Membrane proteins are synthesized and folded in the endoplasmic reticulum (ER), and continue their path to their site of residence along the secretory pathway. The COPII system has been identified as a key player for selecting and directing the fate of membrane and secretory cargo proteins. Selection of cargo proteins within the COPII vesicles is achieved by cargo receptors. The cornichon cargo receptor belongs to a conserved protein family found in eukaryotes that has been demonstrated to participate in the selection of integral membrane proteins as cargo for their correct targeting. Here it is demonstrated at the cellular level that rice cornichon OsCNIH1 interacts with OsHKT1;3 and, in yeast cells, enables the expression of the sodium transporter to the Golgi apparatus. Physical and functional HKT-cornichon interactions are confirmed by the mating-based split ubiquitin system, bimolecular fluorescence complementation, and Xenopus oocyte and yeast expression systems. The interaction between the two proteins occurs in the ER of plant cells and their co-expression in oocytes leads to the sequestration of the transporter in the ER. In the yeast cornichon mutant erv14, OsHKT1;3 is mistargeted, preventing the toxic effects of sodium transport in the cell observed in wild-type cells or in the erv14 mutant that co-expressed OsHKT1;3 with either OsCNIH1 or Erv14p. Identification and characterization of rice cornichon as a possible cargo receptor opens up the opportunity to improve our knowledge on membrane protein targeting in plant cells.

• MeSH
• Biological Transport MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Golgi Apparatus metabolism   MeSH
• Protein Interaction Mapping MeSH
• Membrane Transport Proteins genetics   metabolism   physiology   MeSH
• Molecular Sequence Data MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Plant Proteins genetics   metabolism   physiology   MeSH
• Oryza genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, Protein MeSH
• Sequence Alignment MeSH
• Sodium metabolism   MeSH
• Xenopus MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The plant selective autophagy cargo receptor neighbour of breast cancer 1 gene (NBR1) has been scarcely studied in the context of abiotic stress. We wanted to expand this knowledge by using Arabidopsis thaliana lines with constitutive ectopic overexpression of the AtNBR1 gene (OX lines) and the AtNBR1 Knock-Out (KO lines). Transcriptomic analysis of the shoots and roots of one representative OX line indicated differences in gene expression relative to the parental (WT) line. In shoots, many differentially expressed genes, either up- or down-regulated, were involved in responses to stimuli and stress. In roots the most significant difference was observed in a set of downregulated genes that is mainly related to translation and formation of ribonucleoprotein complexes. The link between AtNBR1 overexpression and abscisic acid (ABA) signalling was suggested by an interaction network analysis of these differentially expressed genes. Most hubs of this network were associated with ABA signalling. Although transcriptomic analysis suggested enhancement of ABA responses, ABA levels were unchanged in the OX shoots. Moreover, some of the phenotypes of the OX (delayed germination, increased number of closed stomata) and the KO lines (increased number of lateral root initiation sites) indicate that AtNBR1 is essential for fine-tuning of the ABA signalling pathway. The interaction of AtNBR1 with three regulatory proteins of ABA pathway (ABI3, ABI4 and ABI5) was observed in planta. It suggests that AtNBR1 might play role in maintaining the balance of ABA signalling by controlling their level and/or activity.

• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Autophagy * MeSH
• Plants, Genetically Modified MeSH
• Germination MeSH
• Abscisic Acid metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Seeds genetics   MeSH
• Seedlings MeSH
• Signal Transduction * MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• Publication type
• Meeting Abstract MeSH

Lipids are secreted into milk as bilayer-coated structures: milk fat globules (MFGs). Adipophilin (ADRP) and perilipin 3 (TIP47) are associated with MFGs in human breast milk; however, the role of these proteins in milk lipid secretion is not fully understood. The study aimed to investigate levels of ADRP, TIP47 and total lipid content in human breast milk, their mutual correlations, and dynamics during lactation. Milk samples from 22 healthy lactating women (Caucasian, Central European) were collected at five time points during lactation (1-3, 12-14, 29-30, 88-90 and 178-180 days postpartum). Mass spectrometry-based method was used for quantification of ADRP and TIP47 in the samples. The gravimetric method was used to determine milk total lipid content. We observed distinctive trends in ADRP, TIP47 levels and lipid content in human breast milk during the first six months of lactation. We also found a significant association between lipid content and ADRP, lipid content and TIP47, and ADRP and TIP47 concentrations in breast milk at all sampling points. A mass spectrometry-based method was developed for quantifying ADRP and TIP47 in human breast milk. Strong mutual correlations were found between ADRP, TIP47 and total lipid content in human breast milk.

• MeSH
• Adult MeSH
• Glycolipids metabolism   MeSH
• Glycoproteins metabolism   MeSH
• Lactation metabolism   MeSH
• Humans MeSH
• Lipid Droplets MeSH
• Milk, Human metabolism   MeSH
• Perilipin-2 metabolism   MeSH
• Perilipin-3 metabolism   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND AND PURPOSE: Extracellular vesicles (EVs) are constitutively shed from cells and released by various stimuli. Their protein and RNA cargo are modified by the stimulus, and in disease conditions can carry pathological cargo involved in disease progression. Neutral sphingomyelinase 2 (nSMase2) is a major regulator in at least one of several independent routes of EV biogenesis, and its inhibition is a promising new therapeutic approach for neurological disorders. Unfortunately, known inhibitors exhibit μM potency, poor physicochemical properties, and/or limited brain penetration. Here, we sought to identify a drug-like inhibitor of nSMase2. EXPERIMENTAL APPROACH: We conducted a human nSMase2 high throughput screen (>365,000 compounds). Selected hits were optimized focusing on potency, selectivity, metabolic stability, pharmacokinetics, and ability to inhibit EV release in vitro and in vivo. KEY RESULTS: We identified phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC), a potent (pIC50  = 6.57) and selective non-competitive inhibitor of nSMase2. PDDC was metabolically stable, with excellent oral bioavailability (%F = 88) and brain penetration (AUCbrain /AUCplasma  = 0.60). PDDC dose-dependently (pEC50  = 5.5) inhibited release of astrocyte-derived extracellular vesicles (ADEV). In an in vivo inflammatory brain injury model, PDDC robustly inhibited ADEV release and the associated peripheral immunological response. A closely related inactive PDDC analogue was ineffective. CONCLUSION AND IMPLICATIONS: PDDC is a structurally novel, potent, orally available, and brain penetrant inhibitor of nSMase2. PDDC inhibited release of ADEVs in tissue culture and in vivo. PDDC is actively being tested in animal models of neurological disease and, along with closely related analogues, is being considered for clinical translation.

• MeSH
• Astrocytes chemistry   metabolism   MeSH
• Extracellular Vesicles drug effects   metabolism   MeSH
• Microsomes, Liver chemistry   metabolism   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Molecular Structure MeSH
• Brain drug effects   metabolism   MeSH
• Mice MeSH
• Rats, Sprague-Dawley MeSH
• High-Throughput Screening Assays MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Parkinson's disease (PD) is an α-synucleinopathy characterized by the progressive loss of specific neuronal populations. Here, we develop a novel approach to transvascularly deliver proteins of complex quaternary structures, including α-synuclein preformed fibrils (pff). We show that a single systemic administration of α-synuclein pff triggers pathological transformation of endogenous α-synuclein in non-transgenic rats, which leads to neurodegeneration in discrete brain regions. Specifically, pff-exposed animals displayed a progressive deterioration in gastrointestinal and olfactory functions, which corresponded with the presence of cellular pathology in the central and enteric nervous systems. The α-synuclein pathology generated was both time dependent and region specific. Interestingly, the most significant neuropathological changes were observed in those brain regions affected in the early stages of PD. Our data therefore demonstrate for the first time that a single, transvascular administration of α-synuclein pff can lead to selective regional neuropathology resembling the premotor stage of idiopathic PD. Furthermore, this novel delivery approach could also be used to deliver a range of other pathogenic, as well as therapeutic, protein cargos transvascularly to the brain.

• MeSH
• alpha-Synuclein metabolism   MeSH
• Humans MeSH
• Brain metabolism   MeSH
• Neurons metabolism   MeSH
• Parkinson Disease * MeSH
• Enteric Nervous System * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The lack of cellular and tissue specificities in conventional chemotherapies along with the generation of a complex tumor microenvironment (TME) limits the dosage of active agents that reaches tumor sites, thereby resulting in ineffective responses and side effects. Therefore, the development of selective TME-responsive nanomedicines is of due relevance toward successful chemotherapies, albeit challenging. In this framework, we have synthesized novel, ready-to-use ROS-responsive amphiphilic block copolymers (BCs) with two different spacer chemistry designs to connect a hydrophobic boronic ester-based ROS sensor to the polymer backbone. Hydrodynamic flow focusing nanoprecipitation microfluidics (MF) was used in the preparation of well-defined ROS-responsive PSs; these were further characterized by a combination of techniques [1H NMR, dynamic light scattering (DLS), static light scattering (SLS), transmission electron microscopy (TEM), and cryogenic TEM (cryo-TEM)]. The reaction with hydrogen peroxide releases an amphiphilic phenol or a hydrophilic carboxylic acid, which affects polymersome (PS) stability and cargo release. Therefore, the importance of the spacer chemistry in BC deprotection and PS stability and cargo release is herein highlighted. We have also evaluated the impact of spacer chemistry on the PS-specific release of the chemotherapeutic drug doxorubicin (DOX) into tumors in vitro and in vivo. We demonstrate that by spacer chemistry design one can enhance the efficacy of DOX treatments (decrease in tumor growth and prolonged animal survival) in mice bearing EL4 T cell lymphoma. Side effects (weight loss and cardiotoxicity) were also reduced compared to free DOX administration, highlighting the potential of the well-defined ROS-responsive PSs as TME-selective nanomedicines. The PSs could also find applications in other environments with high ROS levels, such as chronic inflammations, aging, diabetes, cardiovascular diseases, and obesity.

• MeSH
• Doxorubicin * MeSH
• Micelles MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Tumor Microenvironment MeSH
• Neoplasms * drug therapy   MeSH
• Drug Carriers MeSH
• Reactive Oxygen Species MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Early diagnosis of severe infectious diseases is essential for timely implementation of lifesaving therapies. In a search for novel biomarkers in sepsis diagnosis we focused on polymorphonuclear neutrophils (PMNs). Notably, PMNs have their protein cargo readily stored in granules and following systemic stimulation, an immediate increase of neutrophil-borne proteins can be observed into the circulation of sepsis patients. We applied a combination of mass spectrometry (MS) based approaches, LC-MS/MS and selected reaction monitoring (SRM), to characterise and quantify the neutrophil proteome in healthy or disease conditions. With this approach we identified a neutrophil-derived protein abundance pattern in blood plasma consisting of 20 proteins that can be used as a protein signature for severe infectious diseases. Our results also show that SRM is highly sensitive, specific, and reproducible and, thus, a promising technology to study a complex, dynamic and multifactorial disease such as sepsis.

• MeSH
• Biomarkers metabolism   MeSH
• Chromatography, Liquid * MeSH
• Blood Proteins * metabolism   MeSH
• Humans MeSH
• Neutrophils * immunology   metabolism   microbiology   MeSH
• Predictive Value of Tests MeSH
• Disease Progression MeSH
• Proteomics * methods   MeSH
• Sepsis * MeSH
• Case-Control Studies MeSH
• Severity of Illness Index MeSH
• Tandem Mass Spectrometry * MeSH
• Check Tag
• Humans MeSH

GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aβ, a component of senile plaques in Alzheimer's disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aβ formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer's disease increases Aβ formation.

• MeSH
• Amyloid metabolism   MeSH
• Amyloid beta-Peptides chemistry   metabolism   MeSH
• Axonal Transport * MeSH
• Axons metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Dendrites metabolism   MeSH
• Epitopes metabolism   MeSH
• HEK293 Cells MeSH
• Kinesins metabolism   MeSH
• Humans MeSH
• Cell Adhesion Molecules chemistry   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Nerve Tissue Proteins chemistry   metabolism   MeSH
• GTP-Binding Proteins metabolism   MeSH
• Proteomics MeSH
• Receptors, GABA-B metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction MeSH
• Protein Stability MeSH
• Synapses metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH