Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum.

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.

Copper-independent lysosomal localisation of the Wilson disease protein ATP7B.

In hepatocytes, the Wilson disease protein ATP7B resides on the trans-Golgi network (TGN) and traffics to peripheral lysosomes to export excess intracellular copper through lysosomal exocytosis. We found that in basal copper or even upon copper chelation, a significant amount of ATP7B persists in the endolysosomal compartment of hepatocytes but not in non-hepatic cells. These ATP7B-harbouring lysosomes lie in close proximity of ~10 nm to the TGN. ATP7B constitutively distributes itself between the sub-domain of the TGN with a lower pH and the TGN-proximal lysosomal compartments. The presence of ATP7B on TGN-lysosome colocalising sites upon Golgi disruption suggested a possible exchange of ATP7B directly between the TGN and its proximal lysosomes. Manipulating lysosomal positioning significantly alters the localisation of ATP7B in the cell. Contrary to previous understanding, we found that upon copper chelation in a copper-replete hepatocyte, ATP7B is not retrieved back to TGN from peripheral lysosomes; rather, ATP7B recycles to these TGN-proximal lysosomes to initiate the next cycle of copper transport. We report a hitherto unknown copper-independent lysosomal localisation of ATP7B and the importance of TGN-proximal lysosomes but not TGN as the terminal acceptor organelle of ATP7B in its retrograde pathway.

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane.

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11-positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245-dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97- vs. Golgin-245-dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin-mediated cell polarity and cell-cell junctions.

Thymic Stromal Lymphopoietin (TSLP) Is Cleaved by Human Mast Cell Tryptase and Chymase.

Thymic stromal lymphopoietin (TSLP), mainly expressed by epithelial cells, plays a central role in asthma. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP). Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play key roles in asthma. We evaluated the early proteolytic effects of tryptase and chymase released by HLMCs on TSLP by mass spectrometry. We also investigated whether TSLP and its fragments generated by these enzymes induce angiogenic factor release from HLMs. Mass spectrometry (MS) allowed the identification of TSLP cleavage sites caused by tryptase and chymase. Recombinant human TSLP treated with recombinant tryptase showed the production of 1-97 and 98-132 fragments. Recombinant chymase treatment of TSLP generated two peptides, 1-36 and 37-132. lfTSLP induced the release of VEGF-A, the most potent angiogenic factor, from HLMs. By contrast, the four TSLP fragments generated by tryptase and chymase failed to activate HLMs. Long-term TSLP incubation with furin generated two peptides devoid of activating property on HLMs. These results unveil an intricate interplay between mast cell-derived proteases and TSLP. These findings have potential relevance in understanding novel aspects of asthma pathobiology.

Sphingolipid metabolic flow controls phosphoinositide turnover at the trans-Golgi network.

Sphingolipids are membrane lipids globally required for eukaryotic life. The sphingolipid content varies among endomembranes with pre- and post-Golgi compartments being poor and rich in sphingolipids, respectively. Due to this different sphingolipid content, pre- and post-Golgi membranes serve different cellular functions. The basis for maintaining distinct subcellular sphingolipid levels in the presence of membrane trafficking and metabolic fluxes is only partially understood. Here, we describe a homeostatic regulatory circuit that controls sphingolipid levels at the trans-Golgi network (TGN). Specifically, we show that sphingomyelin production at the TGN triggers a signalling pathway leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol and sphingolipid transport to the trans-Golgi network, PtdIns(4)P consumption interrupts this transport in response to excessive sphingomyelin production. Based on this evidence, we envisage a model where this homeostatic circuit maintains a constant lipid composition in the trans-Golgi network and post-Golgi compartments, thus counteracting fluctuations in the sphingolipid biosynthetic flow.

Design of Gelatin-Capped Plasmonic-Diatomite Nanoparticles with Enhanced Galunisertib Loading Capacity for Drug Delivery Applications.

Inorganic diatomite nanoparticles (DNPs) have gained increasing interest as drug delivery systems due to their porous structure, long half-life, thermal and chemical stability. Gold nanoparticles (AuNPs) provide DNPs with intriguing optical features that can be engineered and optimized for sensing and drug delivery applications. In this work, we combine DNPs with gelatin stabilized AuNPs for the development of an optical platform for Galunisertib delivery. To improve the DNP loading capacity, the hybrid platform is capped with gelatin shells of increasing thicknesses. Here, for the first time, full optical modeling of the hybrid system is proposed to monitor both the gelatin generation, degradation, and consequent Galunisertib release by simple spectroscopic measurements. Indeed, the shell thickness is optically estimated as a function of the polymer concentration by exploiting the localized surface plasmon resonance shifts of AuNPs. We simultaneously prove the enhancement of the drug loading capacity of DNPs and that the theoretical modeling represents an efficient predictive tool to design polymer-coated nanocarriers.

Self-assembly of two hydrophobins from marine fungi affected by interaction with surfaces.

Hydrophobins are amphiphilic fungal proteins endowed with peculiar characteristics, such as a high surface activity and an interface triggered self-assembly. Several applications of these proteins have been proposed in the food, cosmetics and biomedical fields. Moreover, their use as proteinaceous coatings can be effective for materials and nanomaterials applications. The discovery of novel hydrophobins with diverse properties may be advantageous from both the scientific and industrial points of view. Stressful environmental conditions of fungal growth may induce the production of proteins with peculiar features. Two Class I hydrophobins from fungi isolated from marine environment have been recently purified. Herein, their propensity to aggregate forming nanometric fibrillar structures has been compared, using different techniques, such as circular dichroism, dynamic light scattering and Thioflavin T fluorescence assay. Furthermore, TEM and AFM images indicate that the interaction of these proteins with specific surfaces, are crucial in the formation of amyloid fibrils and in the assembly morphologies. These self-assembling proteins show promising properties as bio-coating for different materials via a green process. Biotechnol. Bioeng. 2017;114: 2173-2186. © 2017 Wiley Periodicals, Inc.

COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression.

During corticogenesis, late-born callosal projection neurons (CPNs) acquire their laminar position through glia-guided radial migration and then undergo final differentiation. However, the mechanisms controlling radial migration and final morphology of CPNs are poorly defined. Here, we show that in COUP-TFI mutant mice CPNs are correctly specified, but are delayed in reaching the cortical plate and have morphological defects during migration. Interestingly, we observed that the rate of neuronal migration to the cortical plate normally follows a low-rostral to high-caudal gradient, similar to that described for COUP-TFI. This gradient is strongly impaired in COUP-TFI(-/-) brains. Moreover, the expression of the Rho-GTPase Rnd2, a modulator of radial migration, is complementary to both these gradients and strongly increases in the absence of COUP-TFI function. We show that COUP-TFI directly represses Rnd2 expression at the post-mitotic level along the rostrocaudal axis of the neocortex. Restoring correct Rnd2 levels in COUP-TFI(-/-) brains cell-autonomously rescues neuron radial migration and morphological transitions. We also observed impairments in axonal elongation and dendritic arborization of COUP-TFI-deficient CPNs, which were rescued by lowering Rnd2 expression levels. Thus, our data demonstrate that COUP-TFI modulates late-born neuron migration and favours proper differentiation of CPNs by finely regulating Rnd2 expression levels.

TSLP is localized in and released from human lung macrophages activated by T2-high and T2-low stimuli: relevance in asthma and COPD.

BACKGROUND: Macrophages are the predominant immune cells in the human lung and play a central role in airway inflammation, including asthma and chronic obstructive pulmonary disease (COPD). Thymic stromal lymphopoietin (TSLP), a pleiotropic cytokine mainly expressed by bronchial epithelial cells, plays a key role in asthma and COPD pathobiology. TSLP exists in two variants: the long form (lfTSLP) and a shorter TSLP isoform (sfTSLP). We aimed to localize TSLP in human lung macrophages (HLMs) and investigate the mechanisms of its release from these cells. We also evaluated the effects of the two variants of TSLP on the release of angiogenic factor from HLMs. METHODS: We employed immunofluorescence and Western blot to localize intracellular TSLP in HLMs purified from human lung parenchyma. HLMs were activated by T2-high (IL-4, IL-13) and T2-low (lipopolysaccharide: LPS) immunological stimuli. RESULTS: TSLP was detected in HLMs and subcellularly localized in the cytoplasm. IL-4 and LPS induced TSLP release from HLMs. Preincubation of macrophages with brefeldin A, known to disrupt the Golgi apparatus, inhibited TSLP release induced by LPS and IL-4. lfTSLP concentration-dependently induced the release of vascular endothelial growth factor-A (VEGF-A), the most potent angiogenic factor, from HLMs. sfTSLP neither activated nor interfered with the activating property of lfTSLP on macrophages. CONCLUSIONS: Our results highlight a novel immunologic circuit between HLMs and TSLP. Given the central role of macrophages in airway inflammation, this autocrine loop holds potential translational relevance in understanding innovative aspects of the pathobiology of asthma and chronic inflammatory lung disorders.

Paper-based electrochemical device for early detection of integrin αvß6 expressing tumors.

Despite progress in the prevention and diagnosis of cancer, current technologies for tumor detection present several limitations including invasiveness, toxicity, inaccuracy, lengthy testing duration and high cost. Therefore, innovative diagnostic techniques that integrate knowledge from biology, oncology, medicinal and analytical chemistry are now quickly emerging in the attempt to address these issues. Following this approach, here we developed a paper-based electrochemical device for detecting cancer-derived Small Extracellular Vesicles (S-EVs) in fluids. S-EVs were obtained from cancer cell lines known to express, at a different level, the αvß6 integrin receptor, a well-established hallmark of numerous epithelial cancer types. The resulting biosensor turned out to recognize αvß6-containing S-EVs down to a limit of 0.7*103 S-EVs/mL with a linear range up to 105 S-EVs /mL, and a relative standard deviation of 11%, thus it may represent a novel opportunity for αvß6 expressing cancers detection.

Role of Annexin A1 in mouse myoblast cell differentiation.

Annexin A1 (ANXA1) is a calcium- and phospholipid-binding protein involved in a broad range of cellular events. This study used molecular and microscopy approaches to explore the role of ANXA1 in mouse myoblast C2C12 cell differentiation. We report that ANXA1 expression increases during differentiation and that the down-regulation of ANXA1 significantly inhibits the differentiation process. ANXA1 is expressed in vivo in both quiescent and activated satellite cells and is highly localized in the cells that migrate in the lumen of regenerating fibers after an acute injury. Endogenous ANXA1 co-localizes with actin fibers at the protruding ends of undifferentiated but not differentiated cells suggesting a role of the protein in cell migration. Furthermore, ANXA1 neutralizing antibody reduces MyHC expression, decreases myotube formation and significantly inhibits cell migration. The data reported here suggest for the first time that ANXA1 plays a role in myogenic differentiation.

Expression of Serum Exosomal miRNA 122 and Lipoprotein Levels in Dogs Naturally Infected by Leishmania infantum: A Preliminary Study.

Current knowledge on the role of exosomal microRNA (miRNA) in canine leishmaniasis (CL), with particular regards to the interaction between miR-122 and lipid alterations, is limited. The aim of this study was to isolate/characterize exosomes in canine serum and evaluate the expression of miR-122 in ten healthy and ten leishmaniotic dogs. Serum exosomes were isolated using a polymer-based kit, ExoQuick® and characterized by flow cytometry and transmission electron microscopy, whereas miR-122-5p expression was evaluated by quantitative reverse-transcriptase polymerase chain reaction. A significant decreased expression of exosomal miR-122-5p, decreased serum levels of high-density lipoproteins, and increased serum levels of low-density lipoproteins were seen in leishmaniotic dogs when compared with healthy dogs. These results suggest that hepatic dysfunctions induced by the parasite interfere with lipoprotein status. The decreased expression of exosomal miR122 represents an additional effect of Leishmania infection in dogs as in people.

Gold Nanoparticles Modulate BCG-Induced Innate Immune Memory in Human Monocytes by Shifting the Memory Response towards Tolerance.

Innate immune memory is characterized by a modulation in the magnitude with which innate immune cells such as monocytes and macrophages respond to potential dangers, subsequent to previous exposure to the same or unrelated agents. In this study, we have examined the capacity of gold nanoparticles (AuNP), which are already in use for therapeutic and diagnostic purposes, to modulate the innate memory induced by bacterial agents. The induction of innate memory was achieved in vitro by exposing human primary monocytes to bacterial agents (lipopolysaccharide -LPS-, or live Bacille Calmette-Guérin -BCG) in the absence or presence of AuNP. After the primary activation, cells were allowed to return to a resting condition, and eventually re-challenged with LPS. The induction of memory was assessed by comparing the response to the LPS challenge of unprimed cells with that of cells primed with bacterial agents and AuNP. The response to LPS was measured as the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra). While ineffective in directly inducing innate memory per se, and unable to influence LPS-induced tolerance memory, AuNP significantly affected the memory response of BCG-primed cells, by inhibiting the secondary response in terms of both inflammatory and anti-inflammatory factor production. The reprogramming of BCG-induced memory towards a tolerance type of reactivity may open promising perspectives for the use of AuNP in immunomodulatory approaches to autoimmune and chronic inflammatory diseases.

Intramuscular viral delivery of paraplegin rescues peripheral axonopathy in a model of hereditary spastic paraplegia.

Degeneration of peripheral motor axons is a common feature of several debilitating diseases including complicated forms of hereditary spastic paraplegia. One such form is caused by loss of the mitochondrial energy-dependent protease paraplegin. Paraplegin-deficient mice display a progressive degeneration in several axonal tracts, characterized by the accumulation of morphological abnormal mitochondria. We show that adenoassociated virus-mediated (AAV-mediated) intramuscular delivery of paraplegin halted the progression of neuropathological changes and rescued mitochondrial morphology in the peripheral nerves of paraplegin-deficient mice. One single injection before onset of symptoms improved the motor performance of paraplegin-deficient mice for up to 10 months, indicating that the peripheral neuropathy contributes to the clinical phenotype. This study provides a proof of principle that gene transfer may be an effective therapeutic option for patients with paraplegin deficiency and demonstrates that AAV vectors can be successfully employed for retrograde delivery of an intracellular protein to spinal motor neurons, opening new perspectives for several hereditary axonal neuropathies of the peripheral nerves.

GRASP55 and UPR Control Interleukin-1ß Aggregation and Secretion.

Signal-sequence-lacking interleukin (IL)-1ß, is cleaved by caspase-1 to mature mIL-1ß, which is secreted, without entering the endoplasmic reticulum. We report that macrophages of GRASP55-/- mice are defective in mIL-1ß secretion and retain it as intracellular aggregates. Intriguingly, GRASP55-/- macrophages are defective in the IRE1α branch of the unfolded protein response. This finding fits well with our data that inhibition of IRE1α also impairs mIL-1ß secretion and causes its accumulation in intracellular aggregates. PERK inhibition, on the other hand, controls caspase-1-mediated conversion of proIL-1ß to mIL-1ß. These findings reveal translation-independent functions of PERK and IRE1α: PERK controls the production of mIL-1ß, which is then followed by GRASP55 and IRE1α activity to keep mIL-1ß in a secretion-competent form.

Compositional complexity of rods and rings.

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood. Here we show that the regulatory GTPase, ARL2, a subset of its binding partners, and several resident proteins at the endoplasmic reticulum (ER) also localize to RRs. We also have identified two new inducers of RR formation: AICAR and glucose deprivation. We demonstrate that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, we show that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. These findings suggest that RRs are considerably more complex than previously thought and that the function(s) of RRs may include involvement of a regulatory GTPase, its effectors, and potentially contacts with intracellular membranes.

Akap1 Regulates Vascular Function and Endothelial Cells Behavior.

MitoAKAPs (mitochondrial A kinase anchoring proteins), encoded by the Akap1 gene, regulate multiple cellular processes governing mitochondrial homeostasis and cell viability. Although mitochondrial alterations have been associated to endothelial dysfunction, the role of mitoAKAPs in the vasculature is currently unknown. To test this, postischemic neovascularization, vascular function, and arterial blood pressure were analyzed in Akap1 knockout mice (Akap1-/- ) and their wild-type (wt) littermates. Primary cultures of aortic endothelial cells (ECs) were also obtained from Akap1-/- and wt mice, and ECs migration, proliferation, survival, and capillary-like network formation were analyzed under different experimental conditions. After femoral artery ligation, Akap1-/- mice displayed impaired blood flow and functional recovery, reduced skeletal muscle capillary density, and Akt phosphorylation compared with wt mice. In Akap1-/- ECs, a significant enhancement of hypoxia-induced mitophagy, mitochondrial dysfunction, reactive oxygen species production, and apoptosis were observed. Consistently, capillary-like network formation, migration, proliferation, and AKT phosphorylation were reduced in Akap1-/- ECs. Alterations in Akap1-/- ECs behavior were also confirmed in Akap1-/- mice, which exhibited a selective reduction in acetylcholine-induced vasorelaxation in mesenteric arteries and a mild but significant increase in arterial blood pressure levels compared with wt. Finally, overexpression of a constitutively active Akt mutant restored vascular reactivity and ECs function in Akap1-/- conditions. These results demonstrate the important role of mitoAKAPs in the modulation of multiple ECs functions in vivo and in vitro, suggesting that mitochondria-dependent regulation of ECs might represent a novel therapeutic approach in cardiovascular diseases characterized by endothelial dysfunction.

Axonal degeneration in paraplegin-deficient mice is associated with abnormal mitochondria and impairment of axonal transport.

In several neurodegenerative diseases, axonal degeneration occurs before neuronal death and contributes significantly to patients' disability. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous condition characterized by selective degeneration of axons of the corticospinal tracts and fasciculus gracilis. HSP may therefore be considered an exemplary disease to study the local programs mediating axonal degeneration. We have developed a mouse model for autosomal recessive HSP due to mutations in the SPG7 gene encoding the mitochondrial ATPase paraplegin. Paraplegin-deficient mice are affected by a distal axonopathy of spinal and peripheral axons, characterized by axonal swelling and degeneration. We found that mitochondrial morphological abnormalities occurred in synaptic terminals and in distal regions of axons long before the first signs of swelling and degeneration and correlated with onset of motor impairment during a rotarod test. Axonal swellings occur through massive accumulation of organelles and neurofilaments, suggesting impairment of anterograde axonal transport. Retrograde axonal transport is delayed in symptomatic mice. We speculate that local failure of mitochondrial function may affect axonal transport and cause axonal degeneration. Our data suggest that a timely therapeutic intervention may prevent the loss of axons.

TANGO1 assembles into rings around COPII coats at ER exit sites.

TANGO1 (transport and Golgi organization 1) interacts with CTAGE5 and COPII components Sec23/Sec24 and recruits ERGIC-53 (endoplasmic reticulum [ER]-Golgi intermediate compartment 53)-containing membranes to generate a mega-transport carrier for export of collagens and apolipoproteins from the ER. We now show that TANGO1, at the ER, assembles in a ring that encircles COPII components. The C-terminal, proline-rich domains of TANGO1 molecules in the ring are initially tilted onto COPII coats but appear to be pushed apart as the carrier grows. These findings lend support to our suggestion that growth of transport carriers for exporting bulky cargoes requires addition of membranes and not simply COPII-mediated accretion of a larger surface of ER. TANGO1 remains at the neck of the newly forming transport carrier, which grows in size by addition of ERGIC-53-containing membranes to generate a transport intermediate for the export of bulky collagens.