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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.

The SMART (MLKL-activation reporter) system FRET biosensor allows for necroptosis assessment in primary cells and in a mouse model of cisplatininduced AKI.

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.

Nakabayashi et al find that the E3 ligase MIB2 ubiquitylates the apoptosis-inhibitor cFLIP and that deletion of MIB2 enhances both RIPK1 kinase-dependent and -independent apoptosis through an increase in RIPK1 kinase activity and impairment of ubiquitylation of cFLIPL, respectively.

Unlike canonical macroautophagy, alternative autophagy does not require the factors Atg5 and Atg7, but does require Ulk1. Here the authors show that phosphorylation of Ulk1 at Ser746 by RIPK3 is required for alternative autophagy initiation at the Golgi in response to genotoxic stress.

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.

Summary: A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner. : Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology Subject Areas: Immunology, Immune Response, Cell Biology, Functional Aspects of Cell Biology

Receptor interacting protein kinase 3 (RIPK3) induces necroptosis, a type of regulated necrosis, through its kinase domain and receptor interacting protein (RIP) homotypic interaction motif (RHIM). In addition, RIPK3 has been shown to regulate NLRP3 inflammasome and nuclear factor κB (NF-κB) activation. However, the relative contribution of these signaling pathways to RIPK3-dependent inflammation in distinct immune effectors is unknown. To investigate these questions, we generated RIPK3-GFP reporter mice. We found that colonic CD11c+CD11b+CD14+ mononuclear phagocytes (MNPs) expressed the highest level of RIPK3 in the lamina propria. Consequently, deletion of the RIPK3 RHIM in CD11c+ cells alone was sufficient to impair dextran sodium sulfate (DSS)-induced interleukin (IL)-23 and IL-1β expression, leading to severe intestinal inflammation. In contrast, mice expressing kinase inactive RIPK3 were not hypersensitive to DSS. Thus, a key physiological function of RIPK3 is to promote reparative cytokine expression through intestinal CD11c+ MNPs in a kinase- and necroptosis-independent manner.

BIG1, an activator protein of the small GTPase, Arf, and encoded by the Arfgef1 gene, is one of candidate genes for epileptic encephalopathy. To know the involvement of BIG1 in epileptic encephalopathy, we analyzed BIG1-deficient mice and found that BIG1 regulates neurite outgrowth and brain development in vitro and in vivo. The loss of BIG1 decreased the size of the neocortex and hippocampus. In BIG1-deficient mice, the neuronal progenitor cells (NPCs) and the interneurons were unaffected. However, Tbr1+ and Ctip2+ deep layer (DL) neurons showed spatial-temporal dependent apoptosis. This apoptosis gradually progressed from the piriform cortex (PIR), peaked in the neocortex, and then progressed into the hippocampus from embryonic day 13.5 (E13.5) to E17.5. The upper layer (UL) and DL order in the neocortex was maintained in BIG1-deficient mice, but the excitatory neurons tended to accumulate before their destination layers. Further pulse-chase migration assay showed that the migration defect was non-cell autonomous and secondary to the progression of apoptosis into the BIG1-deficient neocortex after E15.5. In BIG1-deficient mice, we observed an ectopic projection of corticothalamic axons from the primary somatosensory cortex (S1) into the dorsal lateral geniculate nucleus (dLGN). The thalamocortical axons were unable to cross the diencephalon-telencephalon boundary (DTB). In vitro, BIG1-deficient neurons showed a delay in neuronal polarization. BIG1-deficient neurons were also hypersensitive to low dose glutamate (5 μM), and died via apoptosis. This study showed the role of BIG1 in the survival of DL neurons in developing embryonic brain and in the generation of neuronal polarity.

Oligosaccharides, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. Recent advances in glycomics have identified several types of glyco-biomarkers containing fucosylation that are linked to certain types of cancer. Fucosylated alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular carcinoma because it is more specific than alpha-fetoprotein. High levels of fucosylated haptoglobin have also been found in sera of patients with various carcinomas. We have recently established a simple lectin-antibody ELISA to measure fucosylated haptoglobin and to investigate its clinical use. Cellular fucosylation is dependent upon fucosyltransferase activity and the level of its donor substrate, guanosine diphosphate (GDP)-fucose. GDP-mannose-4,6-dehydratase (GMDS) is a key enzyme involved in the synthesis of GDP-fucose. Mutations of GMDS found in colon cancer cells induced a malignant phenotype, leading to rapid growth in athymic mice resistant to natural killer cells. This review describes the role of fucosylated haptoglobin as a cancer biomarker, and discusses the possible biological role of fucosylation in cancer development.

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-1β 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-1β and pro–IL-18 processing in bone marrow–derived dendritic cells (BMDCs) in response to LPS stimulation. Caspase 8–mediated pro–IL-1β 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-1β 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-1β secretion. Hence, RIPK3 is an unexpected positive regulator of caspase 8 activity that promotes IL-1β maturation in BMDCs. [ABSTRACT FROM AUTHOR]

Changes in the levels of fucosylation regulate the biological phenotype of cancer cells and a specific fucosylation, such as fucosylated α-fetoprotein (AFP-L3) has been clinically used as a tumor marker for hepatocellular carcinoma (HCC). However, detailed molecular mechanisms that explain the increased fucosylation in HCC remain unknown despite 10 years of study by these researchers. Fucosylation is regulated by complicated mechanisms that involve several factors: fucosyltransferases, GDP-fucose transporter (GDP-Fuc Tr), and synthetic enzymes of GDP-fucose, such as GDP-mannose 4, 6-dehydratase (GMD), GDP-4-keto-6-deoxy-mannose-3, 5-epimerase-4-reductase (FX), and GDP-fucose pyrophosphorylase. In this study, the expression of fucosylation-related genes in HCC tissues was studied and it was found that GDP-Fuc Tr is a key factor for increases in fucosylation. A real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed significant increases in GDP-Fuc Tr and FX expression in HCC, and levels of the GMD protein were upregulated by posttranslational modification in HCC tissues. In vitro cell experiments showed that the level of GDP-Fuc Tr was the most significantly correlated with the level of cellular fucosylation and the overexpression of GDP-Fuc Tr dramatically increased fucosylation in Hep3B cells. The importance of GDP-Fuc Tr in the increase of fucosylation was also confirmed with immunohistochemical analyses. These findings suggest that the upregulation of GDP-Fuc Tr plays a pivotal role in increased fucosylation in HCC and represents an attractive target for new treatments and diagnosis for HCC. [ABSTRACT FROM AUTHOR]

Fucose (6-deoxy-L-galactose) is a monosaccharide that is found on glycoproteins and glycolipids in verte-brates, invertebrates, plants, and bacteria. Fucosylation, which comprises the transfer of a fucose residue to oligosaccharides and proteins, is regulated by many kinds of molecules, including fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporter(s). Dramatic changes in the expression of fucosylated oligosaccharides have been observed in cancer and inflammation. Thus, monoclonal antibodies and lectins recognizing cancer-associated fucosylated oligosaccharides have been clinically used as tumor markers for the last few decades. Recent advanced glycomic approaches allow us to identify novel fucosylation-related tumor markers. Moreover, a growing body of evidence supports the functional significance of fucosylation at various pathophysiological steps of carcinogenesis and tumor progression. This review highlights the biological and medical significance of fucosylation in gastrointestinal cancer.