Manganese regulation of COPII condensation controls circulating lipid homeostasis.

Precise control of circulating lipids is instrumental in health and disease. Bulk lipids, carried by specialized lipoproteins, are secreted into the circulation, initially via the coat protein complex II (COPII). How the universal COPII machinery accommodates the abundant yet unconventional lipoproteins remains unclear, let alone its therapeutic translation. Here we report that COPII uses manganese-tuning, self-constrained condensation to selectively drive lipoprotein delivery and set lipid homeostasis in vivo. Serendipitously, adenovirus hijacks the condensation-based transport mechanism, thus enabling the identification of cytosolic manganese as an unexpected control signal. Manganese directly binds the inner COPII coat and enhances its condensation, thereby shifting the assembly-versus-dynamics balance of the transport machinery. Manganese can be mobilized from mitochondria stores to signal COPII, and selectively controls lipoprotein secretion with a distinctive, bell-shaped function. Consequently, dietary titration of manganese enables tailored lipid management that counters pathological dyslipidaemia and atherosclerosis, implicating a condensation-targeting strategy with broad therapeutic potential for cardio-metabolic health.

Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.

Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.

Colocalization pattern of cocaine- and amphetamine-regulated transcript peptide and parvalbumin immunoreactivity in the hippocampus proper of the chinchilla.

This study set out to investigate, for the first time, the distribution and colocalization pattern of cocaine-and amphetamine-regulated transcript (CART) and one of the calcium binding- -proteins: parvalbumin (PV) in the chinchilla's hippocampus proper (HP). HP, consisting of Ammon's horn (CA) and the dentate gyrus (DG), is an important component of the limbic system, involved in learning and memory processes. CA showed a higher immunoreactivity of CART (-IR) compared to DG. CART-IR neurons were mainly observed in the molecular layer of DG and in the pyramidal layer of CA. CART-IR fibers were present in the granular layer; in the hilus numerous mossy fibers were detected, while in the molecular layer CART-IR fibers were not found. In all CA fields (CA1-CA3), CART-IR fibers were only present in the lacuno- sum-molecular layer. Immunofluorescence with double- labeling showed that only CART-IR cells stained positive for PV, whereas in CART-IR fibers there was no PV-positive reaction. Our research supplements missing knowledge about the distribution and colocalization pattern of CART with PV in the chinchilla's hippocampus, and also provides a better understanding of the similarities and differences among individuals of the same species and also with other mammals.

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy.

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

BioID screen of Salmonella type 3 secreted effectors reveals host factors involved in vacuole positioning and stability during infection.

Many bacterial pathogens express virulence proteins that are translocated into host cells (herein referred to as effectors), where they can interact with target proteins to manipulate host cell processes. These effector-host protein interactions are often dynamic and transient in nature, making them difficult to identify using traditional interaction-based methods. Here, we performed a systematic comparison between proximity-dependent biotin labelling (BioID) and immunoprecipitation coupled with mass spectrometry to investigate a series of Salmonella type 3 secreted effectors that manipulate host intracellular trafficking (SifA, PipB2, SseF, SseG and SopD2). Using BioID, we identified 632 candidate interactions with 381 unique human proteins, collectively enriched for roles in vesicular trafficking, cytoskeleton components and transport activities. From the subset of proteins exclusively identified by BioID, we report that SifA interacts with BLOC-2, a protein complex that regulates dynein motor activity. We demonstrate that the BLOC-2 complex is necessary for SifA-mediated positioning of Salmonella-containing vacuoles, and affects stability of the vacuoles during infection. Our study provides insight into the coordinated activities of Salmonella type 3 secreted effectors and demonstrates the utility of BioID as a powerful, complementary tool to characterize effector-host protein interactions.

Silica nanoparticles disrupt OPT-2/PEP-2-dependent trafficking of nutrient peptides in the intestinal epithelium.

Despite of the increasing application of silica nanoparticles and identification of oral exposure as a major entry portal, we lack understanding of nanosilica effects in the gut. Thus, we investigated biointeractions of nanosilica with single intestinal cells. The invertebrate nematode Caenorhabditis elegans was chosen as model organism with a tractable intestine and realistic target organism of nanomaterials in the environment. We found that nanosilica impairs the intestinal uptake of oligopeptides. Downstream to absorption by the apical OPT-2/PEP-2 transporter dipeptides were trapped in aberrant vesicles that grow over time and reach diameters of ≥6 µm. The peptide vesicles do not correspond to known organelles such as gut granules and form independently of related gene products GLO-1 or GLO-3. Formation of aberrant peptide vesicles also occurred independently of insulin/IGF-I receptor (DAF-2) signaling and daf-2 loss of function mutants showed specific vesicle patterns including distinct localization along the apical membrane of single intestinal cells. As malnutrition of exposed C. elegans manifested in reduced growth and a petite phenotype similar to OPT-2/PEP-2 transporter deficient mutants, we conclude that nanosilica-induced peptide vesicles represent a new compartment of di- and tripeptide trapping which disrupts hydrolysis of nutrient peptides and metabolism.

The BBSome in POMC and AgRP Neurons Is Necessary for Body Weight Regulation and Sorting of Metabolic Receptors.

The BBSome, a complex of eight Bardet-Biedl syndrome (BBS) proteins involved in cilia function, has emerged as an important regulator of energy balance, but the underlying cellular and molecular mechanisms are not fully understood. Here, we show that the control of energy homeostasis by the anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons require intact BBSome. Targeted disruption of the BBSome by Bbs1 gene deletion in POMC or AgRP neurons increases body weight and adiposity. We demonstrate that obesity in mice lacking the Bbs1 gene in POMC neurons is associated with hyperphagia. Mechanistically, we present evidence implicating the BBSome in the trafficking of G protein-coupled neuropeptide Y Y2 receptor (NPY2R) and serotonin 5-hydroxytryptamine (HT)2C receptor (5-HT2CR) to cilia and plasma membrane, respectively. Consistent with this, loss of the BBSome reduced cell surface expression of the 5-HT2CR, interfered with serotonin-evoked increase in intracellular calcium and membrane potential, and blunted the anorectic and weight-reducing responses evoked by the 5-HT2cR agonist, lorcaserin. Finally, we show that disruption of the BBSome causes the 5-HT2CR to be stalled in the late endosome. Our results demonstrate the significance of the hypothalamic BBSome for the control of energy balance through regulation of trafficking of important metabolic receptors.

Development of Simplified in Vitro P-Glycoprotein Substrate Assay and in Silico Prediction Models To Evaluate Transport Potential of P-Glycoprotein.

For efficient drug discovery and screening, it is necessary to simplify P-glycoprotein (P-gp) substrate assays and to provide in silico models that predict the transport potential of P-gp. In this study, we developed a simplified in vitro screening method to evaluate P-gp substrates by unidirectional membrane transport in P-gp-overexpressing cells. The unidirectional flux ratio positively correlated with parameters of the conventional bidirectional P-gp substrate assay ( R2 = 0.941) and in vivo Kp,brain ratio (mdr1a/1b KO/WT) in mice ( R2 = 0.800). Our in vitro P-gp substrate assay had high reproducibility and required approximately half the labor of the conventional method. We also constructed regression models to predict the value of P-gp-mediated flux and three-class classification models to predict P-gp substrate potential (low-, medium-, and high-potential) using 2397 data entries with the largest data set collected under the same experimental conditions. Most compounds in the test set fell within two- and three-fold errors in the random forest regression model (71.3 and 88.5%, respectively). Furthermore, the random forest three-class classification model showed a high balanced accuracy of 0.821 and precision of 0.761 for the low-potential classes in the test set. We concluded that the simplified in vitro P-gp substrate assay was suitable for compound screening in the early stages of drug discovery and that the in silico regression model and three-class classification model using only chemical structure information could identify the transport potential of compounds including P-gp-mediated flux ratios. Our proposed method is expected to be a practical tool to optimize effective central nervous system (CNS) drugs, to avoid CNS side effects, and to improve intestinal absorption.

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.

Arginine inhibits the malignant transformation induced by interferon-gamma through the NF-κB-GCN2/eIF2α signaling pathway in mammary epithelial cells in vitro and in vivo.

Breast cancer is the most common female malignant tumors in the world. It seriously affects women's physical and mental health and the leading cause of cancer death among women. Our previous study demonstrated that diet-derived IFN-γ promoted the malignant transformation of primary bovine mammary epithelial cells by accelerating arginine depletion. The current study aimed to explore whether arginine addition could inhibit the degree of malignant transformation and its molecular mechanism. The results indicate that arginine addition could alleviate the malignant transformation of mammary epithelial cells induced by IFN-γ, including reducing cell proliferation, cell migration and colony formation, through the NF-κB-GCN2/eIF2α pathway. The in vivo experiments also consistently confirmed that arginine supplementation could significantly inhibit tumor growth in tumor-bearing mice. Furthermore, the investigation of the clinical data also revealed that the plasma or tissue from human breast cancer patients owned lower arginine level and higher IFN-γ level than that from patients with benign breast disease, showing IFN-γ may be a potential control target. Our findings demonstrate that arginine supplement could antagonize the malignant transformation of mammary epithelial cells induced by IFN-γ (nutritionally induced) both in vitro and in vivo, and IFN-γ was higher in breast cancer women. This might provide a novel strategy for the prevention and treatment of breast cancer regarding to nutrition.

Unconventional secretion of annexins and galectins.

Eukaryotic cells have a highly evolved system of protein secretion, and dysfunction in this pathway is associated with many diseases including cancer, infection, metabolic disease and neurological disorders. Most proteins are secreted using the conventional endoplasmic reticulum (ER)/Golgi network and as such, this pathway is well-characterised. However, several cytosolic proteins have now been documented as secreted by unconventional transport pathways. This review focuses on two of these proteins families: annexins and galectins. The extracellular functions of these proteins are well documented, as are associations of their perturbed secretion with several diseases. However, the mechanisms and regulation of their secretion remain poorly characterised, and are discussed in this review. This review is part of a Special Issues of SCDB on 'unconventional protein secretion' edited by Walter Nickel and Catherine Rabouille.

Hypothalamic and liver transcriptome from two crucial life-history stages in a migratory songbird.

NEW FINDINGS: What is the central question of this study? What are the molecular underpinnings of the seasonal adaptation in a latitudinal migratory songbird? What is the main finding and its importance? We found changes in mRNA levels after a photoperiod-induced alteration of seasonal state in a captive long-distance latitudinal avian migrant. The hypothalamus and liver transcriptomes revealed genes involved in the regulatory and functional pathways between non-migratory and migratory states. Our results provide insights into mechanisms underlying homeostasis during seasonal changes that are conserved across most species, including humans. ABSTRACT: Very little is understood about genetic mechanisms underlying the onset of spring migration in latitudinal avian migrants. To gain insight into the genetic architecture of the hypothalamus and liver tissues of a long-distance migrant, we examined and compared the transcriptome profile of captive night-migratory black-headed buntings (Emberiza melanocephala) between photoperiod-induced winter non-migratory (WnM) and spring migratory (SM) life-history states under short and long days, respectively. High-throughput 454 pyrosequenced transcripts were mapped initially with reference to the genome of two phylogenetically close species, Taeniopygia guttata and Ficedula albicollis. The F. albicollis genome gave higher annotation results and was used for further analysis. A total of 216 (78 in hypothalamus; 138 in liver) genes were found to be expressed differentially between the WnM and SM life-history states. These genes were enriched for physiological pathways that might be involved in the regulation of seasonal migrations in birds. For example, genes for the ATP binding pathway in the hypothalamus were expressed at a significantly higher level in SM than in the WnM life-history state. Likewise, upregulated genes associated with the myelin sheath and focal adhesion were enriched in the hypothalamus, and those with cell-to-cell junction, intracellular protein transport, calcium ion transport and small GTPase-mediated signal transduction were enriched in the liver. Many of these genes are a part of physiological pathways potentially involved in the regulation of seasonal migration in birds. These results show molecular changes at the regulatory and metabolic levels associated with seasonal transitions in a long-distance migrant and provide the basis for future studies aimed at unravelling the genetic control of migration in birds.

NQO2 inhibition relieves reactive oxygen species effects on mouse oocyte meiotic maturation and embryo development.

NRH: quinone oxidoreductase 2 (NQO2) is a cytosolic and ubiquitously expressed flavoprotein that catalyzes the two-electron reduction of quinone to hydroquinones. Herein, we assessed the protein expression, subcellular localization, and possible functions of NQO2 in mouse oocyte meiotic maturation and embryo development. Western blot analysis detected high and stable protein expression of NQO2 in mouse oocytes during meiotic progression. Immunofluorescence illustrated NQO2 distribution on nuclear membrane, chromosomes, and meiotic spindles. Microtubule poisons treatment (nocodazole and taxol) showed that filamentous assembly of NQO2 and its co-localization with microtubules require microtubule integrity and normal dynamics. Increased levels of NQO2, reactive oxygen species (ROS), malondialdehyde (MDA), and autophagy protein Beclin1 expression were detected in oocytes cultured with ROS stimulator vitamin K3 (VK3), combined with decreased antioxidant glutathione (GSH). These oocytes were arrested at metaphase I with abnormal spindle structure and chromosome configuration. However, this impact was counteracted by melatonin or NQO2 inhibitor S29434, and the spindle configuration and first polar body extrusion were restored. Similarly, morpholino oligo-induced NQO2 knockdown suppressed ROS, MDA, and Beclin1, instead increased GSH in oocytes under VK3. Supplementary S29434 or melatonin limited changes in NQO2, ROS, MDA, Beclin1, and GSH during in vitro aging of ovulated oocytes, thereby maintaining spindle structure, as well as ordered chromosome separation and embryo development potential after parthenogenetic activation with SrCl2. Taken together, NQO2 is involved in ROS generation and subsequent cytotoxicity in oocytes, and its inhibition can restore oocyte maturation and embryo development, suggesting NQO2 as a pharmacological target for infertility cure.

Biosynthesis of proresolving lipid mediators by vascular cells and tissues.

Recent evidence suggests that specialized proresolving lipid mediators (SPMs) generated from docosahexaenoic acid (DHA) can modulate the vascular injury response. However, cellular sources for these autacoids within the vessel wall remain unclear. Here, we investigated whether isolated vascular cells and tissues can produce SPMs and assessed expression and subcellular localization of the key SPM biosynthetic enzyme 5-lipoxygenase (LOX) in vascular cells. Intact human arteries incubated with DHA ex vivo produced 17-hydroxy DHA (17-HDHA) and D-series resolvins, as assessed by liquid chromatography-tandem mass spectrometry. Addition of 17-HDHA to human arteries similarly increased resolvin production. Primary cultures of human saphenous vein endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) converted 17-HDHA to SPMs, including resolvin D1 (RvD1) and other D-series resolvins and protectins. This was accompanied by a rapid translocation of 5-LOX from nucleus to cytoplasm in both ECs and VSMCs, potentially facilitating SPM biosynthesis. Conditioned medium from cells exposed to 17-HDHA inhibited monocyte adhesion to TNF-α-stimulated EC monolayers. These downstream effects were partially reversed by antibodies against the RvD1 receptors ALX/FPR2 and GPR32. These results suggest that autocrine and/or paracrine signaling via locally generated SPMs in the vasculature may represent a novel homeostatic mechanism of relevance to vascular health and disease.-Chatterjee, A., Komshian, S., Sansbury, B. E., Wu, B., Mottola, G., Chen, M., Spite, M., Conte, M. S. Biosynthesis of proresolving lipid mediators by vascular cells and tissues.

Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes.

The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effects of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD90) and 50% (APD50) repolarization, reduced the maximum upstroke velocity (dV/dtmax) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including IKr, IKs, INa, and ICa. Among them, IKr and INa showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K(+) channels.

Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm.

An Arabidopsis pollen grain (male gametophyte) consists of three cells: the vegetative cell, which forms the pollen tube, and two sperm cells enclosed within the vegetative cell. It is still unclear if there is intercellular communication between the vegetative cell and the sperm cells. Here we show that ABA-hypersensitive germination3 (AHG3), encoding a protein phosphatase, is specifically transcribed in the vegetative cell but predominantly translated in sperm cells. We used a series of deletion constructs and promoter exchanges to document transport of AHG3 transcripts from the vegetative cell to sperm and showed that their transport requires sequences in both the 5' UTR and the coding region. Thus, in addition its known role in transporting sperm during pollen tube growth, the vegetative cell also contributes transcripts to the sperm cells.

Ligand-Mediated cis-Inhibition of Receptor Signaling in the Self-Incompatibility Response of the Brassicaceae.

The inhibition of self-pollination in self-incompatible Brassicaceae is based on allele-specific trans-activation of the highly polymorphic S-locus receptor kinase (SRK), which is displayed at the surface of stigma epidermal cells, by its even more polymorphic pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. In an attempt to achieve constitutive activation of SRK and thus facilitate analysis of self-incompatibility (SI) signaling, we coexpressed an Arabidopsis lyrata SCR variant with its cognate SRK receptor in the stigma epidermal cells of Arabidopsis (Arabidopsis thaliana) plants belonging to the C24 accession, in which expression of SRK and SCR had been shown to exhibit a robust SI response. Contrary to expectation, however, coexpression of SRK and SCR was found to inhibit SRK-mediated signaling and to disrupt the SI response. This phenomenon, called cis-inhibition, is well documented in metazoans but has not as yet been reported for plant receptor kinases. We demonstrate that cis-inhibition of SRK, like its trans-activation, is based on allele-specific interaction between receptor and ligand. We also show that stigma-expressed SCR causes entrapment of its SRK receptor in the endoplasmic reticulum, thus disrupting the proper targeting of SRK to the plasma membrane, where the receptor would be available for productive interaction with its pollen coat-derived SCR ligand. Although based on an artificial cis-inhibition system, the results suggest novel strategies of pollination control for the generation of hybrid cultivars and large-scale seed production from hybrid plants in Brassicaceae seed crops and, more generally, for inhibiting cell surface receptor function and manipulating signaling pathways in plants.

Trafficking of Na+/Ca2+ exchanger to the site of persistent inflammation in nociceptive afferents.

Persistent inflammation results in an increase in the amplitude and duration of depolarization-evoked Ca(2+) transients in putative nociceptive afferents. Previous data indicated that these changes were the result of neither increased neuronal excitability nor an increase in the amplitude of depolarization. Subsequent data also ruled out an increase in voltage-gated Ca(2+) currents and recruitment of Ca(2+)-induced Ca(2+) release. Parametric studies indicated that the inflammation-induced increase in the duration of the evoked Ca(2+) transient required a relatively large and long-lasting increase in the concentration of intracellular Ca(2+) implicating the Na(+)/Ca(2+) exchanger (NCX), a major Ca(2+) extrusion mechanism activated with high intracellular Ca(2+) loads. The contribution of NCX to the inflammation-induced increase in the evoked Ca(2+) transient in rat sensory neurons was tested using fura-2 AM imaging and electrophysiological recordings. Changes in NCX expression and protein were assessed with real-time PCR and Western blot analysis, respectively. An inflammation-induced decrease in NCX activity was observed in a subpopulation of putative nociceptive neurons innervating the site of inflammation. The time course of the decrease in NCX activity paralleled that of the inflammation-induced changes in nociceptive behavior. The change in NCX3 in the cell body was associated with a decrease in NCX3 protein in the ganglia, an increase in the peripheral nerve (sciatic) yet no change in the central root. This single response to inflammation is associated with changes in at least three different segments of the primary afferent, all of which are likely to contribute to the dynamic response to persistent inflammation.

The Arg98Trp mutation in human VKORC1 causing VKCFD2 disrupts a di-arginine-based ER retention motif.

Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is an enzyme localized to the endoplasmic reticulum (ER) membrane. VKORC1 catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K and to vitamin K hydroquinone, the latter required by the enzyme γ-carboxylase for γ-carboxylation of all vitamin K-dependent (VKD) proteins. Until now, only 1 human VKORC1 mutation, p.Arg98Trp, is known to cause combined deficiency of VKD clotting factors type 2 (VKCFD2), a disease phenotype reported in 3 unrelated families. VKCFD2 patients suffer from spontaneous bleeding episodes because of decreased levels of γ-carboxylated VKD clotting factors. Daily supraphysiological vitamin K supplementation restores clotting for VKCFD2 patients and results in high serum levels of vitamin K 2,3-epoxide, suggesting that supplemented vitamin K is reduced in vivo. Although the p.Arg98Trp mutation results in reduced vitamin K 2,3-epoxide reductase activity, the molecular mechanism underlying this pathophysiology is unknown. Using a combination of in silico analysis and confocal microscopy, we demonstrate for the first time that VKORC1:p.Arg98Trp disrupts a di-arginine ER retention motif resulting in 20% ER colocalization only. As a consequence, VKORC1 exits the ER membrane by cellular quality control systems and results in the observed VKCFD2 phenotype.

Glycine is a nutritionally essential amino acid for maximal growth of milk-fed young pigs.

Analysis of amino acids in milk protein reveals a relatively low content of glycine. This study was conducted with young pigs to test the hypothesis that milk-fed neonates require dietary glycine supplementation for maximal growth. Fourteen-day-old piglets were allotted randomly into one of four treatments (15 piglets/treatment), representing supplementation with 0, 0.5, 1 or 2% glycine (dry matter basis) to a liquid milk replacer. Food was provided to piglets every 8 h (3 times/day) for 2 weeks. Milk intake (32.0-32.5 g dry matter/kg body weight per day) did not differ between control and glycine-supplemented piglets. Compared with control piglets, dietary supplementation with 0.5, 1 and 2% glycine increased (P < 0.05) plasma concentrations of glycine and serine, daily weight gain, and body weight without affecting body composition, while reducing plasma concentrations of ammonia, urea, and glutamine, in a dose-dependent manner. Dietary supplementation with 0.5, 1 and 2% glycine enhanced (P < 0.05) small-intestinal villus height, glycine transport (measured using Ussing chambers), mRNA levels for GLYT1, and anti-oxidative capacity (indicated by increased concentrations of reduced glutathione and a decreased ratio of oxidized glutathione to reduced glutathione). These novel results indicate, for the first time, that glycine is a nutritionally essential amino acid for maximal protein accretion in milk-fed piglets. The findings not only enhance understanding of protein nutrition, but also have important implications for designing improved formulas to feed human infants, particularly low birth weight and preterm infants.