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.

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.

Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3.

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

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.

The Diffusion Model of Intra-Golgi Transport Has Limited Power.

The Golgi complex (GC) is the main station along the cell biosecretory pathway. Until now, mechanisms of intra-Golgi transport (IGT) have remained unclear. Herein, we confirm that the goodness-of-fit of the regression lines describing the exit of a cargo from the Golgi zone (GZ) corresponds to an exponential decay. When the GC was empty before the re-initiation of the intra-Golgi transport, this parameter of the curves describing the kinetics of different cargoes (which are deleted in Golgi vesicles) with different diffusional mobilities within the GZ as well as their exit from the GZ was maximal for the piecewise nonlinear regression, wherein the first segment was horizontal, while the second segment was similar to the exponential decay. The kinetic curve describing cargo exit from the GC per se resembled a linear decay. The Monte-Carlo simulation revealed that such curves reflect the role of microtubule growth in cells with a central GC or the random hovering of ministacks in cells lacking a microtubule. The synchronization of cargo exit from the GC already filled with a cargo using the wave synchronization protocol did not reveal the equilibration of cargo within a Golgi stack, which would be expected from the diffusion model (DM) of IGT. Moreover, not all cisternae are connected to each other in mini-stacks that are transporting membrane proteins. Finally, the kinetics of post-Golgi carriers and the important role of SNAREs for IGT at different level of IGT also argue against the DM of IGT.

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.

Structure of the enterocyte transcytosis compartments during lipid absorption.

In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view. In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view.

Differential protein modulation by ketoprofen and ibuprofen underlines different cellular response by gastric epithelium.

Ketoprofen L-lysine salt (KLS), is widely used due to its analgesic efficacy and tolerability, and L-lysine was reported to increase the solubility and the gastric tolerance of ketoprofen. In a recent report, L-lysine salification has been shown to exert a gastroprotective effect due to its specific ability to counteract the NSAIDs-induced oxidative stress and up-regulate gastroprotective proteins. In order to derive further insights into the safety and efficacy profile of KLS, in this study we additionally compared the effect of lysine and arginine, another amino acid counterion commonly used for NSAIDs salification, in control and in ethanol challenged human gastric mucosa model. KLS is widely used for the control of post-surgical pain and for the management of pain and fever in inflammatory conditions in children and adults. It is generally well tolerated in pediatric patients, and data from three studies in >900 children indicate that oral administration is well tolerated when administered for up to 3 weeks after surgery. Since only few studies have so far investigated the effect of ketoprofen on gastric mucosa maintenance and adaptive mechanisms, in the second part of the study we applied the cMap approach to compare ketoprofen-induced and ibuprofen-induced gene expression profiles in order to explore compound-specific targeted biological pathways. Among the several genes exclusively modulated by ketoprofen, our attention was particularly focused on genes involved in the maintenance of gastric mucosa barrier integrity (cell junctions, morphology, and viability). The hypothesis was further validated by Real-time PCR.

Identification of p38 MAPK and JNK as new targets for correction of Wilson disease-causing ATP7B mutants.

UNLABELLED: Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans-Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum-retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c-Jun N-terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7B(H1069Q) (as well as that of several other WD-causing mutants) from the endoplasmic reticulum to the trans-Golgi network compartment, in recovery of its Cu-dependent trafficking, and in reduction of intracellular Cu levels. CONCLUSION: Our findings indicate p38 and c-Jun N-terminal kinase as intriguing targets for correction of WD-causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842-1859).

Glycosphingolipid-Protein Interaction in Signal Transduction.

Glycosphingolipids (GSLs) are a class of ceramide-based glycolipids essential for embryo development in mammals. The synthesis of specific GSLs depends on the expression of distinctive sets of GSL synthesizing enzymes that is tightly regulated during development. Several reports have described how cell surface receptors can be kept in a resting state or activate alternative signalling events as a consequence of their interaction with GSLs. Specific GSLs, indeed, interface with specific protein domains that are found in signalling molecules and which act as GSL sensors to modify signalling responses. The regulation exerted by GSLs on signal transduction is orthogonal to the ligand-receptor axis, as it usually does not directly interfere with the ligand binding to receptors. Due to their properties of adjustable production and orthogonal action on receptors, GSLs add a new dimension to the control of the signalling in development. GSLs can, indeed, dynamically influence progenitor cell response to morphogenetic stimuli, resulting in alternative differentiation fates. Here, we review the available literature on GSL-protein interactions and their effects on cell signalling and development.

Correlative video-light-electron microscopy: development, impact and perspectives.

Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the 'history,' or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light-electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular 'context' to the fluorescent dynamic structures of interest. CLEM 'multiplies' the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging.

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.

Salmonella acquires lysosome-associated membrane protein 1 (LAMP1) on phagosomes from Golgi via SipC protein-mediated recruitment of host Syntaxin6.

Several intracellular pathogens have developed diverse strategies to avoid targeting to lysosomes. However, they universally recruit lysosome-associated membrane protein 1 (LAMP1); the mechanism of LAMP1 recruitment remains unclear. Here, we report that a Salmonella effector protein, SipC, specifically binds with host Syntaxin6 through its C terminus and thereby recruits Syntaxin6 and other accessory molecules like VAMP2, Rab6, and Rab8 on Salmonella-containing phagosomes (SCP) and acquires LAMP1 by fusing with LAMP1-containing Golgi-derived vesicles. In contrast, sipC knock-out:SCP (sipC(-):SCP) or sipC(M398K):SCP fails to obtain significant amounts of Syntaxin6 and is unable to acquire LAMP1. Moreover, phagosomes containing respective knock-out Salmonella like sipA(-), sipB(-), sipD(-), sopB(-), or sopE(-) recruit LAMP1, demonstrating the specificity of SipC in this process. In addition, depletion of Syntaxin6 by shRNA in macrophages significantly inhibits LAMP1 recruitment on SCP. Additionally, survival of sipC(-):Salmonella in mice is found to be significantly inhibited in comparison with WT:Salmonella. Our results reveal a novel mechanism showing how Salmonella acquires LAMP1 through a SipC-Syntaxin6-mediated interaction probably to stabilize their niche in macrophages and also suggest that similar modalities might be used by other intracellular pathogens to recruit LAMP1.

Community Detection in Protein-Protein Interaction Networks and Applications.

The ability to identify and characterize not only the protein-protein interactions but also their internal modular organization through network analysis is fundamental for understanding the mechanisms of biological processes at the molecular level. Indeed, the detection of the network communities can enhance our understanding of the molecular basis of disease pathology, and promote drug discovery and disease treatment in personalized medicine. This work gives an overview of recent computational methods for the detection of protein complexes and functional modules in protein-protein interaction networks, also providing a focus on some of its applications. We propose a systematic reformulation of frequently adopted taxonomies for these methods, also proposing new categories to keep up with the most recent research. We review the literature of the last five years (2017-2021) and provide links to existing data and software resources. Finally, we survey recent works exploiting module identification and analysis, in the context of a variety of disease processes for biomarker identification and therapeutic target detection. Our review provides the interested reader with an up-to-date and self-contained view of the existing research, with links to state-of-the-art literature and resources, as well as hints on open issues and future research directions in complex detection and its applications.

Golgi Apparatus Regulates Plasma Membrane Composition and Function.

Golgi apparatus is the central component of the mammalian secretory pathway and it regulates the biosynthesis of the plasma membrane through three distinct but interacting processes: (a) processing of protein and lipid cargoes; (b) creation of a sharp transition in membrane lipid composition by non-vesicular transport of lipids; and (c) vesicular sorting of proteins and lipids at the trans-Golgi network to target them to appropriate compartments. We discuss the molecules involved in these processes and their importance in physiology and development. We also discuss how mutations in these molecules affect plasma membrane composition and signaling leading to genetic diseases and cancer.

Visualizing sphingolipid biosynthesis in cells.

Biosynthetic pathways play a fundamental role in the building and operation of the cell by synthesizing the constituents by which the cell is constructed, and by producing signalling intermediates that play a key role in cell regulation. While a lot is known about the metabolite profile of the cells and about the biochemical pathways through which these metabolites are produced, the cellular localization of the biosynthetic machineries and the importance of this localization to the regulation of the metabolism has often been given less attention. This derives from the fact that, for several of these pathways, the enzymes involved are found colocalized in one compartment where their specific localization is unlikely to influence their function. The sphingolipid (SL) metabolic pathway is a notable exception to this as SL synthetic enzymes are laid out on a specific pattern across the secretory compartments. Such compartmentalized organization of the SL synthesis has functional implications as it makes the fine-tuned regulation of the process possible by allowing cells to regulate specific segments of the pathway in response to stimuli and for adaptation. The organization, dynamics, and regulation of the SLs and their biosynthetic machinery have been investigated using imaging-based methods. Here we provide a brief introduction to the techniques that have been or that could be employed to visualize the SL biosynthetic machinery and SLs themselves and discuss the insights provided by these studies in understanding this metabolism.

Translation of genome to glycome: role of the Golgi apparatus.

Glycans are one of the four biopolymers of the cell and they play important roles in cellular and organismal physiology. They consist of both linear and branched structures and are synthesized in a nontemplated manner in the secretory pathway of mammalian cells with the Golgi apparatus playing a key role in the process. In spite of the absence of a template, the glycans synthesized by a cell are not a random collection of possible glycan structures but a distribution of specific glycans in defined quantities that is unique to each cell type (Cell type here refers to distinct cell forms present in an organism that can be distinguished based on morphological, phenotypic and/or molecular criteria.) While information to produce cell type-specific glycans is encoded in the genome, how this information is translated into cell type-specific glycome (Glycome refers to the quantitative distribution of all glycan structures present in a given cell type.) is not completely understood. We summarize here the factors that are known to influence the fidelity of glycan biosynthesis and integrate them into known glycosylation pathways so as to rationalize the translation of genetic information to cell type-specific glycome.

GOLPH3 and oncogenesis: What is the molecular link?

The Golgi phosphoprotein 3 (GOLPH3) is encoded by a gene that is located in a region of the human genome that is often amplified in different solid tumours. GOLPH3, an evolutionary conserved phosphatidylinositol 4-phosphate (PI4P) binding protein, is mainly localised at trans Golgi network (TGN). It regulates several cellular functions like Golgi vesicular trafficking, Golgi glycosylation and mitochondrial cardiolipin production. Recently, GOLPH3 was discovered to be part of the DNA damage response signalling pathway, with a role in cell survival following DNA damage. In this review, we will explore the cellular functions regulated by GOLPH3 and discuss if and how they contribute to the oncogenic activity of this intriguing Golgi localized oncoprotein.

Rare ER protein misfolding-mistrafficking disorders: Therapeutic developments.

The presence of a functional protein at the appropriate location in the cell is the result of the processes of transcription, translation, folding and trafficking to the correct destination. There are numerous diseases that are caused by protein misfolding, mainly due to mutations in the respective gene. The consequences of this misfolding may be that proteins effectively lose their function, either by being removed by the cellular quality control machinery or by accumulating at the incorrect intracellular or extracellular location. A number of mutations that lead to protein misfolding and affect trafficking to the final destination, e.g. Cystic fibrosis, Wilson's disease, and Progressive Familial Intrahepatic 1 cholestasis, result in proteins that retain partial function if their folding and trafficking is restored either by molecular or pharmacological means. In this review, we discuss several mutant proteins within this class of misfolding diseases and provide an update on the status of molecular and therapeutic developments and potential therapeutic strategies being developed to counter these diseases.