, Meike Vogler, Simone Fulda.

  Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Smac (second mitochondria-derived activator of caspases) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pre-treatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies. This article is protected by copyright. All rights reserved.

Keywords:  Diffuse large B-cell lymphoma; NOXA; Smac mimetics; intrinsic apoptosis; proteasome inhibitor

DOI:  https://doi.org/10.1002/ijc.32976
Nat Commun. 2020 Mar 11. 11(1): 1306

UBB pseudogene 4 encodes functional ubiquitin variants.

Marie-Line Dubois, Anna Meller, Sondos Samandi, Mylène Brunelle, Julie Frion, Marie A Brunet, Amanda Toupin, Maxime C Beaudoin, Jean-François Jacques, Dominique Lévesque, Michelle S Scott, Pierre Lavigne, Xavier Roucou, François-Michel Boisvert.

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.

DOI: https://doi.org/10.1038/s41467-020-15090-6
Curr Opin Chem Biol. 2020 Mar 05. pii: S1367-5931(20)30011-9. [Epub ahead of print]57 8-16

Proteolysis targeting chimeras (PROTACs) for epigenetics research.

Anja Vogelmann, Dina Robaa, Wolfgang Sippl, Manfred Jung.

  Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules and allow selective protein degradation by addressing the natural ubiquitin proteasome system. As this new strategy of chemically induced protein degradation can serve as a biological tool and provides new possibilities for drug discovery, it has been applied to a variety of targets including (nuclear) receptors, kinases, and epigenetic proteins. A lot of PROTACs have already been designed in the field of epigenetics, and their synthesis and characterization highly contributed to structural optimization and improved mechanistic understanding of these molecules. In this review, we will discuss and summarize recent advances in PROTAC discovery with focus on epigenetic targets.

Keywords:  Drug discovery; Epigenetics; PROTACs; Protein degradation

DOI:  https://doi.org/10.1016/j.cbpa.2020.01.010
Curr Opin Struct Biol. 2020 Mar 04. pii: S0959-440X(20)30015-4. [Epub ahead of print]60 167-174

Monobodies as enabling tools for structural and mechanistic biology.

Oliver Hantschel, Matthew Biancalana, Shohei Koide.

Monobodies, built with the scaffold of the fibronectin type III domain, are among the most well-established synthetic binding proteins. They promote crystallization of challenging molecular systems. They have strong tendency to bind to functional sites and thus serve as drug-like molecules that perturb the biological functions of their targets. Monobodies lack disulfide bonds and thus they are particularly suited as genetically encoded reagents to be used intracellularly. This article reviews recent monobody-enabled studies that reveal new structures, molecular mechanisms and potential therapeutic opportunities. A systematic analysis of the crystal structures of monobody-target complexes suggests important attributes that make monobodies effective crystallization chaperones.

DOI: https://doi.org/10.1016/j.sbi.2020.01.015
Cell Death Dis. 2020 Mar 13. 11(3): 185

Combined reduction in the expression of MCL-1 and BCL-2 reduces organismal size in mice.

Francine Ke, Graeme I Lancaster, Stephanie Grabow, Andrew J Murphy, Andreas Strasser.

The intrinsic apoptotic pathway is controlled by the BCL-2 family of proteins, which exhibit either a pro-death or pro-survival function. Gene knockout studies revealed that different pro-survival BCL-2 proteins are critical for the survival of distinct cell types, although overlapping functions amongst such proteins have also been identified. In the process of studying mice lacking single alleles of Mcl-1 (Mcl-1+/-), Bcl-2 (Bcl-2+/-), or both in combination (Mcl-1+/-Bcl-2+/-), we observed that Mcl-1+/-Bcl-2+/- mice weighed less when compared with their wild-type littermates as they aged. Body composition analysis demonstrated that while fat mass was similar to wild-type controls, lean mass was significantly reduced in Mcl-1+/-, Bcl-2+/-, and, most strikingly in Mcl-1+/-Bcl-2+/- mice. The weights of several tissues including the heart, tibialis anterior, and kidney were likewise reduced in Mcl-1+/-Bcl-2+/- mice. When lean mass and specific tissue weights were expressed relative to body weight, these differences were no longer significant, indicating that that Mcl-1+/-Bcl-2+/- mice, and to a lesser extent Mcl-1+/- and Bcl-2+/- mice, are smaller than their wild-type counterparts. Consistently, the anal-naso length was reduced in Mcl-1+/-Bcl-2+/- mice. While minor reductions in size were observed in female Mcl-1+/-Bcl-2+/- mice, these effects were most prominent in males. Notably, Mcl-1+/-Bcl-2+/- males had markedly smaller testes even after accounting for differences in body weight. Collectively, these data reveal that combined loss of a single allele of Mcl-1 and Bcl-2, while not overtly impairing organismal development, leads to a reduction in animal size.

DOI: https://doi.org/10.1038/s41419-020-2376-5
Exp Cell Res. 2020 Mar 07. pii: S0014-4827(20)30140-3. [Epub ahead of print] 111935

Pleiotropic roles of tankyrase/PARP proteins in the establishment and maintenance of human naïve pluripotency.

Ludovic Zimmerlin, Elias T Zambidis.

  Tankyrase 1 (TNKS1; PARP-5a) and Tankyrase 2 (TNKS2; PARP-5b) are poly-ADP-ribosyl-polymerase (PARP)-domain-containing proteins that regulate the activities of a wide repertoire of target proteins via post-translational addition of poly-ADP-ribose polymers (PARylation). Although tankyrases were first identified as regulators of human telomere elongation, important and expansive roles of tankyrase activity have recently emerged in the development and maintenance of stem cell states. Herein, we summarize the current state of knowledge of the various tankyrase-mediated activities that may promote human naïve and 'extended' pluripotency'. We review the putative role of tankyrase and PARP inhibition in trophectoderm specification, telomere elongation, DNA repair and chromosomal segregation, metabolism, and PTEN-mediated apoptosis. Importantly, tankyrases possess PARP-independent activities that include regulation of MDC1-associated DNA repair by homologous recombination (HR) and autophagy/pexophagy, which is an essential mechanism of protein synthesis in the preimplantation embryo. Additionally, tankyrases auto-regulate themselves via auto-PARylation which augments their cellular protein levels and potentiates their non-PARP tankyrase functions. We propose that these non-PARP-related activities of tankyrase proteins may further independently affect both naïve and extended pluripotency via mechanisms that remain undetermined. We broadly outline a hypothetical framework for how inclusion of a tankyrase/PARP inhibitor in small molecule cocktails may stabilize and potentiate naïve and extended pluripotency via pleiotropic routes and mechanisms.

Keywords:  Human stem cell; Naive pluripotent stem cell; PARP; Pluripotency; Regenerative medicine; Tankyrase

DOI:  https://doi.org/10.1016/j.yexcr.2020.111935
Proc Natl Acad Sci U S A. 2020 Mar 10. pii: 202001683. [Epub ahead of print]

Regulation of Cullin-RING E3 ligase dynamics by Inositol hexakisphosphate.

Daniel C Scott, Gary Kleiger.

DOI: https://doi.org/10.1073/pnas.2001683117
Drug Des Devel Ther. 2020 ;14 795-810

Ubiquitin-Fold Modifier-1 Participates in the Diabetic Inflammatory Response by Regulating NF-κB p65 Nuclear Translocation and the Ubiquitination and Degradation of IκBα.

Xiaolei Hu, Hengyan Zhang, Langen Zhuang, Guoxi Jin, Qingqing Yang, Min Li, Weihua Sun, Fengling Chen.

   Background: Ubiquitin-fold modifier-1 (Ufm1) is a recently identified ubiquitin-like protein. We previously confirmed that Ufm1 expression was increased in diabetic mice. However, its role in the development of diabetes remains undefined.
Methods: Lentivirus-mediated gene knockdown and overexpression techniques were used to observe the effect of Ufm1 on the expression of inflammatory factors, adhesion molecules and chemokines, as well as the transcriptional activity of nuclear factor kappa-B (NF-κB) in macrophages. Western blot and immunofluorescence analyses were used to analyse the mechanism by which Ufm1 affects the transcriptional activity of NF-κB. Finally, the effects of Ufm1 on inflammation and pancreatic, renal and myocardial damage were observed in db/db mice.
Results: Knockdown of Ufm1 by lentivirus shRNA targeting Ufm1 (Lv-shUfm1) led to decreased secretion of IL-6, IL-1β, ICAM-1, VCAM-1, MCP-1 and CXCL2 in RAW264.7 cells that were exposed to LPS and TNF-α, while lentiviral overexpression of Ufm1 (Lv-Ufm1) caused the opposite effect. Interestingly, further investigation indicated that Ufm1 induced NF-κB p65 nuclear translocation in RAW264.7 cells via increasing the ubiquitination and degradation of IκBα. In an in vivo experiment, pretreatment of db/db mice with Lv-shUfm1 reduced the mRNA levels of TNF-α, IL-6, IL-1β, ICAM-1, VCAM-1, MCP-1 and CXCL2 in resident peritoneal macrophages (RPMs) and decreased the plasma levels of TNF-α, IL-6, IL-1β, ICAM-1, VCAM-1, MCP-1 and CXCL2. Additionally, in Lv-Ufm1-treated mice, the inverse results were observed. Following treatment with Lv-shUfm1 and Lv-Ufm1, NF-κB p65 nuclear translocation in RPMs was decreased and increased, respectively. Importantly, we observed that Lv-shUfm1 injection led to a decrease in plasma glycaemia, a reduction in urinary albuminuria and cardiomyocyte hypertrophy and an improvement in the histopathological appearance of pancreatic, kidney and myocardial tissue. Pretreatment of the mice with Lv-shUfm1 inhibited macrophage infiltration in the pancreas, kidney and myocardial tissue.
Conclusion: Our data elucidate a new biological function of Ufm1 that mediates inflammatory responses. Ufm1-mediated p65 nuclear translocation occurs by modulating the ubiquitination and degradation of IκBα. Moreover, downregulating Ufm1 is an effective strategy to prevent the development of type 2 diabetes and its complications.

Keywords:  IκBα; NF-κB; diabetes; diabetic complications; macrophage; ubiquitin-fold modifier-1

DOI:  https://doi.org/10.2147/DDDT.S238695
Curr Opin Hematol. 2020 Mar 09.

The many flavors of monocyte/macrophage--endothelial cell interactions.

Gloria E Hernandez, M Luisa Iruela-Arispe.

PURPOSE OF REVIEW: The well recognized plasticity and diversity, typical of monocytes and macrophages have recently been expanded by the knowledge that additional macrophage lineages originated directly from embryonic progenitors, populate and establish residency in all tissues examined so far. This review aims to summarize our current understanding on the diversity of monocyte/macrophage subtypes associated with the vasculature, their specific origins, and nature of their cross-talk with the endothelium.
RECENT FINDINGS: Taking stock of the many interactions between the endothelium and monocytes/macrophages reveals a far more intricate and ever-growing depth. In addition to circulating and surveilling the endothelium, monocytes can specifically be differentiated into patrolling cells that crawl on the surface of the endothelium and promote homeostasis. The conversion of classical to patrolling is endothelium-dependent uncovering an important functional link. In addition to patrolling cells, the endothelium also recruits and harbor an intimal-resident myeloid population that resides in the tunica intima in the absence of pathological insults. Moreover, the adventitia is populated with resident macrophages that support blood vessel integrity and prevent fibrosis.
SUMMARY: The last few years have witnessed a significant expansion in our knowledge of the many subtypes of monocytes and macrophages and their corresponding functional interactions with the vascular wall. In addition to surveying the endothelium for opportunities of diapedeses, monocyte and macrophages take residence in both the intima (as patrolling or resident) and in the adventitia. Their contributions to vascular function are broad and critical to homeostasis, regeneration, and expansion.

DOI: https://doi.org/10.1097/MOH.0000000000000573
Nat Commun. 2020 Mar 10. 11(1): 1299

Decorating bacteria with self-assembled synthetic receptors.

Naama Lahav-Mankovski, Pragati Kishore Prasad, Noa Oppenheimer-Low, Gal Raviv, Tali Dadosh, Tamar Unger, Tomer Meir Salame, Leila Motiei, David Margulies.

The responses of cells to their surroundings are mediated by the binding of cell surface proteins (CSPs) to extracellular signals. Such processes are regulated via dynamic changes in the structure, composition, and expression levels of CSPs. In this study, we demonstrate the possibility of decorating bacteria with artificial, self-assembled receptors that imitate the dynamic features of CSPs. We show that the local concentration of these receptors on the bacterial membrane and their structure can be reversibly controlled using suitable chemical signals, in a way that resembles changes that occur with CSP expression levels or posttranslational modifications (PTMs), respectively. We also show that these modifications can endow the bacteria with programmable properties, akin to the way CSP responses can induce cellular functions. By programming the bacteria to glow, adhere to surfaces, or interact with proteins or mammalian cells, we demonstrate the potential to tailor such biomimetic systems for specific applications.

DOI: https://doi.org/10.1038/s41467-020-14336-7
Cell Rep. 2020 Mar 10. pii: S2211-1247(20)30210-2. [Epub ahead of print]30(10): 3411-3423.e7

Transferrin Receptor Is a Specific Ferroptosis Marker.

Huizhong Feng, Kenji Schorpp, Jenny Jin, Carrie E Yozwiak, Benjamin G Hoffstrom, Aubrianna M Decker, Presha Rajbhandari, Michael E Stokes, Hannah G Bender, Joleen M Csuka, Pavan S Upadhyayula, Peter Canoll, Koji Uchida, Rajesh K Soni, Kamyar Hadian, Brent R Stockwell.

  Ferroptosis is a type of regulated cell death driven by the iron-dependent accumulation of oxidized polyunsaturated fatty acid-containing phospholipids. There is no reliable way to selectively stain ferroptotic cells in tissue sections to characterize the extent of ferroptosis in animal models or patient samples. We address this gap by immunizing mice with membranes from lymphoma cells treated with the ferroptosis inducer piperazine erastin and screening ∼4,750 of the resulting monoclonal antibodies generated for their ability to selectively detect cells undergoing ferroptosis. We find that one antibody, 3F3 ferroptotic membrane antibody (3F3-FMA), is effective as a selective ferroptosis-staining reagent. The antigen of 3F3-FMA is identified as the human transferrin receptor 1 protein (TfR1). We validate this finding with several additional anti-TfR1 antibodies and compare them to other potential ferroptosis-detecting reagents. We find that anti-TfR1 and anti-malondialdehyde adduct antibodies are effective at staining ferroptotic tumor cells in multiple cell culture and tissue contexts.

Keywords:  ROS; biomarker; cancer; cell death; ferroptosis; ferroptosis marker; ferroptosis-specific antibody; iron; tissue staining; transferrin receptor

DOI:  https://doi.org/10.1016/j.celrep.2020.02.049
Proc Natl Acad Sci U S A. 2020 Mar 10. pii: 201916503. [Epub ahead of print]

Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis.

Andrea N Johnston, Yuyong Ma, Hua Liu, Shuzhen Liu, Sarah Hanna-Addams, She Chen, Chuo Chen, Zhigao Wang.

  Necroptosis is a regulated necrotic cell death pathway involved in development and disease. Its signaling cascade results in the formation of disulfide bond-dependent amyloid-like polymers of mixed lineage kinase domain-like protein (MLKL), which mediate proinflammatory cell membrane disruption. We screened compound libraries provided by the National Cancer Institute and identified a small-molecule inhibitor of necroptosis named necroptosis-blocking compound 1 (NBC1). Biotin-labeled NBC1 specifically conjugates to heat shock protein Hsp70. NBC1 and PES-Cl, a known Hsp70 substrate-binding inhibitor, block the formation of MLKL polymers, but not MLKL tetramers in necroptosis-induced cells. In vitro, recombinant Hsp70 interacts with the N-terminal domain (NTD) of MLKL and promotes NTD polymerization, which has been shown to mediate the cell killing activity. Furthermore, the substrate-binding domain (SBD) of Hsp70 is sufficient to promote MLKL polymerization. NBC1 covalently conjugates cysteine 574 and cysteine 603 of the SBD to block its function. In addition, an SBD mutant with both cysteines mutated to serines loses its ability to promote MLKL polymerization. Interestingly, knockdown of Hsp70 in cells leads to MLKL destabilization, suggesting that MLKL might also be a client protein of Hsp70. In summary, using NBC1, an inhibitor of necroptosis, we identified Hsp70 as a molecular chaperone performing dual functions in necroptosis. It stabilizes MLKL protein under normal condition and promotes MLKL polymerization through its substrate-binding domain during necroptosis.

Keywords:  Hsp70; MLKL; NBC1; necroptosis; polymerization

DOI:  https://doi.org/10.1073/pnas.1916503117
J Immunol. 2020 Mar 09. pii: ji1901396. [Epub ahead of print]

Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies.

Simone Nüssing, Imran G House, Conor J Kearney, Amanda X Y Chen, Stephin J Vervoort, Paul A Beavis, Jane Oliaro, Ricky W Johnstone, Joseph A Trapani, Ian A Parish.

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.

DOI: https://doi.org/10.4049/jimmunol.1901396
Semin Cancer Biol. 2020 Mar 06. pii: S1044-579X(20)30063-8. [Epub ahead of print]

Old Drugs, New Uses: Drug Repurposing in Hematological Malignancies.

Vijay P Kale, Hasan Habib, Robert Chitren, Milan Patel, Kartick C Pramanik, Subash Jonnalagadda, Kishore Challagundla, Manoj K Pandey.

  Discovery and development of novel anti-cancer drugs are expensive and time consuming. Systems biology approaches have revealed that a drug being developed for a non-cancer indication can hit other targets as well, which play critical roles in cancer progression. Since drugs for non-cancer indications would have already gone through the preclinical and partial or full clinical development, repurposing such drugs for hematological malignancies would cost much less, and drastically reduce the development time, which is evident in case of thalidomide. Here, we have reviewed some of the drugs for their potential to repurpose for treating the hematological malignancies. We have also enlisted resources that can be helpful in drug repurposing.

Keywords:  Multiple myeloma; drug development; drug repositioning; drug repurposing; hematological malignancies

DOI:  https://doi.org/10.1016/j.semcancer.2020.03.005
Cell Res. 2020 Mar 09.

Toward targeting inflammasomes: insights into their regulation and activation.

Shelbi Christgen, David E Place, Thirumala-Devi Kanneganti.

Inflammasomes are multi-component signaling complexes critical to the initiation of pyroptotic cell death in response to invading pathogens and cellular damage. A number of innate immune receptors have been reported to serve as inflammasome sensors. Activation of these sensors leads to the proteolytic activation of caspase-1, a proinflammatory caspase responsible for the cleavage of proinflammatory cytokines interleukin-1β and interleukin-18 and the effector of pyroptotic cell death, gasdermin D. Though crucial to the innate immune response to infection, dysregulation of inflammasome activation can lead to the development of inflammatory diseases, neurodegeneration, and cancer. Therefore, clinical interest in the modulation of inflammasome activation is swiftly growing. As such, it is imperative to develop a mechanistic understanding of the regulation of these complexes. In this review, we divide the regulation of inflammasome activation into three parts. We discuss the transcriptional regulation of inflammasome components and related proteins, the post-translational mechanisms of inflammasome activation, and advances in the understanding of the structural basis of inflammasome activation.

DOI: https://doi.org/10.1038/s41422-020-0295-8
Curr Opin Rheumatol. 2020 Mar 09.

Treatment of immune checkpoint inhibitor-induced inflammatory arthritis.

Susanna Jeurling, Laura C Cappelli.

PURPOSE OF REVIEW: This review summarizes the current evidence on treatment strategies for inflammatory arthritis because of cancer treatment with immune checkpoint inhibitors (ICI), prognosis of ICI-induced arthritis, and management of patients with preexisting inflammatory arthritis receiving ICI therapy.
RECENT FINDINGS: Inflammatory arthritis is the most common rheumatic immune-related adverse event observed in patients receiving ICI therapy. Most patients can successfully be treated with low doses of corticosteroids or conventional synthetic disease modifying anti-rheumatic drug (DMARDs). A small minority will develop severe symptoms requiring biologic therapy including TNF inhibitors and IL-6 receptor inhibitors. Many cases of inflammatory arthritis will resolve with cessation of ICI therapy. Some patients will develop persistent arthritis despite discontinuation. Patients with preexisting inflammatory arthritis (e.g. rheumatoid arthritis) commonly flare on ICI therapy, but can usually be managed with corticosteroids.
SUMMARY: Inflammatory arthritis following ICI therapy for cancer is relatively common and the practicing rheumatologist should be able to recognize and manage it in conjunction with Oncology. The majority of patients respond to corticosteroids, but some will need treatment with conventional synthetic or biologic DMARDs. Additional studies should investigate the effects of immunosuppression on tumor response and the use of ICI therapy in patients with preexisting autoimmune disease.

DOI: https://doi.org/10.1097/BOR.0000000000000701
Cancers (Basel). 2020 Mar 05. pii: E604. [Epub ahead of print]12(3):

The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis.

Karla L H Feijs, Christopher D O Cooper, Roko Žaja.

  Post-translational modifications (PTM) of proteins are crucial for fine-tuning a cell's response to both intracellular and extracellular cues. ADP-ribosylation is a PTM, which occurs in two flavours: modification of a target with multiple ADP-ribose moieties (poly(ADP-ribosyl)ation or PARylation) or with only one unit (MARylation), which are added by the different enzymes of the PARP family (also known as the ARTD family). PARylation has been relatively well-studied, particularly in the DNA damage response. This has resulted in the development of PARP inhibitors such as olaparib, which are increasingly employed in cancer chemotherapeutic approaches. Despite the fact that the majority of PARP enzymes catalyse MARylation, MARylation is not as well understood as PARylation. MARylation is a dynamic process: the enzymes reversing intracellular MARylation of acidic amino acids (MACROD1, MACROD2, and TARG1) were discovered in 2013. Since then, however, little information has been published about their physiological function. MACROD1, MACROD2, and TARG1 have a 'macrodomain' harbouring the catalytic site, but no other domains have been identified. Despite the lack of information regarding their cellular roles, there are a number of studies linking them to cancer. However, some of these publications oppose each other, some rely on poorly-characterised antibodies, or on aberrant localisation of overexpressed rather than native protein. In this review, we critically assess the available literature on a role for the hydrolases in cancer and find that, currently, there is limited evidence for a role for MACROD1, MACROD2, or TARG1 in tumorigenesis.

Keywords:  ADP-ribosyl hydrolase; ADP-ribosylation; ARTD; MACROD1; MACROD2; PARP; TARG1; cancer; macrodomain

DOI:  https://doi.org/10.3390/cancers12030604
Curr Med Chem. 2020 Mar 12.

Recent Advances in the Design and Development of anticancer molecules based on PROTAC technology.

Zere Mukhamejanova, Yichen Tong, Qi Xiang, Fang Xu, Jiyan Pang.

  (Proteolysis targeting chimera) degraders based on protein knockdown technology now are suggested as a novel option for the treatment of various diseases. Over the last couple of years, application of PROTAC technology has spread in a wide range of disorders, and plenty of PROTAC molecules with high potency have been reported. Mostly developing for anticancer therapy, these molecules show high selectivity to target proteins, ability to significantly induce degradation of oncoproteins, good in vivo and in vitro results. In this review, we summarized the recent development of PROTAC technology in the anticancer therapy field, including molecular design, types of targeted proteins, in vivo and in vitro data results. Additionally, we also discuss on the prospects and challenges for application of candidates based on PROTAC strategy in clinical trials.

Keywords:  PROTAC; anticancer therapy; small molecule degrader; targeted protein degradation

DOI:  https://doi.org/10.2174/0929867327666200312112412
Biochim Biophys Acta Rev Cancer. 2020 Mar 05. pii: S0304-419X(19)30176-3. [Epub ahead of print] 188357

Caspase-8: The double-edged sword.

Ranadip Mandal, Joan Compte Barrón, Izabela Kostova, Sven Becker, Klaus Strebhardt.

Caspase-8 is a cysteine protease that classically triggers the extrinsic apoptotic pathway, in response to the activation of cell surface Death Receptors (DRs) like FAS, TRAIL-R and TNF-R. Besides it's roles in triggering death receptor-mediated apoptosis, Caspase-8 has also been implicated in the onsets of anoikis, Integrin Mediated Death (IMD) and autophagy. Furthermore, Caspase-8 also plays a crucial pro-survival function by inhibiting an alternative form of programmed cell death called necroptosis. Low expression levels of pro-Caspase-8 is therefore associated with the malignant transformation of cancers. However, the long-held notion that pro-Caspase-8 expression/activity is generally lost in most cancers, thereby contributing to apoptotic escape and enhanced resistance to anti-cancer therapeutics, has been found to be true for only a minority of cancers types. In the majority of cases, pro-Caspase-8 expression is maintained and sometimes elevated, while it's apoptotic activity is regulated through different mechanisms. This supports the notion that the non-apoptotic functions of Caspase-8 offer growth advantage in these cancer types and have, therefore, gained renewed interest in the recent years. In light of these reasons, a number of therapeutic approaches have been employed, with the intent of targeting pro-Caspase-8 in cancer cells. In this review, we would attempt to discuss - the classic roles of Caspase-8 in initiating apoptosis; it's non-apoptotic functions; it's the clinical significance in different cancer types; and the therapeutic applications exploiting the ability of pro-Caspase-8 to regulate various cellular functions.

DOI: https://doi.org/10.1016/j.bbcan.2020.188357
Life Sci. 2020 Mar 05. pii: S0024-3205(20)30266-6. [Epub ahead of print] 117518

Ninjurin-1 upregulated by TNFα receptor 1 stimulates monocyte adhesion to human TNFα-activated endothelial cells; benefic effects of amlodipine.

Laura Toma, Gabriela M Sanda, Mina Raileanu, Camelia S Stancu, Loredan S Niculescu, Anca V Sima.

   AIMS: The objectives of the present study were to investigate the mechanisms of Ninj-1 regulation in TNFα-activated human endothelial cells (HEC), and to test if Amlodipine (AML) ameliorates the inflammatory stress by decreasing Ninj-1 expression.
MAIN METHODS: TNFα-activated HEC with/without AML (0.1 μM and 1 μM) were used. TNFα-receptor 1 (TNFR1) was silenced and inhibitors for oxidative stress (N-acetyl cysteine), endoplasmic reticulum stress (salubrinal, 4-phenyl butyric acid), or NF-kB (Bay 11-7085) and p38 MAPK (SB203580) were used. Levels of Ninj-1, TNFR1, monocyte adhesion, endoplasmic reticulum stress (ERS) sensors, NADPH oxidase- and mitochondria-derived oxidative species were evaluated.
KEY FINDINGS: The novel findings that we report here are: (i) silencing the endothelial TNFR1 leads to decreased Ninj-1 expression and diminished monocyte adhesion; (ii) increased oxidative stress, ERS and NF-kB activation enhance Ninj-1 expression and monocyte adhesion; (iii) up-regulation of endothelial Ninj-1 expression stimulates monocytes adhesion to TNFα - activated HEC; (iv) AML diminishes monocyte adhesion by reducing Ninj-1 expression through mechanisms involving the decrease of NADPH oxidase and mitochondria-dependent oxidative stress, ERS and NF-kB. In addition, AML alleviates apoptosis by reducing the pro-apoptotic CHOP expression and re-establishing the mitochondrial transmembrane potential.
SIGNIFICANCE: The results of the present study suggest that Ninj-1 and the proteins involved in its regulation can be considered therapeutic targets for the alleviation of inflammation- dependent disorders. In addition, we demonstrate that some of the benefic effects of AML can be achieved through regulation of Ninj-1.

Keywords:  Amlodipine; Endoplasmic reticulum stress; Endothelial cell; Ninjurin-1; TNFα-receptor1

DOI:  https://doi.org/10.1016/j.lfs.2020.117518
Curr Med Chem. 2020 Mar 12.

A Review on Poly (ADP-ribose) Polymerase (PARP) Inhibitors and Synthetic Methodologies.

Ying Li, Chenfu Liu, Guowu Rao.

  Poly (ADP-ribose) polymerase (PARP) acts as an essential DNA repair enzyme. PARP inhibitors are a novel small molecule targeted drugs based on the principle of 'Synthetic Lethality", which affect the DNA repair process by competitively inhibiting the activity of the PARP enzyme and thereby kill cancer cells. Currently, four PARP inhibitors including olaparib, rucaparib, niraparib, and talazoparib have been approved by FDA for cancer treatment and have achieved great success in the treatment of ovarian cancer, breast cancer, and pancreatic cancer etc. This paper provides a general overview of the research progress of PARP inhibitors including the major structure types, structure-activity relationship (SAR), and synthetic routes, with the aim of providing ideas for the discovery and synthesis of novel PARP inhibitors.

Keywords:  Olaparib; Ovarian cancer; PARP inhibitors; Structure-activity relationship; Synthesis; Synthetic lethality; phthalazinones

DOI:  https://doi.org/10.2174/0929867327666200312113011
J Med Chem. 2020 Mar 11.

Bifunctional Molecules beyond PROTACs.

Stuart J Conway.

Heterobifunctional molecules, which recruit E3 ligases to ubiquitinate a target protein of interest, have found wide application as both biological tools and molecules with the potential to have clinical effects. In their recent paper, Yamazoe et al. report a heterobifunctional molecule that recruits the phosphatase PP1 to promote the dephosphorylation of pAKT to give AKT. This Viewpoint seeks to place this work in the wider context of heterobifunctional molecules and looks ahead to new possibilities presented by these results.

DOI: https://doi.org/10.1021/acs.jmedchem.0c00293
Structure. 2020 Mar 09. pii: S0969-2126(20)30071-X. [Epub ahead of print]

An Extended Conformation for K48 Ubiquitin Chains Revealed by the hRpn2:Rpn13:K48-Diubiquitin Structure.

Xiuxiu Lu, Danielle L Ebelle, Hiroshi Matsuo, Kylie J Walters.

  Rpn13/Adrm1 is recruited to the proteasome by PSMD1/Rpn2, where it serves as a substrate receptor that binds preferentially to K48-linked ubiquitin chains, an established signal for protein proteolysis. Here, we use NMR to solve the structure of hRpn13 Pru:hRpn2 (940-953):K48-diubiquitin. Surprisingly, hRpn2-bound hRpn13 selects a dynamic, extended conformation of K48-diubiquitin that is unique from previously determined structures. NMR experiments on free K48-diubiquitin demonstrate the presence of the reported "closed" conformation observed by crystallography, but also this more extended state, in which the hRpn13-binding surface is exposed. This extended K48-diubiquitin conformation is defined by interactions between L73 from G76-linked (distal) ubiquitin and a Y59-centered surface of K48-linked (proximal) ubiquitin. Furthermore, hRpn13 exchanges between the two ubiquitins within 100 ms, although prefers the proximal ubiquitin due to interactions with the K48 linker region. Altogether, these data lead to a revised model of how ubiquitinated substrates interact with the proteasome.

Keywords:  ADRM1; K48 ubiquitin; NMR; PSMD1; Rpn13; Rpn2; dynamics; proteasome; ubiquitin signaling

DOI:  https://doi.org/10.1016/j.str.2020.02.007
Oncogenesis. 2020 Mar 10. 9(3): 33

BET protein inhibitor JQ1 downregulates chromatin accessibility and suppresses metastasis of gastric cancer via inactivating RUNX2/NID1 signaling.

Siqi Zhou, Shu Zhang, Lei Wang, Shuling Huang, Yue Yuan, Jie Yang, Hui Wang, Xihan Li, Pin Wang, Lin Zhou, Jun Yang, Yuemei Xu, Huan Gao, Yixuan Zhang, Ying Lv, Xiaoping Zou.

Chromatin accessibility is critical for tumor development, whose mechanisms remain unclear. As a crucial regulator for chromatin remodeling, BRD4 promotes tumor progression by regulating multiple genes. As a small-molecule inhibitor of BRD4, JQ1 has potent chemotherapeutic activity against various human cancers. However, whether JQ1 has potential anti-tumor effects and how JQ1 regulates global transcription in gastric cancer (GC) remain largely unknown. In this research, we found BRD4 was highly expressed in GC tissues and was significantly associated with poor prognosis. JQ1 inhibited the proliferation and induced apoptosis of GC cells in vitro. Besides, JQ1 suppressed the migration and invasion of cancer cells by inducing MET. Notably, an assay for transposase-accessible chromatin using sequencing (ATAC-seq) data showed that JQ1 obviously downregulated the chromatin accessibility of GC cells and differentially accessible regions were highly enriched for RUNX2-binding motifs. Combinational analysis of ATAC-seq and RNA-seq data discovered NID1 as the downstream target of JQ1 and JQ1 reduced NID1 expression in GC cells. Chromatin immunoprecipitation, luciferase reporter gene assay, and rescue experiments all confirmed that RUNX2/NID1 axis was responsible for JQ1-inhibiting metastasis of GC cells. What's more, high expression of NID1 in GC tissues also predicted poor survival outcome of cancer patients and NID1 knockdown prohibited migration and invasion of cancer cells via partially inducing MET. Finally, in vivo models showed that JQ1 prevented GC growth and suppressed cancer metastasis. In conclusion, JQ1 inhibits the malignant progression of GC by downregulating chromatin accessibility and inactivating RUNX2/NID1 signaling. In addition, NID1 is also a novel therapeutic target for progressive GC patients.

DOI: https://doi.org/10.1038/s41389-020-0218-z
Cell. 2020 Mar 06. pii: S0092-8674(20)30262-2. [Epub ahead of print]

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.

Alexandra C Walls, Young-Jun Park, M Alejandra Tortorici, Abigail Wall, Andrew T McGuire, David Veesler.

  The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

Keywords:  SARS-CoV; SARS-CoV-2; antibodies; coronavirus; cryo-EM; neutralizing antibodies; spike glycoprotein; viral receptor

DOI:  https://doi.org/10.1016/j.cell.2020.02.058
Nature. 2020 Mar;579(7798): 177

A hack allows microscopes to capture the ultrafast and ultrasmall.



Keywords:  Atomic and molecular physics

DOI:  https://doi.org/10.1038/d41586-020-00644-x
Proc Natl Acad Sci U S A. 2020 Mar 13. pii: 201909546. [Epub ahead of print]

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture.

Hawa Racine Thiam, Siu Ling Wong, Rong Qiu, Mark Kittisopikul, Amir Vahabikashi, Anne E Goldman, Robert D Goldman, Denisa D Wagner, Clare M Waterman.

  Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.

Keywords:  innate immunity; microscopy; neutrophil

DOI:  https://doi.org/10.1073/pnas.1909546117
Clin Lab. 2020 Mar 01. 66(3):

The Trend of ripk1/ripk3 and mlkl Mediated Necroptosis Pathway in Patients with Different Stages of Prostate Cancer as Promising Progression Biomarkers.

Farnaz Heidaryan, Hadi Bamehr, Babak Babaabasi, Alireza Imamverdi, Nima Mohammadzadeh, Ahmad Khalili.

BACKGROUND: Programmed cell death is critical to maintain tissue homeostasis. Necroptosis, as well as apoptosis, has been considered as another form of regulated cell death which can be used as an effective way to overcome apoptosis-resistant tumor tissue growth. The aim of present study was to test whether or not ripk1, ripk3, or mlkl expression levels, as the key necroptotic modulators in different stages of prostate tumor growth.
METHODS: Sixty-seven prostate tissues representing histologically confirmed cancer were selected. The cancer samples were categorized into 4 different stages based on cellular differentiation, tumor growth rate, and extra tissue expansion to regional lymph nodes, average PSA levels, and tumor volume. RNA extraction, cDNA synthesis and quantitative real time PCR were done based on standard guidelines.
RESULTS: No statistically significant changes in ripk1 expression showed in all three stages (stage II to IV). The expression pattern of ripk3 represented a remarkable elevation in early stage, while, predominantly repressed in final cancer stage (IV). Also, there has been a significant negative correlation between ripk3 gene expression and tumor size and PSA levels.
CONCLUSIONS: We cannot exclude the importance of the key regulator proteins in development and progression of prevalent lethal disease like prostate cancer. The ripk1/ripk3 mediated necroptosis pathway is more activated in early stages of prostate cancer via induced ripk3 expression, while repressed during prostate cancer final stages. Also, the repression of ripk3 is related to elevation of both PSA levels and tumor volume which represented the tumor progression in final stages.

DOI: https://doi.org/10.7754/Clin.Lab.2019.190439
Am J Physiol Cell Physiol. 2020 Mar 11.

Purinergic signalling, DAMPs and inflammation.

Francesco Di Virgilio, Alba Clara Sarti, Robson Coutinho-Silva.

  Danger sensing is one of the most fundamental evolutionary features enabling multicellular organisms to perceive potential threats, escape from risky situations, fight actual intruders and repair damage. Several endogenous molecules are used to "signal damage", currently referred to as "alarmins" or "Damage-Associated Molecular Patterns" (DAMPs), most being already present within all cells (pre-formed DAMPs), and thus ready to be released, and others neo-synthesized following injury. Over recent years it has become overwhelmingly clear that adenosine 5'-triphosphate (ATP) is a ubiquitous and extremely efficient DAMP (thus promoting inflammation) and its main metabolite, adenosine, is a strong immunosuppressant (thus dampening inflammation). Extracellular ATP ligates and activates the P2 purinergic receptors (P2Rs) and is then degraded by soluble and plasma membrane ecto-nucleotidases to generate adenosine acting at P1 purinergic receptors (P1Rs). Extracellular ATP, P2Rs, ecto-nucleotidases, adenosine and P1Rs are basic elements of the purinergic signalling network and fundamental pillars of inflammation.

Keywords:  adenosine; extracellular ATP; inflammation

DOI:  https://doi.org/10.1152/ajpcell.00053.2020
J Immunol. 2020 Mar 13. pii: ji1901410. [Epub ahead of print]

IFN-Stimulated Gene 15 Is an Alarmin that Boosts the CTL Response via an Innate, NK Cell-Dependent Route.

Victoria Iglesias-Guimarais, Tomasz Ahrends, Evert de Vries, Klaus-Peter Knobeloch, Andriy Volkov, Jannie Borst.

Type I IFN is produced upon infection and tissue damage and induces the expression of many IFN-stimulated genes (ISGs) that encode host-protective proteins. ISG15 is a ubiquitin-like molecule that can be conjugated to proteins but is also released from cells in a free form. Free, extracellular ISG15 is suggested to have an immune-regulatory role, based on disease phenotypes of ISG15-deficient humans and mice. However, the underlying mechanisms by which free ISG15 would act as a "cytokine" are unclear and much debated. We, in this study, demonstrate in a clinically relevant mouse model of therapeutic vaccination that free ISG15 is an alarmin that induces tissue alert, characterized by extracellular matrix remodeling, myeloid cell infiltration, and inflammation. Moreover, free ISG15 is a potent adjuvant for the CTL response. ISG15 produced at the vaccination site promoted the vaccine-specific CTL response by enhancing expansion, short-lived effector and effector/memory differentiation of CD8+ T cells. The function of free ISG15 as an extracellular ligand was demonstrated, because the equivalents in murine ISG15 of 2 aa recently implicated in binding of human ISG15 to LFA-1 in vitro were required for its adjuvant effect in vivo. Moreover, in further agreement with the in vitro findings on human cells, free ISG15 boosted the CTL response in vivo via NK cells in the absence of CD4+ T cell help. Thus, free ISG15 is part of a newly recognized innate route to promote the CTL response.

DOI: https://doi.org/10.4049/jimmunol.1901410
Front Immunol. 2020 ;11 242

Innate Lymphocytes in Psoriasis.

Barbara Polese, Hualin Zhang, Bavanitha Thurairajah, Irah L King.

  Skin is a fundamental component of our host defense system that provides a dynamic physical and chemical barrier against pathogen invasion and environmental insults. Cutaneous barrier function is mediated by complex interactions between structural cells such as keratinocytes and diverse lineages of immune cells. In contrast to the protective role of these intercellular interactions, uncontrolled immune activation can lead to keratinocyte dysfunction and psoriasis, a chronic inflammatory disease affecting 2% of the global population. Despite some differences between human and murine skin, animal models of psoriasiform inflammation have greatly informed clinical approaches to disease. These studies have helped to identify the interleukin (IL)-23-IL-17 axis as a central cytokine network that drives disease. In addition, they have led to the recent description of long-lived, skin-resident innate lymphocyte and lymphoid cells that accumulate in psoriatic lesions. Although not completely defined, these populations have both overlapping and unique functions compared to antigen-restricted αβ T lymphocytes, the latter of which are well-known to contribute to disease pathogenesis. In this review, we describe the diversity of innate lymphocytes and lymphoid cells found in mammalian skin with a special focus on αβ T cells, Natural Killer T cells and Innate Lymphoid cells. In addition, we discuss the effector functions of these unique leukocyte subsets and how each may contribute to different stages of psoriasis. A more complete understanding of these cell types that bridge the innate and adaptive immune system will hopefully lead to more targeted therapies that mitigate or prevent disease progression.

Keywords:  disease; innate; lymphocyte; psoriasis; skin

DOI:  https://doi.org/10.3389/fimmu.2020.00242
PLoS Pathog. 2020 Mar 09. 16(3): e1008364

Interferon inducible GBPs restrict Burkholderia thailandensis motility induced cell-cell fusion.

David E Place, Benoit Briard, Parimal Samir, Rajendra Karki, Anannya Bhattacharya, Clifford S Guy, Jennifer L Peters, Sharon Frase, Peter Vogel, Geoffrey Neale, Masahiro Yamamoto, Thirumala-Devi Kanneganti.

Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promote bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of bacterial Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with the effects in in vitro macrophages, Gbp2-/-, Gbp5-/-, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion and bacteria-induced pathology during infection.

DOI: https://doi.org/10.1371/journal.ppat.1008364
Br J Dermatol. 2020 Mar 09.

"Mind the gap": What patients and clinicians believe is "unknown" about psoriasis.

R Majeed-Ariss, M McPhee, H McAteer, C E M Griffiths, H S Young.

A Priority Setting Partnership (PSP) provides an opportunity for clinicians and patients to jointly decide the research priorities for a specific disease area. The James Lind Alliance (JLA), a non-profit making initiative established in 2004 which is funded by the National Institute for Health Research (NIHR), have developed methodology to facilitate PSPs. The JLA believe that medical research often overlooks the questions that patients, carers and health-care professionals consider important and that many areas of potentially important research are therefore neglected and limited research funding wasted.

DOI: https://doi.org/10.1111/bjd.19023
Cardiovasc Res. 2020 Mar 10. pii: cvaa034. [Epub ahead of print]

RUNX1: an emerging therapeutic target for cardiovascular disease.

Alexandra Riddell, Martin McBride, Thomas Braun, Stuart A Nicklin, Ewan Cameron, Christopher M Loughrey, Tamara P Martin.

  Runt-related transcription factor-1 (RUNX1), also known as acute myeloid leukaemia 1 protein (AML1), is a member of the core-binding factor family of transcription factors which modulate cell proliferation, differentiation, and survival in multiple systems. It is a master-regulator transcription factor, which has been implicated in diverse signalling pathways and cellular mechanisms during normal development and disease. RUNX1 is best characterized for its indispensable role for definitive haematopoiesis and its involvement in haematological malignancies. However, more recently RUNX1 has been identified as a key regulator of adverse cardiac remodelling following myocardial infarction. This review discusses the role RUNX1 plays in the heart and highlights its therapeutic potential as a target to limit the progression of adverse cardiac remodelling and heart failure.

Keywords:  Adverse cardiac remodelling; Calcium; Cardiovascular diseases; Excitation–contraction coupling; Heart failure; Myocardial infarction; Runx1

DOI:  https://doi.org/10.1093/cvr/cvaa034