Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

AbstractRIPK1 is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation. RIPK1 activation is known to be regulated by its DD-mediated interaction and ubiquitination, though underlying mechanisms remain incompletely understood. Here we show that K627 in human RIPK1-DD and its equivalent K612 in murine RIPK1-DD is a key ubiquitination site that regulates the overall ubiquitination pattern of RIPK1 and its DD-mediated interactions with other DD-containing proteins. K627R/K612R mutation inhibits the activation of RIPK1 and blocks both apoptosis and necroptosis mediated by TNFR1 signaling. However, Ripk1K612R/K612R mutation sensitizes cells to necroptosis and caspase-1 activation in response to TLRs signaling. Ripk1K612R/K612R mice are viable, but develop age-dependent reduction of RIPK1 expression, spontaneous intestinal inflammation and splenomegaly, which can be rescued by antibiotic treatment and partially by Ripk3 deficiency. Furthermore, we show that the interaction of RIPK1 with FADD contributes to suppressing the activation of RIPK3 mediated by TLRs signaling. Our study demonstrates the distinct roles of K612 ubiquitination in mRIPK1/K627 ubiquitination in hRIPK1 in regulating its pro-death kinase activity in response to TNFα and pro-survival activity in response to TLRs signaling.

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Long-chain PUFA ameliorate ETEC-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in IPEC-1 cells

British Journal Of Nutrition ◽

10.1017/s0007114521005092 ◽

2021 ◽

pp. 1-36

Author(s):

Kan Xiao ◽

Yang Yang ◽

Yang Zhang ◽

Qingqing Lv ◽

Feifei Huang ◽

...

Keyword(s):

Protein Expression ◽

Mrna Expression ◽

Signaling Pathways ◽

Cell Injury ◽

Intestinal Inflammation ◽

Fluorescein Isothiocyanate ◽

Receptor Protein ◽

Phosphoglycerate Mutase ◽

Death Domain ◽

Caspase 1

Abstract This study was aimed to investigate whether eicosapentaenoic acid (EPA) and arachidonic acid (ARA), the representative n-3 or n-6 polyunsaturated fatty acids (PUFA), could alleviate enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation and injury of intestinal porcine epithelial cells 1 (IPEC-1) by modulating pyroptosis and necroptosis signaling pathways. IPEC-1 cells were cultured with or without EPA or ARA in the presence or absence of ETEC K88. EPA and ARA reduced ETEC K88 adhesion and endotoxin content in the supernatant. EPA and ARA increased transepithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-labeled dextran (FD4), and increased membrane protein expression of occludin, ZO-1 and claudin-1, and relieved disturbed distribution of these proteins. EPA and ARA also reduced cell necrosis ratio. EPA or ARA reduced mRNA and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8, and decreased mRNA abundances of intestinal toll-like receptors 4 (TLR4) and its downstream signals. Moreover, EPA and ARA downregulated mRNA expression of nod-like receptor protein 3 (NLRP3), caspase 1 and IL-18, and inhibited protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D and caspase-1. Finally, EPA and ARA reduced mRNA expression of fas-associated death domain protein (FADD), caspase 8, receptor interacting protein kinase (RIP) 1, mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), motility related protein 1 (Drp1) and high mobility protein 1 (HMGB1), and inhibited protein expression of phosphorylated-RIP1 (p-RIP1), p-RIP3, p-MLKL and HMGB1. These data demonstrate that EPA and ARA prevent ETEC K88-induced cell inflammation and injury, which is partly through inhibiting pyroptosis and necroptosis signaling pathways.

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Metabolomics of aging in primary fibroblasts from small and large breed dogs

GeroScience ◽

10.1007/s11357-021-00388-0 ◽

2021 ◽

Author(s):

Paul S. Brookes ◽

Ana Gabriela Jimenez

Keyword(s):

Aging Process ◽

Tca Cycle ◽

Therapeutic Strategies ◽

Targeted Metabolomics ◽

The Tca Cycle ◽

Age Dependent ◽

Primary Fibroblasts ◽

Aging Rates ◽

Underlying Mechanisms ◽

Coenzyme A Biosynthesis

AbstractAmong several animal groups (eutherian mammals, birds, reptiles), lifespan positively correlates with body mass over several orders of magnitude. Contradicting this pattern are domesticated dogs, with small dog breeds exhibiting significantly longer lifespans than large dog breeds. The underlying mechanisms of differing aging rates across body masses are unclear, but it is generally agreed that metabolism is a significant regulator of the aging process. Herein, we performed a targeted metabolomics analysis on primary fibroblasts isolated from small and large breed young and old dogs. Regardless of size, older dogs exhibited lower glutathione and ATP, consistent with a role for oxidative stress and bioenergetic decline in aging. Furthermore, several size-specific metabolic patterns were observed with aging, including the following: (i) An apparent defect in the lower half of glycolysis in large old dogs at the level of pyruvate kinase. (ii) Increased glutamine anaplerosis into the TCA cycle in large old dogs. (iii) A potential defect in coenzyme A biosynthesis in large old dogs. (iv) Low nucleotide levels in small young dogs that corrected with age. (v) An age-dependent increase in carnitine in small dogs that was absent in large dogs. Overall, these data support the hypothesis that alterations in metabolism may underlie the different lifespans of small vs. large breed dogs, and further work in this area may afford potential therapeutic strategies to improve the lifespan of large dogs.

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Chloroplast Hsp70 Isoform Is Required for Age-Dependent Tissue Preference of Bamboo mosaic virus in Mature Nicotiana benthamiana Leaves

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0012-r ◽

2017 ◽

Vol 30 (8) ◽

pp. 631-645 ◽

Cited By ~ 3

Author(s):

Ying Wen Huang ◽

Chung Chi Hu ◽

Ching Hsiu Tsai ◽

Na Sheng Lin ◽

Yau Heiu Hsu

Keyword(s):

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Transit Peptide ◽

Plant Viruses ◽

Age Dependent ◽

Efficient Replication ◽

Underlying Mechanisms ◽

Lines Of Evidence ◽

Suitable Environment

Plant viruses may exhibit age-dependent tissue preference in their hosts but the underlying mechanisms are not well understood. In this study, we provide several lines of evidence to reveal the determining role of a protein of the Nicotiana benthamiana chloroplast Hsp70 (NbcpHsp70) family, NbcpHsp70-2, involved in the preference of Bamboo mosaic virus (BaMV) to infect older tissues. NbcpHsp70 family proteins were identified in complexes pulled down with BaMV replicase as the bait. Among the isoforms of NbcpHsp70, only the specific silencing of NbcpHsp70-2 resulted in the significant decrease of BaMV RNA in N. benthamiana protopalsts, indicating that NbcpHsp70-2 is involved in the efficient replication of BaMV RNA. We further identified the age-dependent import regulation signal contained in the transit peptide of NbcpHsp70-2. Deletion, overexpression, and substitution experiments revealed that the signal in the transit peptide of NbcpHsp70-2 is crucial for both the import of NbcpHsp70-2 into older chloroplasts and the preference of BaMV for infecting older leaves of N. benthamiana. Together, these data demonstrated that BaMV may exploit a cellular age-dependent transportation mechanism to target a suitable environment for viral replication.

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CCR6–CCL20-Mediated Immunologic Pathways in Inflammatory Bowel Disease

Gastrointestinal Disorders ◽

10.3390/gidisord1010003 ◽

2018 ◽

Vol 1 (1) ◽

pp. 15-29 ◽

Cited By ~ 3

Author(s):

Ranmali Ranasinghe ◽

Rajaraman Eri

Keyword(s):

Inflammatory Bowel Disease ◽

Bowel Disease ◽

Intestinal Inflammation ◽

Systematic Investigation ◽

Protective Mechanism ◽

Food Antigens ◽

Gut Mucosa ◽

T Helper Lymphocyte ◽

Inflammatory Bowel ◽

Underlying Mechanisms

Inflammatory bowel disease (IBD) has evoked significant interest in human immunobiology given its tactical immune evasion methodologies resulting in acute immune destabilization. IBD comprising Crohn’s disease and Ulcerative colitis manifests as chronic inflammation in the gut mucosa, leading to complexities involving immune dysregulation in the T helper lymphocyte arm, effecting disease pathogenicity. The mucosa of the alimentary canal is constantly exposed to a myriad of food antigens and luminal microorganisms for which a consistent host-protective mechanism is operative in healthy people. Lowered mucosal immune expression which allows penetration of the epithelial barrier by infective pathogenic microbes elicits both innate and adaptive immune responses in the gut, culminating in aberrant intestinal inflammation. Interestingly, the IBD leukocyte repertoire is significantly entwined with chemokine-assisted chemotactic navigation into the sites of inflammation, which is also thought to generate favorable immune-suppressive responses. The functions of the cognate chemokine receptor, CCR6, which binds with its unique ligand CCL20, are expected to tilt the balance between upregulation of homeostatic tolerance and inflammatory pathophysiology. This review aims to critically examine the CCR6-driven immune pathways: TH1/TH2, TH1/TH17, TH17/Treg, IL-23/IL-17, Akt/ERK-1/2, ILC3, and TH9/TH2 for systematic investigation of its underlying mechanisms in the future and to underpin its importance in resolving IBD pathology. Thus, CCR6 occupies an exclusive position in gut immunology which renders it an invaluable therapeutic tool for the production of novel medicaments to treat IBD.

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LRR protein RNH1 inhibits inflammasome activation through proteasome-mediated degradation of Caspase-1 and is associated with adverse clinical outcomes in COVID-19 patients.

10.1101/2021.04.12.438219 ◽

2021 ◽

Author(s):

Giuseppe Bombaci ◽

Mayuresh A Sarangdhar ◽

Nicola Andina ◽

Aubry Tardivel ◽

Eric Chi-Wang Yu ◽

...

Keyword(s):

Inflammatory Diseases ◽

Neutrophil Infiltration ◽

Inflammasome Activation ◽

Ribonuclease Inhibitor ◽

Protein Levels ◽

Caspase 1 ◽

Pyrin Domain ◽

Underlying Mechanisms ◽

Lrr Protein ◽

Receptor Family

Inflammasomes are cytosolic innate immune sensors that, upon activation, induce caspase-1 mediated inflammation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and is also detrimental in COVID-19 infection. However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine rich repeat (LRR) protein Ribonuclease inhibitor (RNH1), which shares homology with LRRs of NOD-like receptor family pyrin domain (PYD)-containing (NLRP) proteins, attenuates inflammasome activation. Mechanistically, RNH1 decreased pro-IL1b expression and induced proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and LPS-induced endotoxemia, which are dependent on caspase-1, respectively showed increased neutrophil infiltration and lethality in Rnh1-/- mice compared to WT mice. Further, RNH1 protein levels were negatively correlated with inflammation and disease severity in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.

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Replicating infant astrocyte behavior in the adult after brain injury improves outcomes

10.1101/2020.05.14.096974 ◽

2020 ◽

Author(s):

Leon Teo ◽

Anthony G. Boghdadi ◽

Jihane Homman-Ludiye ◽

Iñaki Carril-Mundiñano ◽

William C. Kwan ◽

...

Keyword(s):

Ischemic Stroke ◽

Brain Injury ◽

Brain Injuries ◽

Adult Patients ◽

Long Term Outcomes ◽

Neuroprotective Effects ◽

Age Dependent ◽

Underlying Mechanisms ◽

Glial Scarring

AbstractInfants and adults respond differently to brain injuries. Specifically, improved neuronal sparing along with reduced astrogliosis and glial scarring often observed earlier in life, likely contributes to improved long-term outcomes. Understanding the underlying mechanisms could enable the recapitulation of neuroprotective effects, observed in infants, to benefit adult patients after brain injuries. We reveal that in primates, Eph/ ephrin signaling contributes to age-dependent reactive astrocyte behavior. Ephrin-A5 expression on astrocytes was more protracted in adults, whereas ephrin-A1 was associated only with infant astrocytes. Furthermore, ephrin-A5 exacerbated major hallmarks of astrocyte reactivity via EphA2 and EphA4 receptors, which was subsequently alleviated by ephrin-A1. Rather than suppressing reactivity, ephrin-A1 signaling shifted astrocytes towards GAP43+ neuroprotection, accounting for improved neuronal sparing in infants. Reintroducing ephrin-A1 after middle-aged ischemic stroke significantly attenuated glial scarring, improved neuronal sparing and preserved circuitry. Therefore, beneficial infant mechanisms can be recapitulated in adults to improve outcomes after CNS injuries.

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Death-domain dimerization-mediated activation of RIPK1 controls necroptosis and RIPK1-dependent apoptosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722013115 ◽

2018 ◽

Vol 115 (9) ◽

pp. E2001-E2009 ◽

Cited By ~ 30

Author(s):

Huyan Meng ◽

Zhen Liu ◽

Xingyan Li ◽

Huibing Wang ◽

Taijie Jin ◽

...

Keyword(s):

Cell Death ◽

Proinflammatory Cytokine ◽

Kinase Activity ◽

Kinase Domain ◽

Death Domain ◽

Degenerative Diseases ◽

Intermediate Domain ◽

A Charge ◽

Mutant Cells

RIPK1 is a critical mediator of cell death and inflammation downstream of TNFR1 upon stimulation by TNFα, a potent proinflammatory cytokine involved in a multitude of human inflammatory and degenerative diseases. RIPK1 contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The kinase activity of RIPK1 promotes cell death and inflammation. Here, we investigated the involvement of RIPK1-DD in the regulation of RIPK1 kinase activity. We show that a charge-conserved mutation of a lysine located on the surface of DD (K599R in human RIPK1 or K584R in murine RIPK1) blocks RIPK1 activation in necroptosis and RIPK1-dependent apoptosis and the formation of complex II. Ripk1K584R/K584R knockin mutant cells are resistant to RIPK1 kinase-dependent apoptosis and necroptosis. The resistance of K584R cells, however, can be overcome by forced dimerization of RIPK1. Finally, we show that the K584R RIPK1 knockin mutation protects mice against TNFα-induced systematic inflammatory response syndrome. Our study demonstrates the role of RIPK1-DD in mediating RIPK1 dimerization and activation of its kinase activity during necroptosis and RIPK1-dependent apoptosis.

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Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

Journal of Cell Science ◽

10.1242/jcs.114.20.3749 ◽

2001 ◽

Vol 114 (20) ◽

pp. 3749-3757 ◽

Cited By ~ 6

Author(s):

Patrick Meraldi ◽

Erich A. Nigg

Keyword(s):

Protein Phosphatase ◽

Kinase Activity ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Microtubule Depolymerization ◽

Drug Induced ◽

Microtubule Network ◽

Similar Phenotype ◽

Underlying Mechanisms

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.

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Phonological span in children and adults: Does response format matter?

International Journal of Behavioral Development ◽

10.1177/0165025419840709 ◽

2019 ◽

Vol 43 (5) ◽

pp. 393-402 ◽

Cited By ~ 1

Author(s):

Eva Oesterlen ◽

Katja Seitz-Stein

Keyword(s):

Visual Search ◽

Digit Span ◽

Fourth Graders ◽

Age Groups ◽

Verbal Response ◽

Response Format ◽

Verbal Recall ◽

Age Dependent ◽

Span Tasks ◽

Underlying Mechanisms

In contrast to classical phonological span tasks, which require verbal recall, those used in self-reliant, group-administrable working memory measurement contain a visuospatial response format. As a consequence, these tasks involve recoding, executive, and visual search requirements in addition to encoding and storage processes. To examine age-dependent effects of these special requirements, tablet-based word and digit span tasks with a visuospatial response format were compared to corresponding tasks with a verbal response format in first and second graders, third and fourth graders, and adults ( N = 105). Whereas children’s performance was impaired by the visuospatial response format, adults’ performance was not. Whether the arrangement of stimuli in the visuospatial response format—either variable or fixed—has differential effects on performance in children and adults ( N = 434) was examined in a second experiment. No substantial differences between variable and fixed response format were found in any of the age groups. Possible underlying mechanisms for age-dependent effects of response format and the role of executive functions and visual search in automatized phonological span tasks are discussed.

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Orchestration of ErbB3 signaling through heterointeractions and homointeractions

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1114 ◽

2015 ◽

Vol 26 (22) ◽

pp. 4109-4123 ◽

Cited By ~ 12

Author(s):

Meghan McCabe Pryor ◽

Mara P. Steinkamp ◽

Adam M. Halasz ◽

Ye Chen ◽

Shujie Yang ◽

...

Keyword(s):

Binding Sites ◽

Tyrosine Kinases ◽

Kinase Activity ◽

Erbb Family ◽

Downstream Signaling ◽

Activating Mutations ◽

Function Point ◽

Dimerization Interface ◽

Underlying Mechanisms ◽

Erbb3 Signaling

Members of the ErbB family of receptor tyrosine kinases are capable of both homointeractions and heterointeractions. Because each receptor has a unique set of binding sites for downstream signaling partners and differential catalytic activity, subtle shifts in their combinatorial interplay may have a large effect on signaling outcomes. The overexpression and mutation of ErbB family members are common in numerous human cancers and shift the balance of activation within the signaling network. Here we report the development of a spatial stochastic model that addresses the dynamics of ErbB3 homodimerization and heterodimerization with ErbB2. The model is based on experimental measures for diffusion, dimer off-rates, kinase activity, and dephosphorylation. We also report computational analysis of ErbB3 mutations, generating the prediction that activating mutations in the intracellular and extracellular domains may be subdivided into classes with distinct underlying mechanisms. We show experimental evidence for an ErbB3 gain-of-function point mutation located in the C-lobe asymmetric dimerization interface, which shows enhanced phosphorylation at low ligand dose associated with increased kinase activity.

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