Unconventional Protein Secretion Research Articles

Noncanonical Inflammasome-Induced release of Alarmins during Sepsis

Abstract Sepsis is a life-threatening disease caused by the immune system’s response to infection that results in excessive inflammation, organ damage and often death. In fact, sepsis is the leading cause of death in intensive care units and is responsible for more than 250,000 deaths every year in the USA. Activation of inflammatory caspases, such as caspase-11 by lipopolysaccharide (LPS) from Gram-negative bacteria, is a key mechanism of innate immune defense against infection. Intracellular sensing of LPS by caspase-11 results in pyroptosis, maturation of caspase-1, IL-1β and IL-18 and unconventional secretion of intracellular proteins that lack a leader sequence for conventional secretion via endoplasmic reticulum-Golgi route. These proteins can act as alarmins or damage associated molecular patterns (DAMPs) to regulate the inflammatory response and therefore play an important role during infectious diseases. However, caspase-11-dependent release of alarmins and DAMPs following noncanonical inflammasome activation is poorly characterized. Using a proteomics approach involving ProteomeLab’s PF2D fractionation system followed by mass spectrometry, we have profiled several new alarmins released in a caspase-11 dependent manner following Gram-negative bacterial infection. By employing various mutant mice and cells, we have defined the molecular and cellular mechanisms underlying the release of a candidate alarmin as a consequence of noncanonical inflammasome activation in vitro and in vivo. Importantly, our in vivo studies demonstrate a critical role for this alarmin in LPS shock. Overall, these findings provide new insights into potential biomarkers and therapeutic targets for blocking the lethal inflammation in sepsis.

Diverse Role of SNARE Protein Sec22 in Vesicle Trafficking, Membrane Fusion, and Autophagy.

Protein synthesis begins at free ribosomes or ribosomes attached with the endoplasmic reticulum (ER). Newly synthesized proteins are transported to the plasma membrane for secretion through conventional or unconventional pathways. In conventional protein secretion, proteins are transported from the ER lumen to Golgi lumen and through various other compartments to be secreted at the plasma membrane, while unconventional protein secretion bypasses the Golgi apparatus. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are involved in cargo vesicle trafficking and membrane fusion. The ER localized vesicle associated SNARE (v-SNARE) protein Sec22 plays a major role during anterograde and retrograde transport by promoting efficient membrane fusion and assisting in the assembly of higher order complexes by homodimer formation. Sec22 is not only confined to ER–Golgi intermediate compartments (ERGIC) but also facilitates formation of contact sites between ER and plasma membranes. Sec22 mutation is responsible for the development of atherosclerosis and symptoms in the brain in Alzheimer’s disease and aging in humans. In the fruit fly Drosophila melanogaster, Sec22 is essential for photoreceptor morphogenesis, the wingless signaling pathway, and normal ER, Golgi, and endosome morphology. In the plant Arabidopsis thaliana, it is involved in development, and in the nematode Caenorhabditis elegans, it is in involved in the RNA interference (RNAi) pathway. In filamentous fungi, it affects cell wall integrity, growth, reproduction, pathogenicity, regulation of reactive oxygen species (ROS), expression of extracellular enzymes, and transcriptional regulation of many development related genes. This review provides a detailed account of Sec22 function, summarizes its domain structure, discusses its genetic redundancy with Ykt6, discusses what is known about its localization to discrete membranes, its contributions in conventional and unconventional autophagy, and a variety of other roles across different cellular systems ranging from higher to lower eukaryotes, and highlights some of the surprises that have originated from research on Sec22.

Early endosome autoantigen 1 regulates IL-1\u03b2 release upon caspase-1 activation independently of gasdermin D membrane permeabilization

Unconventional protein secretion represents an important process of the inflammatory response. The release of the pro-inflammatory cytokine interleukin (IL)-1β which burst during pyroptosis as a consequence of gasdermin D plasma membrane pore formation, can also occur through other unconventional secretion pathways dependent on caspase-1 activation. However, how caspase-1 mediates cytokine release independently of gasdermin D remains poorly understood. Here we show that following caspase-1 activation by different inflammasomes, caspase-1 cleaves early endosome autoantigen 1 (EEA1) protein at Asp127/132. Caspase-1 activation also results in the release of the endosomal EEA1 protein in a gasdermin D-independent manner. EEA1 knock-down results in adecreased release of caspase-1 and IL-1β, but the pyroptotic release of other inflammasome components and lactate dehydrogenase was not affected. This study shows how caspase-1 control the release of EEA1 and IL-1β in a pyroptotic-independent manner.

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion.

When studying how HIV-1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor-α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase-regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)-dependent but was not associated with ER stress. These data indicate that Hck-activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom-dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.

Vesicular Pathways for Unconventional Secretion of Transmembrane Proteins

Vesicular Pathways for Unconventional Secretion of Transmembrane Proteins

Exploring the context of diacidic motif DE as a signal for unconventional protein secretion in eukaryotic proteins

Unconventional protein secretion (UPS) is an important phenomenon with fundamental implications to cargo export. How eukaryotic proteins transported by UPS are recognized without a conventional signal peptide has been an open question. It was recently observed that a diacidic amino acid motif (ASP-GLU or DE) is necessary for the secretion of superoxide dismutase 1 (SOD1) from yeast under nutrient starvation. Taking cue from this discovery, we explore the hypothesis of whether the diacidic motif DE, which can occur fairly ubiquitously, along with its context, can be a generic signal for unconventional secretion of proteins. Four different contexts were evaluated: a physical context encompassing the structural order and charge signature in the neighbourhood of DE, two signalling contexts reflecting the presence of either a phosphorylatable amino acid (‘X’ in XDE, DXE, DEX) or an LC3 interacting region (LIR) which can trigger autophagy and a co-evolutionary constraint relative to other amino acids in the protein interpreted by examining sequences across different species. Among the 100 proteins we curated from different physiological or pathological conditions, we observe a pattern in the unconventional secretion of heat shock proteins in the cancer secretome, where DE in an ordered structural region has higher odds of being a UPS signal.

The Chemical Chaperone 4-Phenylbutyrate Increases Secretion and Activity of Missense and Elongated Factor VII Mutants

The F7 gene mutations p.Q160R and p.A354V-p.P464Hfs are associated with very low factor VII (FVII) activity and antigen levels and a bleeding phenotype. We have previously demonstrated reduced secretion of the recombinant (r) variants rFVII-160R and rFVII-354V-464Hfs in vitro, possibly caused by a misfolding mechanism resulting in intracellular retention and increased endoplasmic reticulum (ER) stress. Chemical chaperones are compounds that can enable proteins to recover from a misfolded state and rescue intracellular processing. The aim of the study was to determine whether chemical chaperones could increase the secretion and enhance the biological activity of the above mentioned FVII mutants.Chinese hamster ovary (CHO-K1) cells stably expressing rFVII-160R or rFVII-354V-464Hfs were treated with the chemical chaperones 4-phenylbutyrate (4-PBA), betaine, taurine, taurourosdeoxycholic acid (TUDCA), trimethylamine N-oxide (TMAO) or lumacaftor (VX-809). The intracellular and secreted levels of FVII antigen were measured by ELISA and F7 mRNA levels were assessed by quantitative RT-PCR. Treatment of cells with 4-PBA for 48 hours increased F7 mRNA levels and increased the ratio of secreted/intracellular of both FVII mutants by ~2-2.5-fold, indicating an effect of 4-PBA FVII expression and secretion.4-PBA is known to inhibit the activity of histone deacetylases, thereby inducing hyperacetylation of histones and increasing gene transcription. To measure histone acetylation, we next performed Western blot analysis of acetylated histone H3 (AcH3). We found that the levels of AcH3 were significantly increased after treatment with 4-PBA, indicating that induced transcription of F7 by 4-PBA could be a result of increased global histone acetylation. Since 4-PBA treatment increased the secretion of the FVII mutants, we questioned whether this also would alleviate ER stress. Using an ER stress luciferase assay, we observed that the ER stress levels in 4-PBA treated cells were significantly lower than in non-treated cells.To determine whether 4-PBA treatment could restore the intracellular trafficking of the FVII mutants from the ER to Golgi, confocal immunofluorescence microscopy was utilized The studies revealed that upon treatment with 4-PBA, the p.Q160R mutant had a strong co-localization with the Golgi marker GM130 and with the Golgi stacking protein GRASP55, known to be associated with unconventional protein secretion. A vesicular pattern of staining could be observed in cells expressing the p.A354V-p.P464Hfs. However, these structures did not overlap with endosomal markers or markers of the ER-Golgi transport remaining the secretory route of this FVII mutant elusive.Finally, to assess the activity of FVII mutants, we measured the generation of activated (a) factor X (FXaG) in FVII deficient plasma supplemented with conditioned medium from cells stably expressing rFVII mutants before and after 4-PBA treatment. To increase the assay sensitivity to very low FVII activity levels we also boosted the coagulation initiation phase by inhibiting the tissue factor pathway inhibitor (TFPI) through an anti-TFPI RNA aptamer. We found that treatment of cells with 4-PBA gave rise to a modest, but measurable FXaG activity in conditioned medium for both FVII mutants. Furthermore, inhibition of TFPI proportionally potentiated FXaG activity of both 4-PBA-induced FVII variants.The present study demonstrates that chemical chaperones, such as 4-PBA, can increase intracellular trafficking through the secretory pathway of misfolded FVII mutants. Importantly, the secreted mutant proteins were found to be biologically regulated and active in the coagulation pathway. Thus, 4-PBA could have therapeutic effect in FVII deficiency caused by misfolded FVII mutants. DisclosuresNo relevant conflicts of interest to declare.

Exploring the context of diacidic motif DE as a signal for unconventional protein secretion in eukaryotic proteins.

Unconventional protein secretion (UPS) is an important phenomenon with fundamental implications to cargo export. How eukaryotic proteins transported by UPS are recognized without a conventional signal peptide has been an open question. It was recently observed that a diacidic amino acid motif (ASP-GLU or DE) is necessary for the secretion of superoxide dismutase 1 (SOD1) from yeast under nutrient starvation. Taking cue from this discovery, we explore the hypothesis of whether the diacidic motif DE, which can occur fairly ubiquitously, along with its context, can be a generic signal for unconventional secretion of proteins. Four different contexts were evaluated: a physical context encompassing the structural order and charge signature in the neighbourhood of DE, two signalling contexts reflecting the presence of either a phosphorylatable amino acid ('X' in XDE, DXE, DEX) or an LC3 interacting region (LIR) which can trigger autophagy and a co-evolutionary constraint relative to other amino acids in the protein interpreted by examining sequences across different species. Among the 100 proteins we curated from different physiological or pathological conditions, we observe a pattern in the unconventional secretion of heat shock proteins in the cancer secretome, where DE in an ordered structural region has higher odds of being a UPS signal.

The Roles of Endo-Lysosomes in Unconventional Protein Secretion.

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.

Evaluation of Unconventional Protein Secretion by Saccharomyces cerevisiae and other Fungi

Development of proteome analysis of extracellular proteins has revealed that a wide variety of proteins, including fungal allergens are present outside the cell. These secreted allergens often do not contain known secretion signal sequences. Recent research progress shows that some fungal allergens are secreted by unconventional secretion pathways, including autophagy- and extracellular-vesicle-dependent pathways. However, secretion pathways remain unknown for the majority of extracellular proteins. This review summarizes recent data on unconventional protein secretion in Saccharomyces cerevisiae and other fungi. Particularly, methods for evaluating unconventional protein secretion are proposed for fungal species, including S. cerevisiae, a popular model organism for investigating protein secretion pathways.

Abstract 24: Extracellular HMGA1 promotes tumor invasion and metastasis in breast cancer

Abstract The recent in-depth study of the cancer secretome suggests that a fraction of the intracellular proteome might be playing alternative roles in the extracellular space during tumorigenesis through its unconventional secretion. A proteomic study aimed at evaluating the role of unconventional protein secretion in the context of tumor invasion led us to investigate the high mobility group A1 (HMGA1) chromatin-binding protein whose overexpression has been correlated with high-grade carcinomas and reduced patient-survival. Here we show that HMGA1 is not only oversecreted in invasive breast tumor cells, but its blockage with specific antibodies in the extracellular space impairs tumor cell migration and invasion. The invasive phenotype caused by extracellular HMGA1 (eHMGA1) is dependent on ERK1/2 signaling and a blocking-Ab against eHMGA1 decreases pERK and re-localizes HMGA1 to the nucleus concomitantly to the loss of invasion capacity. Remarkably, pre-treatment of invasive breast tumor cells with an HMGA1-blocking Ab reduces their metastatic ability in an orthotopic breast cancer xenograft model. HMGA1 also shows a cytoplasmic localization in primary human triple-negative breast cancer (TNBC) tumors that developed distant metastasis while displaying a nuclear localization in TNBC tumors that did not progress to metastatic disease. Thus, eHMGA1 could be a novel drug target in aggressive breast cancer and a biomarker predicting the onset of distant metastasis. Citation Format: Olga Méndez, Vicente Peg, Candida Salvans, Mireia Pujals, Yolanda Fernández, Ibane Abasolo, Jose Perez, Ana Matres, Josep Tabernero, Javier Cortés, Joaquín Arribas, Josep Villanueva. Extracellular HMGA1 promotes tumor invasion and metastasis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 24.

Unconventional protein secretion - new insights into the pathogenesis and therapeutic targets of human diseases.

Most secretory proteins travel through a well-documented conventional secretion pathway involving the endoplasmic reticulum (ER) and the Golgi complex. However, recently, it has been shown that a significant number of proteins reach the plasma membrane or extracellular space via unconventional routes. Unconventional protein secretion (UPS) can be divided into two types: (i) the extracellular secretion of cytosolic proteins that do not bear a signal peptide (i.e. leaderless proteins) and (ii) the cell-surface trafficking of signal-peptide-containing transmembrane proteins via a route that bypasses the Golgi. Understanding the UPS pathways is not only important for elucidating the mechanisms of intracellular trafficking pathways but also has important ramifications for human health, because many of the proteins that are unconventionally secreted by mammalian cells and microorganisms are associated with human diseases, ranging from common inflammatory diseases to the lethal genetic disease of cystic fibrosis. Therefore, it is timely and appropriate to summarize and analyze the mechanisms of UPS involvement in disease pathogenesis, as they may be of use for the development of new therapeutic approaches. In this Review, we discuss the intracellular trafficking pathways of UPS cargos, particularly those related to human diseases. We also outline the disease mechanisms and the therapeutic potentials of new strategies for treating UPS-associated diseases.

Analysis of an acyl-CoA binding protein in Aspergillus oryzae that undergoes unconventional secretion

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.

The endoplasmic reticulum is a hub to sort proteins toward unconventional traffic pathways and endosymbiotic organelles.

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.

Merging roads: chemical tools and cell biology to study unconventional protein secretion.

The endomembrane trafficking network is highly complex and dynamic, with both conventional and so-called unconventional routes which are in essence recently discovered pathways that are poorly understood in plants. One approach to dissecting endomembrane pathways that we have pioneered is the use of chemical biology. Classical genetic manipulations often deal with indirect pleiotropic phenotypes resulting from the perturbation of key players of the trafficking routes. Many of these difficulties can be circumvented using small molecules to modify or disrupt the function or localization of key proteins regulating these pathways. In this review, we summarize how small molecules have been used as probes to define these pathways, and how they could be used to increase current knowledge of unconventional protein secretion pathways.

Protein secretion in plants: conventional and unconventional pathways and new techniques.

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.

Caspase-8 controls the secretion of inflammatory lysyl-tRNA synthetase in exosomes from cancer cells.

Aminoacyl-tRNA synthetases (ARSs), enzymes that normally control protein synthesis, can be secreted and have different activities in the extracellular space, but the mechanism of their secretion is not understood. This study describes the secretion route of the ARS lysyl-tRNA synthetase (KRS) and how this process is regulated by caspase activity, which has been implicated in the unconventional secretion of other proteins. We show that KRS is secreted from colorectal carcinoma cells within the lumen of exosomes that can trigger an inflammatory response. Caspase-8 cleaved the N-terminal of KRS, thus exposing a PDZ-binding motif located in the C terminus of KRS. Syntenin bound to the exposed PDZ-binding motif of KRS and facilitated the exosomic secretion of KRS dissociated from the multi-tRNA synthetase complex. KRS-containing exosomes released by cancer cells induced macrophage migration, and their secretion of TNF-α and cleaved KRS made a significant contribution to these activities, which suggests a novel mechanism by which caspase-8 may promote inflammation.

Unconventional Secretion of Heat Shock Proteins in Cancer.

Heat shock proteins (HSPs) are abundant cellular proteins involved with protein homeostasis. They have both constitutive and inducible isoforms, whose expression levels are further increased by stress conditions, such as temperature elevation, reduced oxygen levels, infection, inflammation and exposure to toxic substances. In these situations, HSPs exert a pivotal role in offering protection, preventing cell death and promoting cell recovery. Although the majority of HSPs functions are exerted in the cytoplasm and organelles, several lines of evidence reveal that HSPs are able to induce cell responses in the extracellular milieu. HSPs do not possess secretion signal peptides, and their secretion was subject to widespread skepticism until the demonstration of the role of unconventional secretion forms such as exosomes. Secretion of HSPs may confer immune system modulation and be a cell-to-cell mediated form of increasing stress resistance. Thus, there is a wide potential for secreted HSPs in resistance of cancer therapy and in the development new therapeutic strategies.

Loss of Cln3 impacts protein secretion in the social amoeba Dictyostelium

Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3− cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3− cells displayed delayed streaming and aggregation, and interestingly, cln3− cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3− CM, of which 3 were absent in cln3− CM. Moreover, 12 proteins that were present in cln3− CM were absent in WT CM. Label-free quantification identified 42 proteins that were present in significantly higher amounts in cln3− CM compared to WT, and 3 proteins that were present in significantly reduced amounts. A GO term enrichment analysis showed that a majority of the affected proteins are linked to endocytosis, vesicle-mediated transport, proteolysis, and metabolism. In total, the results of this study indicate that Cln3 functions in both conventional and unconventional protein secretion and that loss of Cln3 results in deregulated secretion during early development. Importantly, this is the first evidence in any system linking CLN3 function to protein secretion.

Domain architecture of vasohibins required for their chaperone-dependent unconventional extracellular release.

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.