Unconventional Secretory Pathway Research Articles

Necrotic cell death increases the release of macrophage migration inhibitory factor by monocytes/macrophages.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory molecule with both cytokine and noncytokine activity. MIF is constitutively released from multiple cell types via an unconventional secretory pathway that is not well defined. Here, we looked at MIF release from human and mouse monocytes/macrophages in response to different stimuli. While MIF release was not significantly altered in response to lipopolysaccharide or heat-killed Escherichia coli, cytotoxic stimuli strongly promoted release of MIF. MIF release was highly upregulated in cells undergoing necrosis, necroptosis and NLRP3 inflammasome-dependent pyroptosis. Our data suggest that cell death represents a major route for MIF release from myeloid cells. The functional significance of these findings and their potential importance in the context of autoimmune and inflammatory diseases warrant further investigation.

GRASP55: A Multifunctional Protein.

GRASP55 was first found as Golgi cisternae stacking protein. Due to the crucial role of Golgi in vesicular trafficking and protein modification, GRASP55 was found to function in these two aspects. Further investigation revealed that GRASP55 also participates in the unconventional secretory pathway under stress. Moreover, GRASP55 is involved in autophagy initiation and autophagosome maturation, as well as cell activity.

Gasdermin D-independent release of interleukin-1β by living macrophages in response to mycoplasmal lipoproteins and lipopeptides.

Interleukin-1β (IL-1β) plays pivotal roles in controlling bacterial infections and is produced after the processing of pro-IL-1β by caspase-1, which is activated by the inflammasome. In addition, caspase-1 cleaves the cytosolic protein, gasdermin-D (GSDMD), whose N-terminal fragment subsequently forms a pore in the plasma membrane, leading to the pyroptic cell-death-mediated release of IL-1β. Living cells can also release IL-1β via GSDMD pores or other unconventional secretory pathways. However, the precise mechanisms are poorly defined. Here, we show that lipoproteins from Mycoplasma salivarium (MsLP) and Mycoplasma pneumoniae (MpLP) and an M.salivarium-derived lipopeptide (FSL-1), which are activators of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, induce IL-1β release from mouse bone-marrow-derived macrophages (BMMs) without inducing cell death. The levels of IL-1β release induced by MsLP, MpLP and FSL-1 were more than 100 times lower than those induced by the canonical NLRP3 activator nigericin. The IL-1β release-inducing activities of MsLP, MpLP and FSL-1 were not attenuated in BMMs from GSDMD-deficient mice. Furthermore, both active caspase-1 and cleaved GSDMD were detected in response to transfection of FSL-1 into the cytosol of BMMs, but the release of IL-1β was unaffected by GSDMD deficiency. Meanwhile, punicalagin, a membrane-stabilizing agent, drastically down-regulated the release of IL-1β in response to FSL-1. These results suggest that mycoplasmal lipoprotein/lipopeptide-induced IL-1β release by living macrophages is not mediated via GSDMD but rather through changes in membrane permeability.

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1.

Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis, for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments indicate that the two proteins might interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor for Cts1.

Investigating the mechanisms of BK polyomavirus egress and virus-host interactions

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that infects 70-90% of the world's population and causes a lifelong, silently persistent infection. In immunocompromised individuals, BKPyV replication can result in serious pathology. Bone marrow transplant patients can develop a haemorrhagic cystitis, and in kidney transplant patients BKPyV replication can provoke a nephropathy that leads to deterioration of allograft function and eventual loss of the transplanted organ. There are currently no antiviral treatments with clinical efficacy against BKPyV associated nephropathy. While the life cycles of non-enveloped viruses are often assumed to require cell lysis to release progeny virions, we have evidence to suggest that BKPyV exits the cell via non-lytic means using an unconventional secretory pathway. We have investigated the effects of knocking out cellular genes thought to be involved in unconventional secretory pathways that bypass the Golgi apparatus on the release of BKPyV. We observe decreased BKPyV release from cells that have undergone CRISPR-mediated knockout of Golgi Reassembly Stacking Protein (GORASP) 1 or 2. Investigation of BKPyV-induced changes to the plasma membrane of infected cells demonstrated increased cell surface expression of transmembrane proteins normally resident in the endoplasmic reticulum. This appears to be inhibited by the knockout of GORASP1 or 2, suggesting that virions and ER markers are secreted via a common pathway in infected cells. These experiments are uncovering novel virus-host interactions that, when targeted, could help prevent BKPyV-associated nephropathy and allograft loss.

Oligodendrocytes Provide Antioxidant Defense Function for Neurons by Secreting Ferritin Heavy Chain

An evolutionarily conserved function of glia is to provide metabolic and structural support for neurons. To identify molecules generated by glia and with vital functions for neurons, we used Drosophila melanogaster as a screening tool, and subsequently translated the findings to mice. We found that a cargo receptor operating in the secretory pathway of glia was essential to maintain axonal integrity by regulating iron buffering. Ferritin heavy chain was identified as the critical secretory cargo, required for the protection against iron-mediated ferroptotic axonal damage. In mice, ferritin heavy chain is highly expressed by oligodendrocytes and secreted by employing an unconventional secretion pathway involving extracellular vesicles. Disrupting the release of extracellular vesicles or the expression of ferritin heavy chain in oligodendrocytes causes neuronal loss and oxidative damage in mice. Our data point to a role of oligodendrocytes in providing an antioxidant defense system to support neurons against iron-mediated cytotoxicity.

Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

ABSTRACT The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between Krt14+/+-Atg5f/f KCs and Krt14Cre/+-atg5f/f KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in Krt14Cre/+-atg5f/f KCs in vivo. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that Krt14Cre/+-hmgb1f/f mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AGER: advanced glycosylation end-product specific receptor; Anti-HMGB1: anti-HMGB1 neutralizing antibody; Anti-IL18: anti-IL18 neutralizing antibody; Anti-IL1B: anti-IL1B neutralizing antibody; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CASP1: caspase 1; CCL: C-C motif chemokine ligand; CsA: cyclosporine A; ctrl shRNA: lentivirus harboring shRNA against control; CXCL: C-X-C motif chemokine ligand; DCs: dendritic cells; DMEM: dulbecco’s modified Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; FBS: fetal bovine serum; GORASP2 shRNA: lentivirus harboring shRNA against GORASP2; GORASP2/GRASP55: golgi reassembly stacking protein 2; GR1: a composite epitope between LY6 (lymphocyte antigen 6 complex) locus C1 and LY6 locus G6D antigens; H&E: hematoxylin and eosin; HMGB1: high mobility group box 1; HMGB1 shRNA: lentivirus harboring shRNA against HMGB1; IFNG/IFN-γ: interferon gamma; IL17A: interleukin 17A; IL18: interleukin 18; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL22/IL-22: interleukin 22; IL23A: interleukin 23 subunit alpha; IL23R: interleukin 23 receptor; IMQ: imiquimod; ITGAM/CD11B: integrin subunit alpha M; ITGAX/CD11C: integrin subunit alpha X; IVL: involucrin; KC: keratinocyte; KD: knockdown; KO: knockout; Krt14+/+-Atg5f/f mice: mice bearing an Atg5 flox allele, in which exon 3 of the Atg5 gene is flanked by two loxP sites; Krt14+/+-Hmgb1f/f : mice bearing an Hmgb1 flox allele, in which exon 2 to 4 of the Hmgb1 gene is flanked by two loxP sites; Krt14Cre/+-atg5f/f mice: keratinocyte-specific atg5 knockout mice generated by mating Atg5-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt4; Krt14Cre/+-hmgb1f/f mice: keratinocyte-specific hmgb1 knockout mice generated by mating Hmgb1-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt14; Krt14-Vegfa mice: mice expressing 164-amino acid Vegfa splice variant recombinase under the control of promoter of Krt14; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; LORICRIN: loricrin cornified envelope precursor protein; M5: TNF, IL1A, IL17A, IL22 and OSM in combination; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MKI67: marker of proliferation Ki-67; MTT: thiazolyl blue tetrazolium bromide; NFKB/NF-κB: nuclear factor kappa B; NHEKs: primary normal human epidermal keratinocytes; NS: not significant; OSM: oncostatin M; PASI: psoriasis area and severity index; PtdIns3K: class III phosphatidylinositol 3-kinase; qRT-PCR: quantitative RT-PCR; RELA/p65: RELA proto-oncogene, NF-kB subunit; rHMGB1: recombinant HMGB1; rIL18: recombinant interleukin 18; rIL1B: recombinant interleukin 1 beta; S100A: S100 calcium binding protein A; SQSTM1/p62: sequestosome 1; T17: IL17A-producing T; TCR: T-cell receptor; tcrd KO mice: tcrd (T cell receptor delta chain) knockout mice, which show deficient receptor expression in all adult lymphoid and epithelial organs; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor; WOR: wortmannin; WT: wild-type; γδT17 cells: IL17A-producing γδ T cells.

The Cardiac Kv7.1-KCNE1 Channel Assembles at ER-PM Junctions before Translocated to the Plasma Membrane

The voltage-dependent potassium channel Kv7.1 assembles with the KCNE1 regulatory subunit recapitulating the cardiac IKs currents. Although the Kv7.1/KCNE1 complex receives much attention, the subcellular compartment hosting the interaction is the subject of intense debate. Two alternative hypothesis have been raised. Thus, the complex forms either earlier in the endoplasmic reticulum or directly at the plasma membrane. The importance of such issue is confirmed by Kv7.1 and KCNE1 mutations, responsible for long-QT syndromes, which impairing association and traffic alter IKs currents. Our work demonstrates that Kv7.1 and KCNE1 do not interact in the first stages of their biogenesis. Data support an unconventional secretory pathway for Kv7.1-KCNE1 that bypasses Golgi. This forward trafficking route drives channels to endoplasmic reticulum-plasma membrane junctions (ER-PM), where the Kv7.1-KCNE1 complex assembles. This mechanism helps to resolve the ongoing controversy about the subcellular compartment hosting the association. Our results provide new insights into IKs channel localization at ER-PM junctions, deciphering an alternative anterograde trafficking route for oligomeric ion channels.Supported by BFU2017-87104-R from the Ministerio de Economia y Competitividad (MINECO, Spain) and Fondo Europeo de Desarrollo Regional (FEDER).

The Multiple Facets of Plant-Fungal Interactions Revealed Through Plant and Fungal Secretomics.

The plant secretome is usually considered in the frame of proteomics, aiming at characterizing extracellular proteins, their biological roles and the mechanisms accounting for their secretion in the extracellular space. In this review, we aim to highlight recent results pertaining to secretion through the conventional and unconventional protein secretion pathways notably those involving plant exosomes or extracellular vesicles. Furthermore, plants are well known to actively secrete a large array of different molecules from polymers (e.g. extracellular RNA and DNA) to small compounds (e.g. ATP, phytochemicals, secondary metabolites, phytohormones). All of these play pivotal roles in plant-fungi (or oomycetes) interactions, both for beneficial (mycorrhizal fungi) and deleterious outcomes (pathogens) for the plant. For instance, recent work reveals that such secretion of small molecules by roots is of paramount importance to sculpt the rhizospheric microbiota. Our aim in this review is to extend the definition of the plant and fungal secretomes to a broader sense to better understand the functioning of the plant/microorganisms holobiont. Fundamental perspectives will be brought to light along with the novel tools that should support establishing an environment-friendly and sustainable agriculture.

OutCyte: a novel tool for predicting unconventional protein secretion

The prediction of protein localization, such as in the extracellular space, from high-throughput data is essential for functional downstream inference. It is well accepted that some secreted proteins go through the classic endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide. However, a large number of proteins have been found to reach the extracellular space by following unconventional secretory pathways. There remains a demand for reliable prediction of unconventional protein secretion (UPS). Here, we present OutCyte, a fast and accurate tool for the prediction of UPS, which for the first time has been built upon experimentally determined UPS proteins. OutCyte mediates the prediction of protein secretion in two steps: first, proteins with N-terminal signals are accurately filtered out; second, proteins without N-terminal signals are classified as UPS or intracellular proteins based on physicochemical features directly generated from their amino acid sequences. We are convinced that OutCyte will play a relevant role in the annotation of experimental data and will therefore contribute to further characterization of the extracellular nature of proteins by considering the commonly neglected UPS proteins.OutCyte has been implemented as a web server atwww.outcyte.com.

Unconventional secretion of \u03b1-Crystallin B requires the Autophagic pathway and is controlled by phosphorylation of its serine 59 residue

α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity. Surprisingly, several reports show that CRYAB exerts a protective role also extracellularly, and it has been recently demonstrated that CRYAB is secreted from human retinal pigment epithelial cells by an unconventional secretion pathway that involves multi-vesicular bodies. Here we show that autophagy is crucial for this unconventional secretion pathway and that phosphorylation at serine 59 residue regulates CRYAB secretion by inhibiting its recruitment to the autophagosomes. In addition, we found that autophagosomes containing CRYAB are not able to fuse with lysosomes. Therefore, CRYAB is capable to highjack and divert autophagosomes toward the exocytic pathway, inhibiting their canonical route leading to the lysosomal compartment. Potential implications of these findings in the context of disease-associated mutant proteins turn-over are discussed.

Complementing the intrinsic repertoire of Ustilago maydis for degradation of the pectin backbone polygalacturonic acid

Microbial valorization of plant biomass is a key target in bioeconomy. A promising candidate for consolidated bioprocessing is the dimorphic fungus Ustilago maydis. It harbors hydrolytic enzymes to degrade biomass components and naturally produces valuable secondary metabolites like itaconic acid, malic acid or glycolipids. However, hydrolytic enzymes are mainly expressed in the hyphal form. This type of morphology should be prevented in industrial fermentation processes. Genetic activation of these enzymes can enable growth on cognate substrates also in the yeast form. Here, strains were engineered for growth on polygalacturonic acid as major component of pectin. Besides activation of intrinsic enzymes, supplementation with heterologous genes for potent enzymes was tested. The presence of an unconventional secretion pathway allowed exploiting fungal and bacterial enzymes. Growth of the engineered strains was evaluated by a recently developed method for online determination of residual substrates based on the respiration activity. This enabled the quantification of the overall consumed substrate as a key asset for the assessment of the enzyme degradation potential even on polymeric substrates. Co-fermentation of endo- and exo-polygalacturonase overexpression strains resulted in efficient growth on polygalacturonic acid. In the future, the approach will be extended to establish efficient degradation and valorization of pectin.

Protein secretion in wild-type and Othac1 mutant strains of thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.

In yeasts, Hac1 transcription factor of the unfolded protein response (UPR) regulates many genes involved in secretory pathways. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a host for heterologous protein secretion. To understand the role of OtHac1 on the secretome of O. thermomethanolica, a comparative proteomic analysis using LC-MS/MS was employed to identify proteins with altered secretion levels when OtHac1 was mutated. 268 proteins were detected in the extracellular medium of O. thermomethanolica wild-type control and Othac1 mutant strains. A number of metabolic enzymes functioning in amino acid, carbohydrate, glycan, and lipid metabolism showed altered secretion in the mutant suggesting that OtHac1 may play a role in mediating extracellular metabolism. Most of the extracellular proteins identified do not contain canonical signal sequences suggesting that they are secreted via unconventional protein secretion pathways. Collectively, the data provide insights into protein secretion and OtHac1 function in O. thermomethanolica which will be useful for developing efficient host for protein production.

Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei

BackgroundFilamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production.ResultRecombinant Trichoderma reesei strains RBGL, RCBH, and RCMC were successfully constructed from T. reesei RUT-C30, expressing red fluorescent protein DsRed-tagged versions of β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion in T. reesei.ConclusionBased on recombinant T. reesei RBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production in T. reesei were first visualized directly in a dynamic way, preliminarily mapping the location and secretion of T. reesei cellulase and providing evidence for revealing the secretion pathways of cellulase in T. reesei. The obtained results suggest that cellulase excretion majorly occurs via the conventional ER–Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.

Unconventional secretory pathway activation restores hair cell mechanotransduction in an USH3A model

The pathogenic variant c.144T>G (p.N48K) in the clarin1 gene (CLRN1) results in progressive loss of vision and hearing in Usher syndrome IIIA (USH3A) patients. CLRN1 is predicted to be an essential protein in hair bundles, the mechanosensory structure of hair cells critical for hearing and balance. When expressed in animal models, CLRN1 localizes to the hair bundle, whereas glycosylation-deficient CLRN1N48K aggregates in the endoplasmic reticulum, with only a fraction reaching the bundle. We hypothesized that the small amount of CLRN1N48K that reaches the hair bundle does so via an unconventional secretory pathway and that activation of this pathway could be therapeutic. Using genetic and pharmacological approaches, we find that clarin1 knockout (clrn1KO/KO ) zebrafish that express the CLRN1c.144T>G pathogenic variant display progressive hair cell dysfunction, and that CLRN1N48K is trafficked to the hair bundle via the GRASP55 cargo-dependent unconventional secretory pathway (GCUSP). On expression of GRASP55 mRNA, or on exposure to the drug artemisinin (which activates GCUSP), the localization of CLRN1N48K to the hair bundles was enhanced. Artemisinin treatment also effectively restored hair cell mechanotransduction and attenuated progressive hair cell dysfunction in clrn1KO/KO larvae that express CLRN1c.144T>G , highlighting the potential of artemisinin to prevent sensory loss in CLRN1c.144T>G patients.

Autophagy-based unconventional secretion of alarmin HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

Abstract The precise mechanism by which autophagy affects psoriasis is poorly understood. Here we show that keratinocytes (KCs) autophagy was activated in psoriatic lesions of patients and mice model, positively correlating with psoriatic severity, and could be inhibited by MAPK family (ERK, p38, JNK) inactivation, implying autophagy was associated with psoriatic inflammation. Indeed, impaired autophagy flux, caused by autophagy inhibitors or KC-specific deletion of Atg5, alleviated psoriasisform inflammation, demonstrating autophagy positively regulated psoriatic inflammation. We then found an autophagy-based unconventional secretory pathway (autosecretion), depended on Atg5 and GRASP55, promoted the psoriasiform KCs inflammation. Moreover, alarmin HMGB1 was more effective than other autosecretory proteins, IL-1β and IL-18, to regulate the psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs vanished the psoriasiform inflammation differences between autophagy-efficient KCs and autophagy-deficient KCs. Conversely, recombinant HMGB1 almost completely restored the psoriasiform inflammation in autophagy-deficient KCs in vivo. These results suggested that HMGB1-associated autosecretion, not other intracellular autophagy pathways, played a pivotal role in cutaneous inflammation. Finally, we demonstrated that KC-specific HMGB1 deficient mice, but not DC- or myeloid cell-specific HMGB1 deficient mice, displayed attenuated psoriatic inflammation, due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, we uncover a novel autophagy mechanism in psoriasis pathogenesis, and imply its clinical significance in psoriasis treatment.

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.

Investigating the mechanisms of BK polyomavirus egress and virus-host interactions

BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that can establish a lifelong, silently persistent infection in the kidney and is estimated to infect 70–90 % of the world’s population. In immunocompromised individuals, particularly bone marrow and kidney transplant patients, increases in BKPyV replication can result in significant pathological conditions. In the case of kidney transplant patients, this can result in nephropathy, which in severe cases can result in the deterioration of allograft function and loss of the transplanted organ. There are currently no antiviral treatments with strong evidence of clinical efficacy against BKPyV. Though little is known about BKPyV egress from infected cells, we have evidence showing that BKPyV can be released in a non-lytic manner by an unconventional cellular secretory pathway that bypasses the Golgi apparatus. Here, we investigate the mechanisms behind BKPyV non-lytic egress through studying the effects of knocking out candidate host proteins involved in unconventional secretory pathways on BKPyV release, examining the effects of BKPyV infection on host cell protein secretion, and ascertaining which host proteins are essential for the BKPyV life cycle via a whole-genome CRISPR screen. These experiments are uncovering novel virus-host interactions that, when targeted, could lead to antiviral effects.

The Dengue Virus Nonstructural Protein 1 (NS1) Is Secreted from Mosquito Cells in Association with the Intracellular Cholesterol Transporter Chaperone Caveolin Complex.

Dengue virus (DENV) is a mosquito-borne virus of the family Flaviviridae The RNA viral genome encodes three structural and seven nonstructural proteins. Nonstructural protein 1 (NS1) is a multifunctional protein actively secreted in vertebrate and mosquito cells during infection. In mosquito cells, NS1 is secreted in a caveolin-1-dependent manner by an unconventional route. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40, and CyA and is responsible for the cholesterol traffic inside the cell. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 associates with and relies on the CCC for secretion. Treatment of mosquito cells with classic secretion inhibitors, such as brefeldin A, Golgicide A, and Fli-06, showed no effect on NS1 secretion but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV-1 or FKBP52 with short interfering RNAs or the inhibition of CyA by cyclosporine resulted in significant decrease in NS1 secretion, again without affecting virion release. Colocalization, coimmunoprecipitation, and proximity ligation assays indicated that NS1 colocalizes and interacts with all proteins of the CCC. In addition, CAV-1 and FKBP52 expression was found augmented in DENV-infected cells. Results obtained with Zika virus-infected cells suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway as that observed for DENV NS1. These results uncover important differences in the dengue virus-cell interactions between the vertebrate host and the mosquito vector as well as novel functions for the chaperone caveolin complex.IMPORTANCE The dengue virus protein NS1 is secreted efficiently from both infected vertebrate and mosquito cells. Previously, our group reported that NS1 secretion in mosquito cells follows an unconventional secretion pathway dependent on caveolin-1. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 secretion takes place in association with the chaperone caveolin complex, a complex formed by caveolin-1 and the chaperones FKBP52, CyA, and Cy40, which are in charge of cholesterol transport inside the cell. Results obtained with ZIKV-infected mosquito cells suggest that ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. These results uncover important differences in the virus-cell interactions between the vertebrate host and the mosquito vector, as well as novel functions for the chaperone caveolin complex. Moreover, manipulation of the NS1 secretory route may prove a valuable strategy to combat these two mosquito-borne diseases.

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.