Membrane interaction and disulphide-bridge formation in the unconventional secretion of Tau.

Misfolded, pathological tau protein propagates from cell to cell causing neuronal degeneration in Alzheimer's disease and other tauopathies. The molecular mechanisms of this process have remained elusive. Unconventional secretion of tau takes place via several different routes, including direct penetration through the plasma membrane. Here, we show that tau secretion requires membrane interaction via disulphide bridge formation. Mutating residues that reduce tau interaction with membranes or formation of disulphide bridges decrease both tau secretion from cells, and penetration through artificial lipid membranes. Our results demonstrate that tau is indeed able to penetrate protein-free membranes in a process independent of active cellular processes and that both membrane interaction and disulphide bridge formation are needed for this process. QUARK-based de novo modelling of the second and third microtubule-binding repeat domains (MTBDs), in which the two cysteine residues of 4R isoforms of tau are located, supports the concept that this region of tau could form transient amphipathic helices for membrane interaction.

Therapeutic potential of targeting intestinal bitter taste receptors in diabetes associated with dyslipidemia.

Intestinal release of incretin hormones after food intake promotes glucose-dependent insulin secretion and regulates glucose homeostasis. The impaired incretin effects observed in the pathophysiologic abnormality of type 2 diabetes have triggered the pharmacological development of incretin-based therapy through the activation of glucagon-like peptide-1 (GLP-1) receptor, including GLP-1 receptor agonists (GLP-1 RAs) and dipeptidyl peptidase 4 (DPP4) inhibitors. In the light of the mechanisms involved in the stimulation of GLP-1 secretion, it is a fundamental question to explore whether glucose and lipid homeostasis can be manipulated by the digestive system in response to nutrient ingestion and taste perception along the gastrointestinal tract. While glucose is a potent stimulant of GLP-1 secretion, emerging evidence highlights the importance of bitter tastants in the enteroendocrine secretion of gut hormones through activation of bitter taste receptors. This review summarizes bitter chemosensation in the intestines for GLP-1 secretion and metabolic regulation based on recent advances in biological research of bitter taste receptors and preclinical and clinical investigation of bitter medicinal plants, including bitter melon, hops strobile, and berberine-containing herbs (e.g. coptis rhizome and barberry root). Multiple mechanisms of action of relevant bitter phytochemicals are discussed with the consideration of pharmacokinetic studies. Current evidence suggests that specific agonists targeting bitter taste receptors, such as human TAS2R1 and TAS2R38, may provide both metabolic benefits and anti-inflammatory effects with the modulation of the enteroendocrine hormone secretion and bile acid turnover in metabolic syndrome individuals or diabetic patients with dyslipidemia-related comorbidities.

Carvacrol, Thymol, and Garlic Essential Oil Promote Skin Innate Immunity in Gilthead Seabream (Sparus aurata) Through the Multifactorial Modulation of the Secretory Pathway and Enhancement of Mucus Protective Capacity.

One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.

Antihormone treatment differentially regulates PSA secretion, PSMA expression and 68Ga-PSMA uptake in LNCaP cells.

BACKGROUND: In recent years, a variety of innovative therapeutics for castration-resistant prostate cancer have been developed, including novel anti-androgenic drugs, such as abiraterone or VPC-13566. Therapeutic monitoring of these pharmaceuticals is performed either by measuring PSA levels in serum or by imaging. PET using PSMA ligands labeled with Fluor-18 or Gallium-68 is the most sensitive and specific imaging modality for detection of metastases in advanced prostate cancer. To date, it remains unclear how PSMA expression is modulated by anti-hormonal treatment and how it correlates with PSA secretion. METHODS: We analyzed modulation of PSMA-mRNA and protein expression, 68Ga-PSMA uptake and regulation of PSA secretion by abiraterone or VPC-13566 in LNCaP cells in vitro. RESULTS: We found that abiraterone and VPC-13566 upregulate PSMA protein and mRNA expression but block PSA secretion in LNCaP cells. Both anti-androgens also enhanced 68Ga-PSMA uptake normalized by the number of cells, whereas abiraterone and VPC-13566 reduced 68Ga-PSMA uptake in total LNCaP monolayers treated due to cell death. CONCLUSION: Our data indicate that PSA secretion and PSMA expression are differentially regulated upon anti-androgen treatment. This finding might be important for the interpretation of 68Ga-PSMA PET images in monitoring therapies with abiraterone and VPC-13566 in prostate cancer patients, but needs to be validated in vivo.

Carbonic anhydrase 8 (CAR8) negatively regulates GLP-1 secretion from enteroendocrine cells in response to long-chain fatty acids.

Glucagon-like peptide-1 (GLP-1) is an incretin secreted from enteroendocrine preproglucagon (PPG)-expressing cells (traditionally known as L cells) in response to luminal nutrients that potentiates insulin secretion. Augmentation of endogenous GLP-1 secretion might well represent a novel therapeutic target for diabetes treatment in addition to the incretin-associated drugs currently in use. In this study, we found that PPG cells substantially express carbonic anhydrase 8 (CAR8), which has been reported to inhibit inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor and subsequent Ca2+ efflux from the endoplasmic reticulum in neuronal cells. In vitro experiments using STC-1 cells demonstrated that Car8 knockdown increases long-chain fatty acid (LCFA)-stimulated GLP-1 secretion. This effect was reduced in the presence of phospholipase C (PLC) inhibitor; in addition, Car8 knockdown increased the intracellular Ca2+ elevation caused by α-linolenic acid, indicating that CAR8 exerts its effect on GLP-1 secretion via the PLC/IP3/Ca2+ pathway. Car8wdl null mutant mice showed significant increase in GLP-1 response to oral corn oil administration compared with that in wild-type littermates, with no significant change in intestinal GLP-1 content. These results demonstrate that CAR8 negatively regulates GLP-1 secretion from PPG cells in response to LCFAs, suggesting the possibility of augmentation of postprandial GLP-1 secretion by CAR8 inhibition.NEW & NOTEWORTHY This study focused on the physiological significance of carbonic anhydrase 8 (CAR8) in GLP-1 secretion from enteroendocrine preproglucagon (PPG)-expressing cells. We found an inhibitory role of CAR8 in LCFA-induced GLP-1 secretion in vitro and in vivo, suggesting a novel therapeutic approach to diabetes and obesity through augmentation of postprandial GLP-1 secretion by CAR8 inhibition.

Regulation of exosome production and cargo sorting.

Cellular communication can be mediated by the exchange of biological information, mainly in the form of proteins and RNAs. This can occur when extracellular vesicles, such as exosomes, secreted by a donor cell are internalized by an acceptor cell. Exosomes bear specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Knowledge about loadings and processes and mechanisms of cargo sorting of exosomes is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. In this review, we will discuss the molecular mechanisms associated with exosome secretion and their specific cargo sorting, with special attention to the sorting of RNAs and proteins, and thus the outcome and the emerging therapeutic opportunities of the communication between the exosome-producer and recipient cells.

Phytochemical analysis of bioactive fraction from the extract of Crocus sativus petal by LC-MS and its effect on secretion of nerve growth factors in rat c6 glioma cells.

This article was aimed to analyze the bioactive ethyl acetate fraction from the extract of Crocus sativus petal by LC-MS and to study its antidepressant effect. Antidepressant test was performed employing rat C6 glioma cells model. The major compounds from ethyl acetate fraction were cinnamic acid derivatives, which primarily included methylcinnamaldehydes, carboxycinnamylic acid carboxyphenethyl ester, carboxyvinylcinnamyl alcohol propinate, 3-acetoxy-4-propoxycinnamylic acid, carboxycinnamylic acid glycol ester, vinnylcinnamylic acid hexylene glycol ester and cinnamylic acid. In C6 glioma cells test, the ethyl acetate extract of C. sativus petal significantly upregulated nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) expression. C. sativus petal has antidepressant effect, and cinnamic acid derivatives shoud be the main antidepressant constituents. The mechanism may be related to the inhibition of NGF and BDNF reuptake.

The Parotoid Gland Secretion from Peruvian Toad Rhinella horribilis (Wiegmann, 1833): Chemical Composition and Effect on the Proliferation and Migration of Lung Cancer Cells.

Since Rhinella sp. toads produce bioactive substances, some species have been used in traditional medicine and magical practices by ancient cultures in Peru. During several decades, the Rhinella horribilis toad was confused with the invasive toad Rhinella marina, a species documented with extensive toxinological studies. In contrast, the chemical composition and biological effects of the parotoid gland secretions (PGS) remain still unknown for R. horribilis. In this work, we determine for the first time 55 compounds from the PGS of R. horribilis, which were identified using HPLC-MS/MS. The crude extract inhibited the proliferation of A549 cancer cells with IC50 values of 0.031 ± 0.007 and 0.015 ± 0.001 µg/mL at 24 and 48 h of exposure, respectively. Moreover, it inhibited the clonogenic capacity, increased ROS levels, and prevented the etoposide-induced apoptosis, suggesting that the effect of R. horribilis poison secretion was by cell cycle blocking before of G2/M-phase checkpoint. Fraction B was the most active and strongly inhibited cancer cell migration. Our results indicate that the PGS of R. horribilis are composed of alkaloids, bufadienolides, and argininyl diacids derivatives, inhibiting the proliferation and migration of A549 cells.

Qingfei oral liquid alleviates airway hyperresponsiveness and mucus hypersecretion via TRPV1 signaling in RSV-infected asthmatic mice.

Pediatric asthma is exacerbated by Respiratory Syncytial Virus (RSV) infection, and Transient Receptor Potential Vanilloid 1 (TRPV1) promotes production of inflammatory cytokines and mucus hypersecretion in the pathology of this disease. Our previous research revealed that Qingfei oral liquid (QF) inhibited airway inflammation and mucus hypersecretion in RSV-infected asthmatic mice models and that this may be associated with the TRPV1-regulation of NF-κB and Mucin 5AC (MUC5AC) expression, but the exact mechanism is unknown. In the present study, LC-MS was used for analyzing the chemicals in QF, ovalbumin (OVA)-induced asthmatic mice inhaled RSV three consecutive times to create an RSV-infected asthmatic model. We found treatment from QF alleviated airway hyperresponsiveness (AHR) and reduced congestion, edema, and infiltration of inflammatory cells into pulmonary tissues. Additionally, QF was found to decrease expression of NF-κB and its downstream inflammatory cytokines IL-1ß, IL-4, IL-5, and IL-13, as well as a decrease in MUC5AC and pro-inflammatory cytokines in PKC via a reduction in Protein Kinase C-dependent signaling. These findings suggest that QF can alleviate AHR and mucus hypersecretion caused by RSV infection in asthmatic mice, and its mechanism may be associated with the regulation of the TRPV1 signaling pathway.

Costus pictus D. Don leaf extract stimulates GLP-1 secretion from GLUTag L-cells and has cytoprotective effects in BRIN-BD11 ß-cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Costus pictus D. Don, commonly known as insulin plant, is a traditional Indian antidiabetic herbal medicine with glucose-lowering and insulin secretory effects having been reported in animal models and humans with Type 2 diabetes. However, its effects on GLP-1 secretion from intestinal endocrine L-cells and potential metabolic and protective effects in insulin secreting pancreatic ß-cells are not yet fully understood. AIM OF THE STUDY: This study is aimed to elucidate the effects of Costus pictus D. Don leaf extract (CPE) on L-cell function and GLP-1 secretion using the established murine GLUTag L-cell model and to investigate its potential cytoprotective effects against detrimental effects of palmitate and cytokines in pancreatic ß-cells using BRIN-BD11 cells. METHODS: Costus pictus D. Don dried leaf powder was extracted by soxhlet method. Cell viability was determined by MTT assay. Changes in gene and protein expression were quantified by qPCR and western blotting, respectively. GLP-1 and insulin secretion were measured by ELISA. RESULTS: CPE significantly enhanced the percentage of viable BRIN-BD11 and GLUTag cells and protected BRIN-BD11 cells against palmitate- and proinflammatory cytokine-induced toxicity. CPE enhanced acute GLP-1 secretion 6.4-16.3-fold from GLUTag cells at both low (1.1 mM) and high (16.7 mM) glucose (P < 0.01) concentrations. Antioxidant (Nrf2, Cat & Gpx1) and pro-proliferative (Erk1 and Jnk1) gene expression were upregulated by 24 h culture with CPE, while proinflammatory transcription factor NF-κB was downregulated. CONCLUSION: Diminished postprandial GLP-1 secretion and loss of insulin secreting ß-cells are known contributors of T2DM. Our data suggests that CPE acutely stimulates GLP-1 secretion from L-cells. Long term exposure of the BRIN-BD11 cells to CPE enhances cell number and may protect against palmitate and proinflammatory cytokines by activating multiple pathways. Thus, the current study suggests that the possible antidiabetic properties of CPE may be linked to enhanced GLP-1 secretion and ß-cell protection which could be beneficial in the management of T2DM.

Leucine improves α-amylase secretion through the general secretory signaling pathway in pancreatic acinar cells of dairy calves.

The present study aimed to elucidate the mechanisms by which leucine impacts the secretion of pancreatic enzymes, especially amylase, by studying the proteomics profiles of pancreatic acinar (PA) cells from dairy cows. PA cells, the experimental model, were treated with four concentrations of leucine (0, 0.23, 0.45, and 0.90 mM). The abundance of different proteins in the four leucine treatment groups was detected. Label-free proteomic analysis enabled the identification of 1,906 proteins in all four treatment groups, and 1,350 of these proteins showed common expression across the groups. The primary effects of leucine supplementation were increased (P < 0.05) citrate synthase and ATPase activity, which enlarged the cytosolic ATP pool, and the upregulation of secretory protein 61 (Sec61) expression, which promoted protein secretion. In summary, these results suggest that leucine increases citrate synthase in the TCA cycle and ATPase activity and promotes the Sec signaling pathway to increase the exocrine function of PA cells.

A GDSL-type esterase/lipase gene, GELP77, is necessary for pollen dissociation and fertility in Arabidopsis.

Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.

The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2.

FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces.

Dynamics of ACTH and Cortisol Secretion and Implications for Disease.

The past decade has seen several critical advances in our understanding of hypothalamic-pituitary-adrenal (HPA) axis regulation. Homeostatic physiological circuits need to integrate multiple internal and external stimuli and provide a dynamic output appropriate for the response parameters of their target tissues. The HPA axis is an example of such a homeostatic system. Recent studies have shown that circadian rhythmicity of the major output of this system-the adrenal glucocorticoid hormones corticosterone in rodent and predominately cortisol in man-comprises varying amplitude pulses that exist due to a subhypothalamic pulse generator. Oscillating endogenous glucocorticoid signals interact with regulatory systems within individual parts of the axis including the adrenal gland itself, where a regulatory network can further modify the pulsatile release of hormone. The HPA axis output is in the form of a dynamic oscillating glucocorticoid signal that needs to be decoded at the cellular level. If the pulsatile signal is abolished by the administration of a long-acting synthetic glucocorticoid, the resulting disruption in physiological regulation has the potential to negatively impact many glucocorticoid-dependent bodily systems. Even subtle alterations to the dynamics of the system, during chronic stress or certain disease states, can potentially result in changes in functional output of multiple cells and tissues throughout the body, altering metabolic processes, behavior, affective state, and cognitive function in susceptible individuals. The recent development of a novel chronotherapy, which can deliver both circadian and ultradian patterns, provides great promise for patients on glucocorticoid treatment.

Central Sfrp5 regulates hepatic glucose flux and VLDL-triglyceride secretion.

OBJECTIVE: Secreted frizzled-related protein 5 (Sfrp5) has been shown to be associated with energy homeostasis and insulin resistance in mouse models of obesity and diabetes. However, its central role in glucose and lipid metabolism is unknown. METHODS: HFD-fed rats received ICV infusions of vehicle or Sfrp5 during a pancreatic euglycemic clamp procedure. To delineate the pathway(s) by which ICV Sfrp5 modulates HGP and VLDL-TG secretion, we inhibited the hypothalamic KATP channel using glibenclamide, the DVC NMDA receptor with MK801, and selectively transected the hepatic branch of the vagal nerve while centrally infusing Sfrp5. RESULTS: ICV Sfrp5 in HFD-fed rats significantly increased the glucose infusion required to maintain euglycemia due to HGP inhibition during the clamp procedure; moreover, hepatic PEPCK and G6Pase expression was decreased, and InsR and Akt phosphorylation was increased in the liver. ICV Sfrp5 also decreased circulating triglyceride levels via inhibiting hepatic VLDL-TG secretion. These changes were accompanied by the inhibition of enzymes related to lipogenesis in the liver. ICV Sfrp5 significantly increased insulin-stimulated phosphorylation of InsR and Akt in the hypothalamus of HFD-fed rats, and insulin-stimulated immunodetectable PIP3 levels were higher in Sfrp5 group than in control group both in vitro and vivo. The glucose- and lipid-lowering effects of ICV Sfrp5 were eliminated by NMDA receptor or DVC KATP channel inhibition or HVAG. CONCLUSIONS: The present study demonstrates that central Sfrp5 signaling activates a previously unappreciated InsR-Akt-PI3k-KATP channel pathway in the hypothalamus and brain-hepatic vagus neurocircuitry to decrease HGP and VLDL-TG secretion.

Autophagy-dependent secretion: mechanism, factors secreted, and disease implications.

Although best understood as a degradative pathway, recent evidence demonstrates pronounced involvement of the macroautophagic/autophagic molecular machinery in cellular secretion. With either overexpression or inhibition of autophagy mediators, dramatic alterations in the cellular secretory profile occur. This affects secretion of a plethora of factors ranging from cytokines, to granule contents, and even viral particles. Encompassing a wide range of secreted factors, autophagy-dependent secretion is implicated in diseases ranging from cancer to neurodegeneration. With a growing body of evidence shedding light onto the molecular mediators, this review delineates the molecular machinery involved in selective targeting of the autophagosome for either degradation or secretion. In addition, we summarize the current understanding of factors and cargo secreted through this unconventional route, and describe the implications of this pathway in both health and disease. Abbreviations: BECN1, beclin 1; CAF, cancer associated fibroblast; CUPS, compartment for unconventional protein secretion; CXCL, C-X-C motif chemokine ligand; ER, endoplasmic reticulum; FGF2, fibroblast growth factor 2; HMGB1, high mobility group box 1; IDE, insulin degrading enzyme; IL, Interleukin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAPS, misfolding associated protein secretion; MEF, mouse embryonic fibroblast; MTORC1, MTOR complex I; PtdIns, phosphatidyl inositol; SEC22B, SEC22 homolog B, vesicle trafficking protein (gene/pseudogene); SFV, Semliki forest virus; SNCA, synuclein alpha; SQSTM1, sequestosome 1; STX, Syntaxin; TASCC, TOR-associated spatial coupling compartment; TGFB, transforming growth factor beta; TRIM16, tripartite motif containing 16; UPS, unconventional protein secretion; VWF, von Willebrand factor.

Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways.

During physiologic stresses, like micronutrient starvation, infection, and cancer, the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is trafficked to the plasma membrane (PM) and extracellular milieu (ECM). Our work demonstrates that GAPDH mobilized to the PM, and the ECM does not utilize the classic endoplasmic reticulum-Golgi route of secretion; instead, it is first selectively translocated into early and late endosomes from the cytosol via microautophagy. GAPDH recruited to this common entry point is subsequently delivered into multivesicular bodies, leading to its membrane trafficking through secretion via exosomes and secretory lysosomes. We present evidence that both pathways of GAPDH membrane trafficking are up-regulated upon iron starvation, potentially by mobilization of intracellular calcium. These pathways also play a role in clearance of misfolded intracellular polypeptide aggregates. Our findings suggest that cells build in redundancy for vital cellular pathways to maintain micronutrient homeostasis and prevent buildup of toxic intracellular misfolded protein refuse.-Chauhan, A. S., Kumar, M., Chaudhary, S., Dhiman, A., Patidar, A., Jakhar, P., Jaswal, P., Sharma, K., Sheokand, N., Malhotra, H., Raje, C. I., Raje. M. Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways.

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion.

When studying how HIV-1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor-α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase-regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)-dependent but was not associated with ER stress. These data indicate that Hck-activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom-dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation.

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

A bio-guided approach for the development of a chestnut-based proanthocyanidin-enriched nutraceutical with potential anti-gastritis properties.

Gastritis is a widely spread inflammatory disease, mostly caused by Helicobacter pylori infection. Release of IL-8 by the stomach epithelium is a hallmark of gastritis and contributes to the amplification of the inflammatory state. Pharmacological modulation of IL-8 release is a strategy to relieve gastric inflammation and prevent more severe clinical outcomes. In search of nutraceuticals with potential anti-gastritis properties we used a bio-guided approach based on IL-8 secretion by gastric cells to characterize extracts from the fruits of different chestnut varieties. We found that the ability to inhibit IL-8 secretion correlated with the amount of proanthocyanidins and was associated to the not edible parts of chestnut in all the tested varieties. We also found that the anti-inflammatory activity is preserved upon mild thermal treatment and after in vitro simulated gastric digestion. By combining a robust bio-guided approach with a comprehensive analysis of the tannin fraction of chestnut extracts, we provide evidence for the potential use of chestnut-based nutraceuticals in human gastritis. The bioactive components of chestnut fruits inhibit IL-8 secretion by impairing NF-κB signaling and by other mechanisms, thus opening new applications of proanthocyanidins for inflammation-based diseases.