A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion.

Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.

TMED10 mediates the trafficking of insulin-like growth factor 2 along the secretory pathway for myoblast differentiation.

The insulin-like growth factor 2 (IGF2) plays critical roles in cell proliferation, migration, differentiation, and survival. Despite its importance, the molecular mechanisms mediating the trafficking of IGF2 along the secretory pathway remain unclear. Here, we utilized a Retention Using Selective Hook system to analyze molecular mechanisms that regulate the secretion of IGF2. We found that a type I transmembrane protein, TMED10, is essential for the secretion of IGF2 and for differentiation of mouse myoblast C2C12 cells. Further analyses indicate that the residues 112-140 in IGF2 are important for the secretion of IGF2 and these residues directly interact with the GOLD domain of TMED10. We then reconstituted the release of IGF2 into COPII vesicles. This assay suggests that TMED10 mediates the packaging of IGF2 into COPII vesicles to be efficiently delivered to the Golgi. Moreover, TMED10 also mediates ER export of TGN-localized cargo receptor, sortilin, which subsequently mediates TGN export of IGF2. These analyses indicate that TMED10 is critical for IGF2 secretion by directly regulating ER export and indirectly regulating TGN export of IGF2, providing insights into trafficking of IGF2 for myoblast differentiation.

Active secretion of IL-33 from astrocytes is dependent on TMED10 and promotes central nervous system homeostasis.

Interleukin-33 (IL-33), secreted by astrocytes, regulates the synapse development in the spinal cord and hippocampus and suppresses autoimmune disease in the central nervous system (CNS). However, the mechanism of unconventional protein secretion of this cytokine remains unclear. In this study, we found that IFN-γ promotes the active secretion of IL-33 from astrocytes, and the active secretion of IL-33 from cytoplasm to extracellular space was dependent on interaction with transmembrane emp24 domain 10 (TMED10) via the IL-1 like cytokine domain in astrocytes. Knockout of Il-33 or its receptor St2 induced hippocampal astrocyte activation and depressive-like disorder in naive mice, as well as increased spinal cord astrocyte activation and polarization to a neurotoxic reactive subtype and aggravated passive experimental autoimmune encephalomyelitis (EAE). Our results have identified that IL-33 is actively secreted by astrocytes through the unconventional protein secretion pathway facilitated by TMED10 channels. This process helps maintain CNS homeostasis by inhibiting astrocyte activation.

A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

The circular RNA CDR1as regulate cell proliferation via TMED2 and TMED10.

BACKGROUND: Circular RNAs (CircRNAs) are biologically active RNAs. CDR1as is one such circRNA previously reported to be a microRNA-7 (miR-7) sponge, thereby regulating associated gene expression. The specific underlying molecular mechanisms of CDR1as biology, however, remain largely unknown. METHODS: We performed CDR1as knockdown in order to explore its function in cell proliferation, migration, the cell cycle, and tumorigenesis. We further employed quantitative proteomic analyses and associated bioinformatics strategies to globally assess CDR1as-regulated proteins (CRPs). Western blotting and immunofluorescence staining were used to validate the proteomic results. We additionally investigated a specific link between TMED2, TMED10, and miR-7 via a dual-luciferase reporter system, and generated CDR1as knockout cell lines via CRISPR/Cas9 editing. RESULTS: We identified 353 proteins dysregulated upon CDR1as knockdown in 293 T cells. These CRPs were found to interact with one another and to play key roles in certain cellular pathways. Two such proteins, TMED2 and TMED10, were found to specifically contribute to the influence of CDR1as on cell proliferation. CDR1as may regulate these two TMED proteins through miR-7 sponging. We were able to further confirm these results using both CRISPRi cell lines and nude mouse models. CONCLUSION: This study suggested that CDR1as may regulate cell proliferation via serving as a miR-7 sponge, thereby regulating TMED2 and TMED10 expression. These results are an invaluable template for future streamlined studies of circRNAs.

Secreted PTEN binds PLXDC2 on macrophages to drive antitumor immunity and tumor suppression.

Loss of phosphatase and tensin homolog (PTEN) has been linked to an immunosuppressive tumor microenvironment, but its underlying mechanisms remain largely enigmatic. Here, we report that PTEN can be secreted by the transmembrane emp24 domain-containing protein 10 (TMED10)-channeled protein secretion pathway. Inhibiting PTEN secretion from tumor cells contributes to immunosuppression and impairs the tumor-suppressive role of PTEN, while intratumoral injection of PTEN protein promotes antitumor immunity and suppresses tumor growth in mice. Mechanistically, extracellular PTEN binds to the plexin domain-containing protein 2 (PLXDC2) on macrophages, triggering subsequent activation of JAK2-STAT1 signaling, which switches tumor-associated macrophages (TAMs) from the immunosuppressive to inflammatory phenotype, leading to enhanced activation of CD8+ T and natural killer cells. Importantly, PTEN treatment also enhances the therapeutic efficacy of anti-PD-1 treatment in mice and reverses the immune-suppressive phenotype of patient-derived primary TAMs. These data identify a cytokine-like role of PTEN in immune activation and tumor suppression and demonstrate the therapeutic potential for extracellular administration of PTEN in cancer immunotherapy.

Transmembrane p24 trafficking protein 10 (TMED10) inhibits mitochondrial damage and protects neurons in ischemic stroke via the c-Jun N-terminal kinase (JNK) signaling pathway.

Stroke, a type of acute cerebrovascular disease, is a global disease with high mortality. Neuronal ischemia and hypoxia are closely related to occurrence and development of cognitive impairment. Transmembrane p24 trafficking protein 10 (TMED10) as a transmembrane protein involves in vesicle protein transport in the secretory pathways. However, the function and mechanism of TMED10 on ischemic stroke and cognitive impairments remain unclear. In current study, TMED10 was highly expressed in cerebral ischemic penumbra of middle cerebral artery occlusion (MCAO) mouse model. Downregulation of TMED10 suppressed cell survival and facilitated apoptosis in primary cortical neurons, which were grown under oxygen glucose deprivation/reoxygenation (OGD/R) condition. Upregulation of TMED10 protected neurons form apoptosis induced by OGD/R. Further research indicated that the decrease of TMED10 resulted in neuronal mitochondrial injury through increasing reactive oxygen species (ROS) production. Meanwhile, TMED10 reduction induced neuronal apoptosis and mitochondrial damage through activating the c-Jun N-terminal kinase (JNK) pathway. Moreover, the knockdown of TMED10 increased cerebral infarction area, aggravated neuronal injury and promoted neuronal apoptosis through activating the JNK pathway in the cerebral ischemic penumbra of MCAO mouse model. Additionally, Morris water maze test verified that the severity of cognitive impairment increased with the decline of TMED10. Collectively, this study reveals that TMED10 inhibits mitochondrial damage, and protects neurons from apoptosis in MCAO-induced ischemic stroke and cognitive impairment via blocking the JNK pathway.

ER-Golgi-localized proteins TMED2 and TMED10 control the formation of plasma membrane lipid nanodomains.

To promote infections, pathogens exploit host cell machineries such as structural elements of the plasma membrane. Studying these interactions and identifying molecular players are ideal for gaining insights into the fundamental biology of the host cell. Here, we used the anthrax toxin to screen a library of 1,500 regulatory, cell-surface, and membrane trafficking genes for their involvement in the intoxication process. We found that endoplasmic reticulum (ER)-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the plasma membrane of human cells, an essential step dependent on localization to cholesterol-rich lipid nanodomains. Biochemical, morphological, and mechanistic analyses showed that TMED2 and TMED10 are essential components of a supercomplex that operates the exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.

Protein translocation into the ERGIC: an upstream event of secretory autophagy.

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.

Down-regulated TMED10 in Alzheimer disease induces autophagy via ATG4B activation.

Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-ß (Aß) production. Treatment with Aß increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aß: amyloid-ß; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

Neurons Derived from Induced Pluripotent Stem Cells of Patients with Down Syndrome Reproduce Early Stages of Alzheimer's Disease Type Pathology in vitro.

People with Down syndrome (DS) are at high risk of developing pathology similar to Alzheimer's disease (AD). Modeling of this pathology in vitro may be useful for studying this phenomenon. In this study, we analyzed three different cultures of neural cells carrying trisomy of chromosome 21, which were generated by directed differentiation from induced pluripotent stem cells (iPS cells). We report here that in vitro generated DS neural cells have abnormal metabolism of amyloid-ß (Aß) manifested by increased secretion and accumulation of Aß granules of Aß42 pathological isoform with upregulated expression of the APP gene. Additionally, we found increased expression levels of genes that are considered to be associated with AD (BACE2, RCAN1, ETS2, TMED10), as compared to healthy controls. Thus, the neural cells generated from induced pluripotent stem cells with DS reproduce initial cellular signs of AD-type pathology and can be useful tools for modeling and studying this variant of AD in vitro.