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1.
Annu Rev Immunol ; 33: 79-106, 2015.
Article in English | MEDLINE | ID: mdl-25493335

ABSTRACT

Cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. Disease pathologies ensue when these processes are disturbed. A plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity, or immunodeficiency. Programmed necrosis or necroptosis is a form of nonapoptotic cell death driven by the receptor interacting protein kinase 3 (RIPK3) and its substrate, mixed lineage kinase domain-like (MLKL). RIPK3 partners with its upstream adaptors RIPK1, TRIF, or DAI to signal for necroptosis in response to death receptor or Toll-like receptor stimulation, pathogen infection, or sterile cell injury. Necroptosis promotes inflammation through leakage of cellular contents from damaged plasma membranes. Intriguingly, many of the signal adaptors of necroptosis have dual functions in innate immune signaling. This unique signature illustrates the cooperative nature of necroptosis and innate inflammatory signaling pathways in managing cell and organismal stresses from pathogen infection and sterile tissue injury.


Subject(s)
Inflammation/metabolism , Inflammation/pathology , Necrosis/metabolism , Signal Transduction , Animals , Bacterial Infections/genetics , Bacterial Infections/metabolism , Bacterial Infections/pathology , Biological Evolution , Cell Death , Humans , Inflammasomes/metabolism , Inflammation/genetics , Interleukin-1beta/metabolism , NF-kappa B/metabolism , Parasitic Diseases/genetics , Parasitic Diseases/metabolism , Parasitic Diseases/pathology , Phosphorylation , Protein Binding , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Ubiquitination , Virus Diseases/genetics , Virus Diseases/metabolism , Virus Diseases/pathology
2.
Cell ; 150(2): 339-50, 2012 Jul 20.
Article in English | MEDLINE | ID: mdl-22817896

ABSTRACT

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of ß-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.


Subject(s)
Necrosis/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Amino Acid Sequence , Amyloid/chemistry , Humans , Molecular Sequence Data , Protein Interaction Domains and Motifs , Receptor-Interacting Protein Serine-Threonine Kinases/chemistry , Sequence Alignment
3.
Biochem Biophys Res Commun ; 675: 71-77, 2023 10 01.
Article in English | MEDLINE | ID: mdl-37453260

ABSTRACT

Fibroblast growth factor 18 (FGF18) is elevated in several human cancers, such as gastrointestinal and ovarian cancers, and stimulates the proliferation of tumor cells. This suggests that FGF18 may be a promising candidate biomarker in cancer patients. However, the lack of a high-sensitivity enzyme-linked immunosorbent assay (ELISA) does not permit testing of this possibility. In this study, we generated monoclonal antibodies against human FGF18 and developed a high-sensitivity ELISA to measure human FGF18 at concentrations as low as 10 pg/mL. Of the eight tumor cell lines investigated, we detected human FGF18 in culture supernatants from four tumor cell lines, including HeLa, OVCAR-3, BxPC-3, and SW620 cells, albeit the production levels were relatively low in the latter two cell lines. Moreover, the in-house ELISA could detect murine FGF18 in sera from mice overexpressing murine Fgf18 in hepatocytes, although the sensitivity in detecting murine FGF18 was relatively low. This FGF18 ELISA could be a valuable tool to validate FGF18 as a potential biomarker for cancer patients and to test the contribution of FGF18 for various disease models invivo and in vitro.


Subject(s)
Apoptosis , Ovarian Neoplasms , Humans , Mice , Animals , Female , Cell Line, Tumor , Ovarian Neoplasms/pathology , Fibroblast Growth Factors/metabolism , Enzyme-Linked Immunosorbent Assay
4.
Osteoporos Int ; 34(10): 1703-1709, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37291359

ABSTRACT

A history of fracture in adulthood and urinary pentosidine levels were independently and significantly associated with fracture occurrence in this prospective observational study of community-dwelling older adults. PURPOSE: This prospective observational study aimed to determine the factors associated with fragility fractures in community-dwelling older adults. METHODS: Overall, 254 older adults who were participants of the Good Aging and Intervention Against Nursing Care and Activity Decline study in 2016 were included in this study. Grip strength, muscle mass, gait speed, calcaneal bone density, and the levels of parathyroid hormone, osteocalcin, 25-hydroxyvitamin D, total procollagen type I N-terminal propeptide, insulin-like growth factor-1 (IGF-1), tartrate-resistant acid phosphatase-5b, and urinary pentosidine were measured at baseline. Participants were classified as fracture ( +) or fracture (-) based on the data collected during a 5-year follow-up period. RESULTS: Excluding those who were lost to follow-up during the observation period, 182 participants (64 men and 118 women, mean age: 74.2 years, range: 47-99 years) were included in the analysis. During the observation period, 23 patients experienced 24 new fractures. In univariate analysis, sex, height, weight, history of fracture in adulthood, baseline grip strength, muscle mass, bone density, and the levels of urinary pentosidine and IGF-1 at baseline were significantly different between patients who developed a fracture during follow-up and those who did not. In multivariate analysis, a history of fracture in adulthood and urinary pentosidine levels were independently and significantly associated with fracture occurrence. CONCLUSION: High urine pentosidine levels and a history of fracture in adulthood are independent risk factors for fracture occurrence in community-dwelling older adults.


Subject(s)
Fractures, Bone , Insulin-Like Growth Factor I , Male , Humans , Female , Aged , Independent Living , Bone Density/physiology
5.
Immunity ; 41(4): 567-78, 2014 Oct 16.
Article in English | MEDLINE | ID: mdl-25367573

ABSTRACT

Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor-interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation by regulating cytokine expression in dendritic cells (DCs). Ripk3(-/-) bone-marrow-derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and by impaired caspase 1-mediated processing of interleukin-1ß (IL-1ß). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3(-/-) mice exhibited an impaired axis of injury-induced IL-1ß, IL-23, and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild-type DCs, but not Ripk3(-/-) DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory-cytokine expression, and injury-induced tissue repair.


Subject(s)
Apoptosis/immunology , Dendritic Cells/immunology , Necrosis/immunology , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Wound Healing/genetics , Adoptive Transfer , Animals , Bone Marrow Cells/immunology , Caspase 1/metabolism , Colitis/genetics , Colitis/immunology , Dendritic Cells/transplantation , Dextran Sulfate , Enzyme Activation/genetics , Enzyme Activation/immunology , Female , Gene Expression Regulation/immunology , Inflammation/immunology , Interleukin-1beta/biosynthesis , Interleukin-1beta/immunology , Interleukin-23/biosynthesis , Interleukin-23/immunology , Interleukins/biosynthesis , Interleukins/immunology , Lipopolysaccharides , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/immunology , RNA, Messenger/biosynthesis , Receptors, Interleukin/biosynthesis , Signal Transduction/immunology , Transcription Factor RelB/genetics , Transcription Factor RelB/immunology , Interleukin-22
6.
Biochem J ; 479(5): 677-685, 2022 03 18.
Article in English | MEDLINE | ID: mdl-35293986

ABSTRACT

Damage-associated molecular patterns (DAMPs) are molecules within living cells that are released when cell membranes are ruptured. Although DAMPs have physiological functions inside the cell, once DAMPs are released extracellularly, they elicit various biological responses, including inflammation, proliferation, tissue damage, and tissue repair, in a context-dependent manner. In past decades, it was assumed that the release of DAMPs was induced by a membrane rupture, caused by passive ATP depletion, or by chemical or mechanical damage to the membrane. However, that concept has been challenged by recent advancements in understanding the regulation of cell death. Necroptosis is a form of regulated cell death, where cells show necrotic morphology. Necroptosis is triggered by death receptors, toll-like receptors, and some viral infections. The membrane rupture is executed by the mixed lineage-like kinase domain-like pseudokinase (MLKL), which forms oligomers that translocate to the plasma membrane during necroptosis. Although the causal relationship between MLKL function and membrane rupture has been extensively investigated, the detailed molecular mechanisms by which oligomerized MLKL induces membrane rupture are not fully understood. This review summarizes recent advances in understanding how MLKL regulates DAMP release and new technologies for visualizing DAMP release at single-cell resolution.


Subject(s)
Apoptosis , Protein Kinases , Apoptosis/physiology , Cell Death , Humans , Necroptosis , Necrosis/metabolism , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism
7.
Mol Cell ; 56(4): 481-95, 2014 Nov 20.
Article in English | MEDLINE | ID: mdl-25459880

ABSTRACT

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.


Subject(s)
Apoptosis , Receptor-Interacting Protein Serine-Threonine Kinases/physiology , Animals , Caspase 8/metabolism , Fas-Associated Death Domain Protein/metabolism , Gene Knock-In Techniques , HT29 Cells , Humans , Mice , Mice, Transgenic , NIH 3T3 Cells , Necrosis/enzymology , Nuclear Pore Complex Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , RNA-Binding Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors
8.
Int J Mol Sci ; 23(13)2022 Jun 23.
Article in English | MEDLINE | ID: mdl-35805980

ABSTRACT

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1ß converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Colonic Neoplasms , Proscillaridin , TNF-Related Apoptosis-Inducing Ligand , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Drug Synergism , Humans , Proscillaridin/administration & dosage , Proscillaridin/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/administration & dosage , TNF-Related Apoptosis-Inducing Ligand/pharmacology
9.
FASEB J ; 34(7): 9450-9465, 2020 07.
Article in English | MEDLINE | ID: mdl-32496646

ABSTRACT

Intestinal epithelial cells (IECs) are not only responsible for the digestion and absorption of dietary substrates but also function as a first line of host defense against commensal and pathogenic luminal bacteria. Disruption of the epithelial layer causes malnutrition and enteritis. Rab6 is a small GTPase localized to the Golgi, where it regulates anterograde and retrograde transport by interacting with various effector proteins. Here, we generated mice with IEC-specific deletion of Rab6a (Rab6a∆IEC mice). While Rab6aΔIEC mice were born at the Mendelian ratio, they started to show IEC death, inflammation, and bleeding in the small intestine shortly after birth, and these changes culminated in early postnatal death. We further found massive lipid accumulation in the IECs of Rab6a∆IEC neonates. In contrast to Rab6a∆IEC neonates, knockout embryos did not show any of these abnormalities. Lipid accumulation and IEC death became evident when Rab6a∆IEC embryos were nursed by a foster mother, suggesting that dietary milk-derived lipids accumulated in Rab6a-deficient IECs and triggered IEC death. These results indicate that Rab6a plays a crucial role in regulating the lipid transport and maintaining tissue integrity.


Subject(s)
Cell Death , Epithelial Cells/pathology , Inflammation/pathology , Intestine, Small/pathology , Lactation , Lipids/chemistry , rab GTP-Binding Proteins/physiology , Animals , Epithelial Cells/metabolism , Female , Glycosylation , Inflammation/etiology , Inflammation/metabolism , Intestine, Small/metabolism , Mice , Mice, Knockout
10.
BMC Musculoskelet Disord ; 22(1): 392, 2021 Apr 26.
Article in English | MEDLINE | ID: mdl-33902533

ABSTRACT

BACKGROUND: Muscle and bone interactions might be associated with osteoporosis and sarcopenia. Urinary pentosidine and serum 25-hydroxyvitamin D (25(OH)D) might affect muscle and bone interactions. It is unclear whether these biomarkers are affected by age and sex or play a role in muscle and physical functions. We aimed to investigate the association between urinary pentosidine and serum 25(OH)D levels with muscle mass, muscle strength, and physical performance in community-dwelling adults. METHODS: Two-hundred and fifty-four middle-aged and elderly adults were enrolled. There was no significant difference in age between 97 men (75.0 ± 8.9 years) and 157 women (73.6 ± 8.1 years). The skeletal muscle mass index (SMI), grip strength, and gait speed were assessed. The urinary pentosidine level was measured. We evaluated the association of urinary pentosidine and serum 25(OH)D levels with age and sex (student's t-test) and correlations between biomarker and each variable (Pearson's correlation coefficients). Multiple regression analysis was performed with grip strength and gait speed as dependent variables and with age, height, weight, body mass index (BMI), speed of sound (SOS), SMI, glycated hemoglobin (HbA1c), estimated glomerular filtration rate (eGFR), 25(OH)D, and pentosidine as independent variables using the stepwise method. RESULTS: The urinary pentosidine level was negatively correlated with grip strength, gait speed, eGFR, and insulin-like growth factor-1 (IGF-1) in men and with SOS, grip strength, and gait speed in women. The serum 25(OH)D level was positively correlated with IGF-1 in women and grip strength in men. Grip strength was associated with age, height, and pentosidine in men and height and pentosidine in women. Gait speed was associated with age, BMI, and pentosidine in men and age, height, and pentosidine in women. CONCLUSION: Urinary pentosidine levels are significantly associated with grip strength and gait speed and may serve as a biomarker of muscle and bone interactions.


Subject(s)
Sarcopenia , Walking Speed , Adult , Aged , Aged, 80 and over , Arginine/analogs & derivatives , Cross-Sectional Studies , Female , Hand Strength , Humans , Independent Living , Lysine/analogs & derivatives , Male , Middle Aged , Muscle Strength , Muscle, Skeletal
11.
Genes Dev ; 27(15): 1640-9, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23913919

ABSTRACT

The receptor-interacting protein kinase 3 (RIP3/RIPK3) has emerged as a critical regulator of programmed necrosis/necroptosis, an inflammatory form of cell death with important functions in pathogen-induced and sterile inflammation. RIP3 activation is tightly regulated by phosphorylation, ubiquitination, and caspase-mediated cleavage. These post-translational modifications coordinately regulate the assembly of a macromolecular signaling complex termed the necrosome. Recently, several reports indicate that RIP3 can promote inflammation independent of its pronecrotic activity. Here, we review our current understanding of the mechanisms that drive RIP3-dependent necrosis and its role in different inflammatory diseases.


Subject(s)
Inflammation/genetics , Necrosis/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Embryonic Development/genetics , Gene Expression Regulation , Humans , Neoplasms/genetics , Protein Processing, Post-Translational , Signal Transduction
12.
J Comput Chem ; 41(11): 1116-1123, 2020 04 30.
Article in English | MEDLINE | ID: mdl-31984537

ABSTRACT

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) fixes atmospheric carbon dioxide into bioavailable sugar molecules. It is also well known that a kinetic isotope effect (KIE; CO2 carbon atoms) accompanies the carboxylation process. To describe the reaction and the KIE α, two different types of molecular dynamics (MD) simulations (ab initio MD and classical MD) have been performed with an Own N-layered Integrated molecular Orbitals and molecular Mechanics (ONIOM)-hybrid model. A channel structure for CO2 transport has been observed during the MD simulation in RubisCO, and assuming the reaction path from the inlet to the product through the coordinate complex with Mg2+ , simulations have been performed on several molecular configuration models fixing several distances between CO2 and ribulose-1,5-bisphosphate along the channel. Free energy analysis and diffusion coefficient analysis have been evaluated for different phases of the process. It is confirmed that the isotopic fractionation effect for CO2 containing either 13 C or 12 C would appear through the transiting path in the channel structure identified in RubisCO. The estimated isotope fractionation constant was quite close to the experimental value.

13.
BMC Musculoskelet Disord ; 20(1): 276, 2019 Jun 05.
Article in English | MEDLINE | ID: mdl-31164134

ABSTRACT

BACKGROUND: Understanding interactions between bone and muscle based on endocrine factors may help elucidate the relationship between osteoporosis and sarcopenia. However, whether the abundance or activity of these endocrine factors is affected by age and sex or whether these factors play a causal role in bone and muscle formation and function is unclear. We aimed to evaluate the association of serum bone- and muscle-derived factors with age, sex, body composition, and physical function in community-dwelling middle-aged and elderly adults. METHODS: In all, 254 residents (97 men, 157 women) participated in this cross-sectional study conducted in Japan. The calcaneal speed of sound (SOS) was evaluated by quantitative ultrasound examination. Skeletal muscle mass index (SMI) was calculated by bioelectrical impedance analysis. Grip strength was measured using a dynamometer. Gait speed was measured by optical-sensitive gait analysis. Serum sclerostin, osteocalcin (OC), insulin-like growth factor-1 (IGF-1), myostatin, and tartrate-resistant acid phosphatase-5b (TRACP-5b) concentrations were measured simultaneously. The difference by sex was determined using t test. Correlations between serum bone- and muscle-derived factors and age, BMI, SOS, SMI, grip strength, gait speed, and TRACP-5b in men and women were determined based on Pearson's correlation coefficients. Multiple regression analysis was performed using the stepwise method. RESULTS: There was no significant difference with regard to age between men (75.0 ± 8.9 years) and women (73.6 ± 8.1 years). Sclerostin was significantly higher in men than in women and tended to increase with age in men; it was significantly associated with SOS and TRACP-5b levels. OC was significantly higher in women than in men and was significantly associated with TRACP-5b levels and age. IGF-1 tended to decrease with age in both sexes and was significantly associated with SOS and body mass index. Myostatin did not correlate with any assessed variables. CONCLUSIONS: Sclerostin was significantly associated with sex, age, and bone metabolism, although there was no discernable relationship between serum sclerostin levels and muscle function. There was no obvious relationship between OC and muscle parameters. This study suggests that IGF-1 is an important modulator of muscle mass and function and bone metabolism in community-dwelling middle-aged and elderly adults.


Subject(s)
Bone and Bones/physiology , Independent Living , Muscle, Skeletal/physiology , Physical Functional Performance , Adaptor Proteins, Signal Transducing , Age Factors , Aged , Aged, 80 and over , Body Composition/physiology , Bone Morphogenetic Proteins/blood , Cross-Sectional Studies , Female , Genetic Markers , Hand Strength/physiology , Humans , Insulin-Like Growth Factor I/analysis , Japan , Male , Osteocalcin/blood , Osteoporosis/blood , Osteoporosis/physiopathology , Sarcopenia/blood , Sarcopenia/physiopathology , Sex Factors , Walking Speed/physiology
14.
J Immunol ; 196(1): 407-15, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26582950

ABSTRACT

The cytokine IL-1ß is intimately linked to many pathological inflammatory conditions. Mature IL-1ß secretion requires cleavage by the inflammasome. Recent evidence indicates that many cell death signal adaptors have regulatory roles in inflammasome activity. These include the apoptosis inducers FADD and caspase 8, and the necroptosis kinases receptor interacting protein kinase 1 (RIPK1) and RIPK3. PGAM5 is a mitochondrial phosphatase that has been reported to function downstream of RIPK3 to promote necroptosis and IL-1ß secretion. To interrogate the biological function of PGAM5, we generated Pgam5(-/-) mice. We found that Pgam5(-/-) mice were smaller compared with wild type littermates, and male Pgam5(-/-) mice were born at sub-Mendelian ratio. Despite these growth and survival defects, Pgam5(-/-) cells responded normally to multiple inducers of apoptosis and necroptosis. Rather, we found that PGAM5 is critical for IL-1ß secretion in response to NLRP3 and AIM2 inflammasome agonists. Moreover, vesicular stomatosis virus-induced IL-1ß secretion was impaired in Pgam5(-/-) bone marrow-derived macrophages, but not in Ripk3(-/-) bone marrow-derived dendritic cells, indicating that PGAM5 functions independent of RIPK3 to promote inflammasome activation. Mechanistically, PGAM5 promotes ASC polymerization, maintenance of mitochondrial integrity, and optimal reactive oxygen species production in response to inflammasome signals. Hence PGAM5 is a novel regulator of inflammasome and caspase 1 activity that functions independently of RIPK3.


Subject(s)
Apoptosis/immunology , Inflammasomes/immunology , Interleukin-1beta/metabolism , Macrophages/immunology , Phosphoric Monoester Hydrolases/genetics , Animals , Carrier Proteins/immunology , Caspase 1/immunology , Caspase 8/immunology , Cells, Cultured , DNA-Binding Proteins/immunology , Dendritic Cells/immunology , Fas-Associated Death Domain Protein/immunology , Inflammation/immunology , Interleukin-1beta/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/immunology , Mitochondria/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Phosphoprotein Phosphatases , Phosphoric Monoester Hydrolases/metabolism , Reactive Oxygen Species/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/immunology , Signal Transduction/immunology , Vesicular stomatitis Indiana virus/immunology
15.
J Biol Chem ; 291(11): 5948-5959, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-26786097

ABSTRACT

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.


Subject(s)
Cell Death/drug effects , Leupeptins/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Cell Line , Cell Line, Tumor , Humans , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Protein Kinases/metabolism , Ubiquitination/drug effects
16.
J Immunol ; 194(4): 1938-44, 2015 Feb 15.
Article in English | MEDLINE | ID: mdl-25567679

ABSTRACT

Caspase 8, the initiator caspase for death receptor-induced apoptosis, functions as a negative regulator of receptor interacting protein kinase 3 (RIPK3), an essential factor for TNF-, TLR3-, and TLR4-induced necroptosis. In certain situations, caspase 8 can also participate in pro-IL-1ß processing. However, the biochemical complex that mediates caspase 8-mediated processing is not defined. In this study, we show that RIPK3 is crucial for caspase 1- and caspase 8-mediated pro-IL-1ß and pro-IL-18 processing in bone marrow-derived dendritic cells (BMDCs) in response to LPS stimulation. Caspase 8-mediated pro-IL-1ß processing requires intact RIPK1, RIPK3, TRIF, and FADD. In response to LPS, a complex that contains RIPK1, RIPK3, FADD, and caspase 8 is formed. Surprisingly, RIPK3-specific kinase inhibitors strongly enhanced caspase 8 activation and pro-IL-1ß processing in LPS-stimulated BMDCs. However, studies in BMDCs expressing the kinase-inactive RIPK3-K51A mutant or RIPK1-K45A mutant showed that the kinase activity of neither RIPK1 nor RIPK3 is required for LPS-induced caspase 8 activation and IL-1ß secretion. Hence, RIPK3 is an unexpected positive regulator of caspase 8 activity that promotes IL-1ß maturation in BMDCs.


Subject(s)
Caspase 8/immunology , Dendritic Cells/immunology , Interleukin-1beta/immunology , Receptor-Interacting Protein Serine-Threonine Kinases/immunology , Animals , Blotting, Western , Bone Marrow Cells/immunology , Enzyme Activation/immunology , Enzyme-Linked Immunosorbent Assay , Gene Knock-In Techniques , Immunoprecipitation , Inflammation/immunology , Mice , Mice, Knockout , Transfection
17.
Cell Mol Life Sci ; 73(11-12): 2325-34, 2016 06.
Article in English | MEDLINE | ID: mdl-27048814

ABSTRACT

Recent advances have identified a signaling cascade involving receptor interacting protein kinase 1 (RIPK1), RIPK3 and the pseudokinase mixed lineage kinase domain-like (MLKL) that is crucial for induction of necroptosis, a non-apoptotic form of cell death. RIPK1-RIPK3-MLKL-mediated necroptosis has been attributed to cause many inflammatory diseases through the release of cellular damage-associated molecular patterns (DAMPs). In addition to necroptosis, emerging evidence suggests that these necroptosis signal adaptors can also facilitate inflammation independent of cell death. In particular, the RIP kinases can drive NF-κB and inflammasome activation independent of cell death. In this review, we will discuss recent discoveries that led to this realization and present arguments why cell death-independent signaling by the RIP kinases may have a more important role in inflammation than necroptosis.


Subject(s)
Inflammation/pathology , Necrosis/pathology , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Apoptosis/physiology , Inflammasomes/metabolism , Mice , Mice, Knockout , NF-kappa B/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics
18.
Proteomics ; 16(24): 3073-3080, 2016 12.
Article in English | MEDLINE | ID: mdl-26949200

ABSTRACT

Most cancers consist of heterogeneous populations of cells with substantial differences in tumorigenicity. Cells that possess self-renewal and tumor-initiating properties are often called cancer stem cells (CSCs). Since CSCs underlie tumor recurrence and metastasis and are resistant to current anti-cancer therapies, novel therapeutic strategies to efficiently target this subset of cells are needed. Aberrant glycosylation is one of the hallmarks of cancer. Many cancer-associated glycans have been reported to be involved in tumor progression and metastasis, and are used as tumor markers. Over the past several years, we have identified characteristic glycans on CSCs by utilizing recent advances in glycoproteomic technologies. In this review, we would like to summarize a series of our recent studies and discuss possible applications of glycomarkers for CSCs.


Subject(s)
Glycoproteins/analysis , Neoplasms/pathology , Neoplastic Stem Cells/pathology , Polysaccharides/analysis , Proteome/analysis , Proteomics/methods , Animals , Biomarkers, Tumor/analysis , Cell Separation/methods , Fucose/analysis , Humans , N-Acetylneuraminic Acid/analysis , Neoplasms/chemistry , Neoplastic Stem Cells/chemistry
19.
Nat Commun ; 15(1): 4514, 2024 May 27.
Article in English | MEDLINE | ID: mdl-38802491

ABSTRACT

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.


Subject(s)
Glycosaminoglycans , Golgi Apparatus , Golgi Apparatus/metabolism , Glycosylation , Humans , Glycosaminoglycans/metabolism , HeLa Cells , CRISPR-Cas Systems , Membrane Proteins/metabolism , Membrane Proteins/genetics , Golgi Matrix Proteins
20.
J Biol Chem ; 287(41): 33973-82, 2012 Oct 05.
Article in English | MEDLINE | ID: mdl-22872641

ABSTRACT

Fucα1-6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1-6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH(2)-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1-6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1-2-, α1-3-, and α1-4-fucosylated glycans. Furthermore, PhoSL bound to α1-6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1-6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1-6 fucosylation and evaluating Fucα1-6 oligosaccharides as cancer biomarkers.


Subject(s)
Fucose/chemistry , Fungal Proteins/chemistry , Lectins/chemistry , Oligosaccharides/chemistry , Pholiota/chemistry , Amino Acid Sequence , Antigens, Neoplasm/metabolism , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Fucose/genetics , Fucose/metabolism , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Humans , Lectins/genetics , Lectins/isolation & purification , Lectins/metabolism , Liver Neoplasms/metabolism , Molecular Sequence Data , Oligosaccharides/genetics , Oligosaccharides/metabolism , Pholiota/genetics , Pholiota/metabolism , Protein Binding , Protein Stability
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