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Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.

The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.

This review highlights current knowledge on membrane trafficking that mediates resistance to powdery mildew fungi and probably involves the release of exosomes. Abstract The ability to ward off filamentous pathogens, such as powdery mildew fungi, is one of the best studied examples of membrane trafficking-dependent disease resistance in plants. Here, papilla formation at the site of attack is essential for the pre-invasive immunity, whereas the encasement can hamper disease post-invasively. Exosomes containing antifungal peptides and small RNAs are thought to play a vital role in forming papillae and encasements that block fungal growth. While exosomes are well described in mammals, and have been shown to play important roles in cell-cell communication regulating development and disease, their function is not well-known in plants. In this review, we focus on some of the recent discoveries on plant exosomes and try to link this information with our current understanding of how plants use this form of unconventional secretion to acquire this durable and effective form of resistance.

Autophagy controls the quality and quantity of the eukaryotic cytoplasm while performing two evolutionarily highly conserved functions: cell‐autonomous provision of energy and nutrients by cytosol autodigestion during starvation, and removal of defunct organelles and large aggregates exceeding the capacity of other cellular degradative systems. In contrast to these autodigestive processes, autophagy in yeast has additional, biogenesis functions. However, no equivalent biosynthetic roles have been described for autophagy in mammals. Here, we show that in mammalian cells, autophagy has a hitherto unappreciated positive contribution to the biogenesis and secretion of the proinflammatory cytokine IL‐1β via an export pathway that depends on Atg5, inflammasome, at least one of the two mammalian Golgi reassembly stacking protein (GRASP) paralogues, GRASP55 (GORASP2) and Rab8a. This process, which is a type of unconventional secretion, expands the functional manifestations of autophagy beyond autodigestive and quality control roles in mammals. It enables a subset of cytosolic proteins devoid of signal peptide sequences, and thus unable to access the conventional pathway through the ER, to enter an autophagy‐based secretory pathway facilitating their exit from the cytoplasm. Autophagy is a central process within the cell that plays roles well beyond autodigestion and quality control. This study reveals that autophagy is involved in unconventional secretion of the proinflammatory cytokine IL‐1β, a pathway that involves Atg5, inflammasome, GRASP55 (GORASP2), and Rab8a.

Recent evidence suggests that autophagy facilitates the unconventional secretion of the pro-inflammatory cytokine interleukin 1β (IL-1β). Here, we reconstituted an autophagy-regulated secretion of mature IL-1β (m-IL-1β) in non-macrophage cells. We found that cytoplasmic IL-1β associates with the autophagosome and m-IL-1β enters into the lumen of a vesicle intermediate but not into the cytoplasmic interior formed by engulfment of the autophagic membrane. In advance of secretion, m-IL-1β appears to be translocated across a membrane in an event that may require m-IL-1β to be unfolded or remain conformationally flexible and is dependent on two KFERQ-like motifs essential for the association of IL-1β with HSP90. A vesicle, possibly a precursor of the phagophore, contains translocated m-IL-1β and later turns into an autophagosome in which m-IL-1β resides within the intermembrane space of the double-membrane structure. Completion of IL-1β secretion requires Golgi reassembly and stacking proteins (GRASPs) and multi-vesicular body (MVB) formation. Cells release a large number of proteins to the extracellular space. The majority of these ‘secreted’ proteins first pass through two structures inside cells called the endoplasmic reticulum and Golgi. However, a growing number of proteins have been identified that are released by an unconventional mechanism that bypasses the endoplasmic reticulum and Golgi. Autophagy is a process that destroys damaged proteins and other unwanted material in cells. It gets triggered when cells are starved of nutrients, leading them to digest their own materials and recycle the resources into new molecules. During autophagy, a cup-like structure with a double layer of membrane forms around the material that is to be digested. This structure then elongates and eventually engulfs the material to form a bubble-like compartment called the autophagosome. Recent evidence has suggested that autophagosomes are involved in the unconventional secretion of a protein called interleukin-1β; this protein is crucial for the body’s immune response against infection. However, it was not clear how these proteins entered the autophagosomes. Zhang et al. have now explored the link between interleukin-1β and autophagy in more detail. The experiments showed that when autophagy was triggered by starvation, the secretion of interleukin-1β was enhanced. Conversely, when autophagy was inhibited, interleukin-1β accumulated inside the cells and could not be secreted. Further experiments then revealed unexpectedly that interleukin-1β was not engulfed by the cup-like structure (as is the case for material that is destined to be removed). Instead, interleukin-1β was found to enter into smaller bubble-like packages (called vesicles) that turn into the autophagosome. Zhang et al. also found that a protein called HSP90 binds to interleukin-1β and enables it to cross the membrane (or translocate) into the vesicles, and that this means that interleukin-1β actually resides in the space between the outer and inner membranes of the autophagosome. How many other proteins share this unusual route out of the cell and what membrane channel is used for this translocation event remain open questions for the future.

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn‐SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione‐treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more O2‐ and less H2O2 than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host‐generated extracellular ROS, and contributes significantly to virulence in V. dahliae. Verticillium dahliae Cu/Zn superoxide dismutase detoxifies self‐ and host‐generated ROS to maximize virulence.

Alpha-synuclein (α-syn), a major component of Lewy bodies found in Parkinson’s disease (PD) patients, has been found exported outside of cells and may mediate its toxicity via cell-to-cell transmission. Here, we reconstituted soluble, monomeric α-syn secretion by the expression of DnaJ homolog subfamily C member 5 (DNAJC5) in HEK293T cells. DNAJC5 undergoes palmitoylation and anchors on the membrane. Palmitoylation is essential for DNAJC5-induced α-syn secretion, and the secretion is not limited by substrate size or unfolding. Cytosolic α-syn is actively translocated and sequestered in an endosomal membrane compartment in a DNAJC5-dependent manner. Reduction of α-syn secretion caused by a palmitoylation-deficient mutation in DNAJC5 can be reversed by a membrane-targeting peptide fusion-induced oligomerization of DNAJC5. The secretion of endogenous α-syn mediated by DNAJC5 is also found in a human neuroblastoma cell line, SH-SY5Y, differentiated into neurons in the presence of retinoic acid, and in human-induced pluripotent stem cell-derived midbrain dopamine neurons. We propose that DNAJC5 forms a palmitoylated oligomer to accommodate and export α-syn.

Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives controlled at 5%, and are also able to classify the limited number of established plant LSPs (SPLpred (3/4, LSPpred 4/4). Until such time as a larger set of bona fide (independently experimentally validated) LSPs is established using imaging technologies (light/fluorescence/electron microscopy) to confirm sub-cellular location, these tools represent a bridging method for predicting and identifying plant putative LSPs for subsequent experimental validation.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

Localized delivery of plasma membrane and cell wall components is an essential process in all plant cells. The vesicletethering complex, the exocyst, an ancient eukaryotic hetero-octameric protein cellular module, assists in targeted delivery of exocytosis vesicles to specific plasma membrane domains. Analyses of Arabidopsis and later other land plant genomes led to the surprising prediction of multiple putative EXO70 exocyst subunit paralogues. All land plant EXO70 exocyst subunits (including those of Bryophytes) form three distinct subfamilies—EXO70.1, EXO70.2, and EXO70.3. Interestingly, while the basal well-conserved EXO70.1 subfamily consists of multiexon genes, the remaining two subfamilies contain mostly single exon genes. Published analyses as well as public transcriptomic and proteomic data clearly indicate that most cell types in plants express and also use several different EXO70 isoforms. Here we sum up recent advances in the characterization of the members of the family of plant EXO70 exocyst subunits and present evidence that members of the EXO70.2 subfamily are often recruited to non-canonical functions in plant membrane trafficking pathways. Engagement of the most evolutionarily dynamic EXO70.2 subfamily of EXO70s in biotic interactions and defence correlates well with massive proliferation and conservation of new protein variants in this subfamily.