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The gene encoding the High Mobility Group A1 (HMGA1) chromatin remodeling protein is upregulated in diverse cancers where high levels portend adverse clinical outcomes. Until recently, HMGA1 was assumed to be a nuclear protein exerting its role in cancer by transcriptionally modulating gene expression and downstream signaling pathways. However, the discovery of an extracellular HMGA1-RAGE autocrine loop in invasive triple-negative breast cancer (TNBC) cell lines implicates HMGA1 as a “moonlighting protein” with different functions depending upon cellular location. Here, we review the role of HMGA1, not only as a chromatin regulator in cancer and stem cells, but also as a potential secreted factor that drives tumor progression. Prior work found that HMGA1 is secreted from TNBC cell lines where it signals through the receptor for advanced glycation end products (RAGE) to foster phenotypes involved in tumor invasion and metastatic progression. Studies in primary TNBC tumors also suggest that HMGA1 secretion associates with distant metastasis in TNBC. Given the therapeutic potential to target extracellular proteins, further work to confirm this role in other contexts is warranted. Indeed, crosstalk between nuclear and secreted HMGA1 could change our understanding of tumor development and reveal novel therapeutic opportunities relevant to diverse human cancers overexpressing HMGA1.

The Golgi Reassembly and Stacking Proteins (GRASPs) are engaged in various functions within the cell, both in unconventional secretion mechanisms and structuring and organizing the Golgi apparatus. Understanding their specific role in each situation still requires more structural and functional data at the molecular level. GRASP55 is one of the GRASP members in mammals, anchored to the membrane via the myristoylation of a Gly residue at its N-terminus. Therefore, co-translational modifications, such as myristoylation, are fundamental when considering a strategy to obtain detailed information on the interactions between GRASP55 and membranes. Despite its functional relevance, the N-terminal myristoylation has been underappreciated in the studies reported to date, compromising the previously proposed models for GRASP-membrane interactions. Here, we investigated the synergy between the presence of the membrane and the formation of oligomeric structures of myristoylated GRASP55, using a series of biophysical techniques to perform the structural characterization of the lipidated GRASP55 and its interaction with biological lipid model membranes. Our data fulfill an unexplored gap: the adequate evaluation of the presence of lipidations and lipid membranes on the structure-function dyad of GRASPs.Communicated by Ramaswamy H. Sarma.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 )-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P 2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling., Competing Interests: FL, JS, RS, DB, JF, SU, SK, PR, SW, HM, XS, SK, CF, MH, RŠ, PC, IV, WN No competing interests declared, (© 2023, Lolicato, Steringer et al.)

Newly synthesized proteins are delivered to the apoplast via conventional or unconventional protein secretion in eukaryotes. In plants, proteins are secreted to perform various biological functions. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been revealed in plants. In the conventional protein secretion pathway, secretory proteins with a signal peptide are translocated into the endoplasmic reticulum and transported to the extracellular region via the endomembrane system. On the contrary, unconventional protein secretion pathways have been demonstrated to mediate the secretion of the leaderless secretory proteins. In this chapter, we summarize the updated findings and provide a comprehensive overview of protein secretion pathways in plants., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To function properly, neurons must maintain a proteome that differs in their somatodendritic and axonal domain. This requires the polarized sorting of newly synthesized secretory and transmembrane proteins into different vesicle populations as they traverse the secretory pathway. Although the trans-Golgi-network is generally considered to be the main sorting hub, this sorting process may already begin at the ER and continue through the Golgi cisternae. At each step in the sorting process, specificity is conferred by adaptors, GTPases, tethers, and SNAREs. Besides this, local synthesis and unconventional protein secretion may contribute to the polarized proteome to enable rapid responses to stimuli. For some transmembrane proteins, some of the steps in the sorting process are well-studied. These will be highlighted here. The universal rules that govern polarized protein sorting remain unresolved, therefore we emphasize the need to approach this problem in an unbiased, top-down manner. Unraveling these rules will contribute to our understanding of neuronal development and function in health and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Kersten and Farías.)

Unconventional protein secretion (UPS) plays important roles in cell physiology. In contrast to canonical secretory routes, UPS does not generally require secretory signal sequences and often bypasses secretory compartments such as the ER and the Golgi apparatus. Giardia lamblia is a protist parasite with reduced subcellular complexity which releases several proteins, some of them virulence factors, without canonical secretory signals. This implicates UPS at the parasite-host interface. No dedicated machinery nor mechanism(s) for UPS in Giardia are currently known, although speculations on the involvement of endocytic organelles called PV/PECs, have been put forth. To begin to address the question of whether PV/PECs are implicated in virulence-associated UPS and to define the composition of molecular machinery involved in protein release, we employed affinity purification and mass spectrometry, coupled to microscopy-based subcellular localization and signal correlation quantification to investigate the interactomes of 11 reported unconventionally secreted proteins, all predicted to be cytosolic. A subset of these are associated with PV/PECs. Extended and validated interactomes point to a core PV/PECs-associated UPS machinery, which includes uncharacterized and Giardia-specific coiled-coil proteins and NEK kinases. Finally, a subset of the alpha-giardin protein family was enriched in all PV/PECs-associated protein interactomes, highlighting a previously unappreciated role for these proteins at PV/PECs and in UPS. Taken together, our results provide the first characterization of a virulence-associated UPS protein complex in Giardia lamblia at PV/PECs, suggesting a novel link between these primarily endocytic and feeding organelles and UPS at the parasite-host interface.

The molecular bases of the host-parasitoid interactions in the biological system Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) and Aphidius ervi (Haliday) (Hymenoptera, Braconidae) have been elucidated allowing the identification of a gamma-glutamyl transpeptidase, the active component of maternal venom secretion, and teratocytes, the embryonic parasitic factors responsible for host physiology regulation after parasitization. Teratocytes, cells deriving from the dissociation of the serosa, the parasitoid embryonic membrane, are responsible for extra-oral digestion of host tissues in order to provide a suitable nutritional environment for the development of parasitoid larvae. Teratocytes rapidly grow in size without undergoing any cell division, synthesize, and release in the host hemolymph two proteins: a fatty acid binding protein (Ae- FABP) and an enolase (Ae -ENO). Ae- FABP is involved in transport of fatty acids deriving from host tissues to the parasitoid larva. Ae -ENO is an extracellular glycolytic enzyme that functions as a plasminogen like receptor inducing its activation to plasmin. Both Ae -FABP and Ae -ENO lack their signal peptides, and they are released in the extracellular environment through an unknown secretion pathway. Here, we investigated the unconventional mechanism by which teratocytes release Ae -FABP and Ae-ENO in the extracellular space. Our results, obtained using immunogold staining coupled with TEM and western blot analyses, show that these two proteins are localized in vesicles released by teratocytes. The specific dimension of these vesicles and the immunodetection of ALIX and HSP70, two exosome markers, strongly support the hypothesis that these vesicles are exosomes. [ABSTRACT FROM AUTHOR]

Stress granules are cytoplasmic compartments, which serve as mRNA storage units during stress, therefore regulating translation. The Arabidopsis thaliana lectin ArathEULS3 has been widely described as a stress inducible gene. This study aimed to examine in detail the localization of ArathEULS3 lectin in normal and stressed cells. Colocalization experiments revealed that the nucleo-cytoplasmic lectin ArathEULS3 relocates to stress granules after stress. The ArathEULS3 sequence encodes a protein with a EUL lectin domain and an N-terminal domain with unknown structure and function. Bioinformatics analyses showed that the N-terminal domain sequence contains intrinsically disordered regions and likely does not exhibit a stable protein fold. Plasmolysis experiments indicated that ArathEULS3 also localizes to the apoplast, suggesting that this protein might follow an unconventional route for secretion. As part of our efforts we also investigated the interactome of ArathEULS3 and identified several putative interaction partners important for the protein translation process.

Macroautophagy/autophagy was recently shown to regulate unconventional protein secretion through a process called secretory autophagy. How the secretory cargo selectively enters into the secretory autophagosome has been a central question. Our recent studies indicate that cargo translocation into the ER-Golgi intermediate compartment, a compartment contributing membranes to the forming autophagosome, acts as a mechanism for secretory cargo entry into the vesicle and may be an early step for secretory autophagy. [ABSTRACT FROM AUTHOR]

Abstract Secretion of proteins lacking leader sequence was deemed rare and unconventional, only accountable for the export of a limited number of clients by mechanisms that are poorly defined. However, recent studies have shown that many leaderless proteins misfolded in the cytoplasm can be selectively exported to extracellular milieu via an unconventional secretory path termed Misfolding-Associated Protein Secretion (MAPS). This process uses the surface of the endoplasmic reticulum (ER) as a platform to enrich abnormally folded polypeptides, and then transport them into the lumen of ER-associated late endosomes for subsequent secretion. Elimination of misfolded proteins via MAPS appears to serve a role in protein homeostasis maintenance, particularly for stressed cells bearing an excess of protein quality control (PQC) burden. [ABSTRACT FROM AUTHOR]

Summary IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion. [ABSTRACT FROM AUTHOR]

Abstract: The pro‐inflammatory cytokine IL‐1β is well known for its role in host defense and the initiation of potent inflammatory responses. It is processed from its inactive pro‐form by the inflammatory caspase‐1 into its mature bioactive form, which is then released from the cell via an unconventional secretion mechanism. Recently, gasdermin‐D has been identified as a new target of caspase‐1. After proteolytical cleavage of gasdermin‐D, the N‐terminal fragment induces pyroptosis, a lytic cell death, by forming large permeability pores in the plasma membrane. Here we show using the murine system that gasdermin‐D is required for IL‐1β secretion by macrophages, dendritic cells and partially in neutrophils, and that secretion is a cell‐lysis‐independent event. Liposome transport assays in vitro further demonstrate that gasdermin‐D pores are large enough to allow the direct release of IL‐1β. Moreover, IL‐18 and other small soluble cytosolic proteins can also be released in a lysis‐independent but gasdermin‐D‐dependent mode, suggesting that the gasdermin‐D pores allow passive the release of cytosolic proteins in a size‐dependent manner. [ABSTRACT FROM AUTHOR]

Contents Summary 1012 I. Introduction 1012 II. The endomembrane system in plant–microbe interactions 1013 III. The cytoskeleton in plant–microbe interactions 1017 IV. Organelles in plant–microbe interactions 1019 V. Inter‐organellar communication in plant–microbe interactions 1022 VI. Conclusions and prospects 1023 Acknowledgements 1024 References 1024 Summary: Plants have evolved a multilayered immune system with well‐orchestrated defense strategies against pathogen attack. Multiple immune signaling pathways, coordinated by several subcellular compartments and interactions between these compartments, play important roles in a successful immune response. Pathogens use various strategies to either directly attack the plant's immune system or to indirectly manipulate the physiological status of the plant to inhibit an immune response. Microscopy‐based approaches have allowed the direct visualization of membrane trafficking events, cytoskeleton reorganization, subcellular dynamics and inter‐organellar communication during the immune response. Here, we discuss the contributions of organelles and the cytoskeleton to the plant's defense response against microbial pathogens, as well as the mechanisms used by pathogens to target these compartments to overcome the plant's defense barrier. [ABSTRACT FROM AUTHOR]

Protein secretion is an essential process in all eukaryotic cells and its mechanisms have been extensively studied. Proteins with an N-terminal leading sequence or transmembrane domain are delivered through the conventional protein secretion (CPS) pathway from the endoplasmic reticulum (ER) to the Golgi apparatus. This feature is conserved in yeast, animals, and plants. In contrast, the transport of leaderless secretory proteins (LSPs) from the cytosol to the cell exterior is accomplished via the unconventional protein secretion (UPS) pathway. So far, the CPS pathway has been well characterized in plants, with several recent studies providing new information about the regulatory mechanisms involved. On the other hand, studies on UPS pathways in plants remain descriptive, although a connection between UPS and the plant defense response is becoming more and more apparent. In this review, we present an update on CPS and UPS. With the emergence of new techniques, a more comprehensive understanding of protein secretion in plants can be expected in the future. [ABSTRACT FROM AUTHOR]

The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

The pathological aggregation of tau protein is a hallmark of a set of neurodegenerative diseases collectively referred to as tauopathies. Tau aggregates independently in each neuron, but this aggregation can also occur in a non-cell autonomous manner in which aggregated tau is transmitted from one cell to another. Such trans-cellular propagation is initiated by the uptake of extracellular tau, which then seeds soluble tau in the recipient cells to spread the tau pathology. Accumulating evidence has demonstrated that tau is not only present in the cytoplasm of neurons but also actively released into the extracellular space. This finding has led to the idea that extracellular tau could be a novel therapeutic target to halt the propagation of tau pathology. From this perspective, the present review article focuses on recent advances in understanding the mechanisms regulating the levels of extracellular tau and discusses the role of such mechanisms in the propagation of tau pathology.

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.

Acyl-CoA binding protein (ACBP) plays important roles in the metabolism of lipids in eukaryotic cells. In the industrially important filamentous fungus Aspergillus oryzae , although we have previously demonstrated that the A. oryzae ACBP (AoACBP) localizes to punctate structures and exhibits long-range motility, which is dependent on autophagy-related proteins, the physiological role of AoACBP remains elusive. Here, we describe identification and characterization of another ACBP from A. oryzae ; we named this ACBP as AoAcb2 and accordingly renamed AoACBP as AoAcb1. The deduced amino acid sequence of AoAcb2 lacked a signal peptide. Phylogenetic analysis classified AoAcb2 into a clade that was same as the ACBP Acb1 of the model yeast Saccharomyces cerevisiae , but was different from that of AoAcb1. In contrast to punctate localization of AoAcb1, AoAcb2 was found to be dispersedly distributed in the cytoplasm, as was previously observed for the S. cerevisiae Acb1. Since we could not generate an Aoacb2 disruptant, we created an Aoacb2 conditional mutant that exhibited less growth under Aoacb2 -repressed condition, suggesting that Aoacb2 is an essential gene for growth. Moreover, we observed that A. oryzae AoAcb2, but not A. oryzae AoAcb1, was secreted under carbon-starved condition, suggesting that AoAcb2 might be secreted via the unconventional protein secretion (UPS) pathway, just like S. cerevisiae Acb1. We also demonstrated that the unconventional secretion of AoAcb2 was dependent on the t-SNARE AoSso1, but was independent of the autophagy-related protein AoAtg1, suggesting that the unconventional secretion of AoAcb2, unlike that of S. cerevisiae Acb1, via the UPS pathway, is not regulated by the autophagy machinery. Thus, the filamentous fungus A. oryzae harbors two types of ACBPs, one of which appears to be essential for growth and undergoes unconventional secretion. [ABSTRACT FROM AUTHOR]

Fibroblast Growth Factor 2 (FGF2) is a potent cell survival factor involved in tumour-induced angiogenesis. FGF2 is secreted from cells through an unconventional secretory mechanism based upon direct translocation across the plasma membrane. The molecular mechanism underlying this process depends on a surprisingly small set of trans-acting factors that are physically associated with the plasma membrane. FGF2 membrane translocation is mediated by the ability of FGF2 to oligomerise and to insert into the plasma membrane in a PI(4,5)P2-dependent manner. Membrane-inserted FGF2 oligomers are dynamic translocation intermediates that are disassembled at the extracellular leaflet mediated by membrane proximal heparan sulphate proteoglycans. This process results in the exposure of FGF2 on cell surfaces as part of its unconventional mechanism of secretion. Although the trans-acting factors and cis-elements in FGF2 required for unconventional secretion have been known for a while, the core mechanism of this mysterious process has now been reconstituted with purified components establishing the molecular basis of FGF2 secretion from tumour cells. [ABSTRACT FROM AUTHOR]

Secretory proteins are conventionally transported through the endoplasmic reticulum to the Golgi and then to the plasma membrane where they are released into the extracellular space. However, numerous substrates also reach these destinations using unconventional pathways. Unconventional protein secretion (UPS) is complex and comprises cargos without a signal peptide or a transmembrane domain that can translocate across the plasma membrane, and cargos that reach the plasma membrane by bypassing the Golgi despite entering the endoplasmic reticulum (ER). With a few exceptions, unconventional secretion is largely triggered by stress. Here I review new results and concepts that are beginning to define these pathways. [ABSTRACT FROM AUTHOR]

Vasohibins (VASH1 and VASH2) are recently identified regulators of angiogenesis and cancer cell functions. They are secreted proteins without any classical secretion signal sequences, and are thought to be secreted instead via an unconventional protein secretion (UPS) pathway in a small vasohibin-binding protein (SVBP)-dependent manner. However, the precise mechanism of SVBP-dependent UPS is poorly understood. In this study, we identified a novel UPS regulatory system in which essential domain architecture (VASH-PS) of VASHs, comprising regions VASH191-180 and VASH280-169, regulate the cytosolic punctate structure formation in the absence of SVBP. We also demonstrate that SVBP form a complex with VASH1 through the VASH1274-282 (SIa), VASH1139-144 (SIb), and VASH1133-137 (SIc), leading to the dispersion in the cytosol and extracellular release of VASH1. The amino acid sequences of VASH-SIa and VASH-PS, containing SIb and SIc, are highly conserved among VASH family members in vertebrates, suggesting that SVBP-dependent UPS may be common within the VASH family. This novel UPS regulatory system may open up new avenues for understanding fundamental protein secretion in vertebrates. [ABSTRACT FROM AUTHOR]

Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes. [ABSTRACT FROM AUTHOR]

DBI/ACBP (diazepam binding protein, acyl-CoA binding protein) participates in the regulation of fatty acid metabolism when it is localized within cells, whereas outside of cells it acts as a diazepam-binding protein. Recent results indicate that many different mammalian cell types release DBI/ACBP upon in vitro or in vivo starvation in a macroautophagy/autophagy-dependent fashion. The autophagy-associated release of DBI/ACBP elicits feedback inhibition of autophagy through 3 independent mechanisms. First, the depletion of DBI/ACBP from cells limits autophagy in a cell-autonomous fashion. Second, extracellular DBI/ACBP acts in a paracrine fashion to inhibit autophagy. Third, DBI/ACBP increasing in the systemic circulation acts as an activator of lipo-anabolism and feeding behavior, thus removing the cause of autophagy induction (starvation) and suppressing the phenomenon. DBI/ACBP expression is upregulated at the mRNA and protein levels in obese mice and humans, and its extracellular neutralization by antibodies controls food intake and increases lipo-catabolism. Current data support the contention that DBI/ACBP is an important pro-obesity factor. Abbreviations: DBI: diazepam binding protein, acyl-CoA binding protein; GABR: gamma-aminobutyric acid type A receptor; TSPO: translocator protein [ABSTRACT FROM AUTHOR]

In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparing the predictive performance against non-secretory proteins indicates that commonly used threshold scores of 0.5 and 0.6 result in false-positive rates in excess of 0.3 when applied to plants proteins. Setting the false-positive rate to 0.05, consistent with the original mammalian performance of SecretomeP, yields only a marginally higher true positive rate compared to false positives. Therefore the use of SecretomeP on plant proteins is not recommended. This study investigates the trade-offs of using SecretomeP on plant proteins and provides insights into predictive features for future development of plant-specific common-feature tools.

Protein secretion in general depends on signal sequence (also named leader sequence), a hydrophobic segment located at or close to the NH2-terminus of a secretory or membrane protein. This sequence guides the entry of nascent polypeptides into the lumen or membranes of the endoplasmic reticulum (ER) for folding, assembly, and export. However, evidence accumulated in recent years has suggested the existence of a collection of unconventional protein secretion (UPS) mechanisms that are independent of the canonical vesicular trafficking route between the ER and the plasma membrane (PM). These UPS mechanisms export soluble proteins bearing no signal sequence. The list of UPS cargos is rapidly expanding, along with the implicated biological functions, but molecular mechanisms accountable for the secretion of leaderless proteins are still poorly defined. This review summarizes our current understanding of UPS mechanisms with an emphasis on the emerging role of endo-lysosomes in this process.

Among multiple distinct isoforms, Nrf1D is synthesized from a de novo translation of an alternatively-spliced transcript of Nrf1 mRNA, as accompanied by a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the generation of a reading frameshift mutation, which results in a constitutive substitution of the intact Nrf1’s C-terminal 72 amino acids (aa, covering the second half of the leucine zipper motif to C-terminal Neh3L domain) by an additional extended 80-aa stretch to generate a unique variant Nrf1D. The C-terminal extra 80-aa region of Nrf1D was herein identified to be folded into a redox-sensitive transmembrane domain, enabling it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D enables it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with a lesser ability than wild-type Nrf1 to mediate target gene expression. Further evidence has also been presented revealing that both mRNA and protein levels of Nrf1D, together with other isoforms similar to those of Nrf1, were detected to varying extents in hemopoietic and somatic tissues. Surprisingly, we found the existence of Nrf1D-derived isoforms in blood plasma, implying that it is a candidate secretory transcription factor, albeit its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies across membranes, before being unleashed from the ER to enter the blood.

In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparing the predictive performance against non-secretory proteins indicates that commonly used threshold scores of 0.5 and 0.6 result in false-positive rates in excess of 0.3 when applied to plants proteins. Setting the false-positive rate to 0.05, consistent with the original mammalian performance of SecretomeP, yields only a marginally higher true positive rate compared to false positives. Therefore the use of SecretomeP on plant proteins is not recommended. This study investigates the trade-offs of using SecretomeP on plant proteins and provides insights into predictive features for future development of plant-specific common-feature tools. [ABSTRACT FROM AUTHOR]

Transglutaminases (denoted TG or TGM) are externalized from cells via an unknown unconventional secretory pathway. Here, we show for the first time that purinergic signaling regulates active secretion of TG2 (also known as TGM2), an enzyme with a pivotal role in stabilizing extracellular matrices and modulating cell-matrix interactions in tissue repair. Extracellular ATP promotes TG2 secretion by macrophages, and this can be blocked by a selective antagonist against the purinergic receptor P2X7 (P2X7R, also known as P2RX7). Introduction of functional P2X7R into HEK293 cells is sufficient to confer rapid, regulated TG2 export. By employing pharmacological agents, TG2 release could be separated from P2X7R-mediated microvesicle shedding. Neither Ca2+ signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement. A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity. These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions. [ABSTRACT FROM AUTHOR]

N-terminal signal peptides are a hallmark of the vast majority of soluble secretory proteins that are transported along the endoplasmic reticulum/Golgi-dependent pathway. They are recognized by signal recognition particle, a process that initiates membrane translocation into the lumen of the endoplasmic reticulum followed by vesicular transport to the cell surface and release into the extracellular space. Beyond this well-established mechanism of protein secretion from eukaryotic cells, a number of extracellular proteins with critical physiological functions in immune surveillance and tissue organization are known to be secreted in a manner independent of signal recognition particle. Such processes have collectively been termed “unconventional protein secretion” and, while known for more than two decades, their underlying mechanisms are only beginning to emerge. Different types of unconventional secretory mechanisms have been described with the best-characterized example being based on direct translocation of cytoplasmic proteins across plasma membranes. The aim of this review is to critically assess our current knowledge of this type of unconventional secretion focusing on fibroblast growth factor 2 (FGF2) as the most established example. [ABSTRACT FROM AUTHOR]