The endolysosomal system in conventional and unconventional protein secretion.

Most secreted proteins are transported through the "conventional" endoplasmic reticulum-Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or "unconventional" secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes.

Conventional and Unconventional Protein Secretion in Yeast and Animal Cells.

Protein secretion mediated by the secretory transport pathway is a sophisticated and highly regulated cellular process in eukaryotic cells. In the conventional secretory transport pathway, newly synthesized proteins pass through several endomembrane compartments to reach their destinations. This transport occurs via small, membrane-enclosed vesicles. To ensure the fidelity of trafficking, eukaryotic cells employ elaborate molecular machinery to accurately sort newly synthesized proteins into specific transport vesicles and precisely deliver them to respective acceptor compartments. Leaderless cargo proteins, lacking a signal peptide, follow an unconventional secretory pathway. This review encompasses the molecular machinery regulating both conventional and unconventional protein secretion in yeast and animal cells.

An Overview of Protein Secretion in Plant Cells.

Newly synthesized proteins are delivered to the apoplast via conventional or unconventional protein secretion in eukaryotes. In plants, proteins are secreted to perform various biological functions. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been revealed in plants. In the conventional protein secretion pathway, secretory proteins with a signal peptide are translocated into the endoplasmic reticulum and transported to the extracellular region via the endomembrane system. On the contrary, unconventional protein secretion pathways have been demonstrated to mediate the secretion of the leaderless secretory proteins. In this chapter, we summarize the updated findings and provide a comprehensive overview of protein secretion pathways in plants.

Discovery of Cell Number-Interstitial Fluid Volume (CIF) Ratio Reveals Secretory Autophagy Pathway to Supply eHsp90α for Wound Healing.

Cell secretion repairs tissue damage and restores homeostasis throughout adult life. The extracellular heat shock protein-90alpha (eHsp90α) has been reported as an exosome cargo and a potential driver of wound healing. However, neither the mechanism of secretion nor the genetic evidence for eHsp90α in wound healing has been substantiated. Herein, we show that tissue injury causes massive deposition of eHsp90α in tissues and secretion of eHsp90α by cells. Sequential centrifugations of conditioned medium from relevant cell lines revealed the relative distributions of eHsp90α in microvesicle, exosome and trypsin-sensitive supernatant fractions to be approximately <2%, <4% and >95%, respectively. Establishing the cell-number-to-interstitial-fluid-volume (CIF) ratio for the microenvironment of human tissues as 1 × 109 cells: 1 mL interstitial fluid enabled us to predict the corresponding tissue concentrations of eHsp90α in these fractions as 3.74 µg/mL, 5.61 µg/mL and 178 µg/mL. Remarkably, the 178 µg/mL eHsp90α matches the previously reported 100-300 µg/mL of recombinant eHsp90α whose topical application promotes maximum wound healing in animal models. More importantly, we demonstrate that two parallel secretory autophagy-regulating gene families, the autophagy-regulating (AR) genes and the Golgi reassembly-stacking protein (GRASP) genes work together to mediate the secretion of the physiological concentration of eHsp90α to promote wound healing. Thus, utilization of the CIF ratio-based extrapolation method may enable investigators to rapidly predict biomarker targets from cell-conditioned-medium data.

DAP12 interacts with RER1 and is retained in the secretory pathway before assembly with TREM2.

DNAX-activating protein of 12 kDa (DAP12) is a transmembrane adapter protein expressed in lymphoid and myeloid lineage cells. It interacts with several immunoreceptors forming functional complexes that trigger intracellular signaling pathways. One of the DAP12 associated receptors is the triggering receptor expressed on myeloid cells 2 (TREM2). Mutations in both DAP12 and TREM2 have been linked to neurodegenerative diseases. However, mechanisms involved in the regulation of subcellular trafficking and turnover of these proteins are not well understood. Here, we demonstrate that proteasomal degradation of DAP12 is increased in the absence of TREM2. Interestingly, unassembled DAP12 is also retained in early secretory compartments, including the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC), thereby preventing its transport to the plasma membrane. We also show that unassembled DAP12 interacts with the retention in ER sorting receptor 1 (RER1). The deletion of endogenous RER1 decreases expression of functional TREM2-DAP12 complexes and membrane proximal signaling, and resulted in almost complete inhibition of phagocytic activity in THP-1 differentiated macrophage-like cells. These results indicate that RER1 acts as an important regulator of DAP12 containing immunoreceptor complexes and immune cell function.

The Construction of an Environmentally Friendly Super-Secreting Strain of Bacillus subtilis through Systematic Modulation of Its Secretory Pathway Using the CRISPR-Cas9 System.

Achieving commercially significant yields of recombinant proteins in Bacillus subtilis requires the optimization of its protein production pathway, including transcription, translation, folding, and secretion. Therefore, in this study, our aim was to maximize the secretion of a reporter α-amylase by overcoming potential bottlenecks within the secretion process one by one, using a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system. The strength of single and tandem promoters was evaluated by measuring the relative α-amylase activity of AmyQ integrated into the B. subtilis chromosome. Once a suitable promoter was selected, the expression levels of amyQ were upregulated through the iterative integration of up to six gene copies, thus boosting the α-amylase activity 20.9-fold in comparison with the strain harboring a single amyQ gene copy. Next, α-amylase secretion was further improved to a 26.4-fold increase through the overexpression of the extracellular chaperone PrsA and the signal peptide peptidase SppA. When the final expression strain was cultivated in a 3 L fermentor for 90 h, the AmyQ production was enhanced 57.9-fold. The proposed strategy allows for the development of robust marker-free plasmid-less super-secreting B. subtilis strains with industrial relevance.

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.

Dengue virus exploits autophagy vesicles and secretory pathways to promote transmission by human dendritic cells.

Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent decades have increasing incidence of dengue-associated disease alongside growing frequency of outbreaks. Although promising progress has been made in anti-DENV immunizations, post-infection treatment remains limited to non-specific supportive treatments. Development of antiviral therapeutics is thus required to limit DENV dissemination in humans and to help control the severity of outbreaks. Dendritic cells (DCs) are amongst the first cells to encounter DENV upon injection into the human skin mucosa, and thereafter promote systemic viral dissemination to additional human target cells. Autophagy is a vesicle trafficking pathway involving the formation of cytosolic autophagosomes, and recent reports have highlighted the extensive manipulation of autophagy by flaviviruses, including DENV, for viral replication. However, the temporal profiling and function of autophagy activity in DENV infection and transmission by human primary DCs remains poorly understood. Herein, we demonstrate that mechanisms of autophagosome formation and extracellular vesicle (EV) release have a pro-viral role in DC-mediated DENV transmission. We show that DENV exploits early-stage canonical autophagy to establish infection in primary human DCs. DENV replication enhanced autophagosome formation in primary human DCs, and intrinsically-heightened autophagosome biogenesis correlated with relatively higher rates of DC susceptibility to DENV. Furthermore, our data suggest that viral replication intermediates co-localize with autophagosomes, while productive DENV infection introduces a block at the late degradative stages of autophagy in infected DCs but not in uninfected bystander cells. Notably, we identify for the first time that approximately one-fourth of DC-derived CD9/CD81/CD63+ EVs co-express canonical autophagy marker LC3, and demonstrate that DC-derived EV populations are an alternative, cell-free mechanism by which DCs promote DENV transmission to additional target sites. Taken together, our study highlights intersections between autophagy and secretory pathways during viral infection, and puts forward autophagosome accumulation and viral RNA-laden EVs as host determinants of DC-mediated DENV infection in humans. Host-directed therapeutics targeting autophagy and exocytosis pathways thus have potential to enhance DC-driven resistance to DENV acquisition and thereby limit viral dissemination by initial human target cells following mosquito-to-human transmission of DENV.

ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway.

Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.

More than meets the eye: knowns and unknowns of the trafficking of small secreted proteins in Arabidopsis.

Small proteins represent a significant portion of the cargo transported through plant secretory pathways, playing crucial roles in developmental processes, fertilization, and responses to environmental stresses. Despite the importance of small secreted proteins, substantial knowledge gaps persist regarding the regulatory mechanisms governing their trafficking along the secretory pathway, and their ultimate localization or destination. To address these gaps, we conducted a comprehensive literature review, focusing particularly on trafficking and localization of Arabidopsis small secreted proteins with potential biochemical and/or signaling roles in the extracellular space, typically those within the size range of 101-200 amino acids. Our investigation reveals that while at least six members of the 21 mentioned families have a confirmed extracellular localization, eight exhibit intracellular localization, including cytoplasmic, nuclear, and chloroplastic locations, despite the presence of N-terminal signal peptides. Further investigation into the trafficking and secretion mechanisms of small protein cargo could not only deepen our understanding of plant cell biology and physiology but also provide a foundation for genetic manipulation strategies leading to more efficient plant cultivation.

Protein condensates in the the secretory pathway: Unraveling biophysical interactions and function.

The emergence of phase separation phenomena among macromolecules has identified biomolecular condensates as fundamental cellular organizers. These condensates concentrate specific components and accelerate biochemical reactions without relying on membrane boundaries. Although extensive studies have revealed a large variety of nuclear and cytosolic membraneless organelles, we are witnessing a surge in the exploration of protein condensates associated with the membranes of the secretory pathway, such as the endoplasmic reticulum and the Golgi apparatus. This review focuses on protein condensates in the secretory pathway and discusses their impact on the organization and functions of this cellular process. Moreover, we explore the modes of condensate-membrane association and the biophysical and cellular consequences of protein condensate interactions with secretory pathway membranes.

Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

A cilia-bound unconventional secretory pathway for Drosophila odorant receptors.

BACKGROUND: Post-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway. RESULTS: In the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity. CONCLUSIONS: Our results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons.

Upregulation of the secretory pathway Ca2+/Mn2+-ATPase isoform 1 in LPS-stimulated microglia and its involvement in Mn2+-induced Golgi fragmentation.

Microglia play an important protective role in the healthy nervous tissue, being able to react to a variety of stimuli that induce different intracellular cascades for specific tasks. Ca2+ signaling can modulate these pathways, and we recently reported that microglial functions depend on the endoplasmic reticulum as a Ca2+ store, which involves the Ca2+ transporter SERCA2b. Here, we investigated whether microglial functions may also rely on the Golgi, another intracellular Ca2+ store that depends on the secretory pathway Ca2+/Mn2+-transport ATPase isoform 1 (SPCA1). We found upregulation of SPCA1 upon lipopolysaccharide stimulation of microglia BV2 cells and primary microglia, where alterations of the Golgi ribbon were also observed. Silencing and overexpression experiments revealed that SPCA1 affects cell morphology, Golgi apparatus integrity, and phagocytic functions. Since SPCA1 is also an efficient Mn2+ transporter and considering that Mn2+ excess causes manganism in the brain, we addressed the role of microglial SPCA1 in Mn2+ toxicity. Our results revealed a clear effect of Mn2+ excess on the viability and morphology of microglia. Subcellular analysis showed Golgi fragmentation and subsequent alteration of SPCA1 distribution from early stages of toxicity. Removal of Mn2+ by washing improved the culture viability, although it did not effectively reverse Golgi fragmentation. Interestingly, pretreatment with curcumin maintained microglia cultures viable, prevented Mn2+-induced Golgi fragmentation, and preserved SPCA Ca2+-dependent activity, suggesting curcumin as a potential protective agent against Mn2+-induced Golgi alterations in microglia.

[Unconventional protein secretion - new perspectives in protein trafficking]. / Sécrétion non conventionnelle - Nouvelles perspectives dans le trafic des protéines.

The characterization of the structural and functional organization of eukaryotic cells has revealed the membrane compartments and machinery required for vesicular protein transport. Most proteins essential for intercellular communication contain an N-terminal signal sequence enabling them to be incorporated into the biosynthetic or conventional secretory pathway, in which proteins are sequentially transported through the endoplasmic reticulum (ER) and the Golgi apparatus. However, major research studies have shown the existence of alternative secretory routes that are independent of the ER-Golgi and designated as unconventional secretory pathways. These pathways involve a large number of players that may divert specific compartments from their primary function in favor of secretory roles. The comprehensive description of these processes is therefore of utmost importance to unveil how proteins secreted through these alternative pathways control cell homeostasis or contribute to disease development.

Title: Sécrétion non conventionnelle - Nouvelles perspectives dans le trafic des protéines. Abstract: L'étude de l'organisation structurale et fonctionnelle des cellules eucaryotes a révélé les compartiments membranaires ainsi que la machinerie nécessaires au trafic vésiculaire des protéines. La plupart des protéines essentielles à la communication intercellulaire contiennent une séquence signal leur permettant d'être incorporées dans la voie de sécrétion conventionnelle, par laquelle les protéines sont transportées séquentiellement dans le réticulum endoplasmique (RE) puis l'appareil de Golgi. Cependant, les cellules eucaryotes sont également dotées de voies de sécrétion alternatives ou voies de sécrétion non conventionnelles, qui mettent en jeu de nombreux acteurs susceptibles de détourner certains compartiments de leurs fonctions principales au profit de fonctions sécrétoires.

Unconventional role of Rab4 in the secretory pathway in Leishmania.

Leishmania donovani is an auxotroph for heme. Parasite acquires heme by clathrin-mediated endocytosis of hemoglobin by specific receptor. However, the regulation of receptor recycling pathway is not known in Leishmania. Here, we have cloned, expressed and characterized the Rab4 homologue from L. donovani. We have found that LdRab4 localizes in both early endosomes and Golgi in L. donovani. To understand the role of LdRab4 in L. donovani, we have generated transgenic parasites overexpressing GFP-LdRab4:WT, GFP-LdRab4:Q67L, and GFP-LdRab4:S22N. Our results have shown that overexpression of GFP-LdRab4:Q67L or GFP-LdRab4:S22N does not alter the cell surface localization of hemoglobin receptor in L. donovani. Surprisingly, we have found that overexpression of GFP-LdRab4:S22N significantly blocks the transport of Ldgp63 to the cell surface whereas the trafficking of Ldgp63 is induced to the cell surface in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites. Consequently, we have found significant inhibition of gp63 secretion by GFP-LdRab4:S22N overexpressing parasites whereas secretion of Ldgp63 is enhanced in GFP-LdRab4:WT and GFP-LdRab4:Q67L overexpressing parasites in comparison to untransfected control parasites. Moreover, we have found that survival of transgenic parasites overexpressing GFP-LdRab4:S22N is severely compromised in macrophages in comparison to GFP-LdRab4:WT and GFP-LdRab4:Q67L expressing parasites. These results demonstrated that LdRab4 unconventionally regulates the secretory pathway in L. donovani.

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification.

Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.

Engineering the Tat-secretion pathway of Bacillus licheniformis for the secretion of cytoplasmic enzyme arginase.

The industrial bacterium Bacillus licheniformis has long been used as a microbial factory for the production of enzymes due to its ability to secrete copious amounts of native extracellular proteins and its generally regarded as safe (GRAS) status. However, most attempts to use B. licheniformis to produce heterologous and cytoplasmic enzymes primarily via the general secretory (Sec) pathway have had limited success. The twin-arginine transport (Tat) pathway offers a promising alternative for the extracellular export of Sec-incompatible proteins because it transports full, correctly folded proteins. However, compared to the Sec pathway, the yields of the Tat pathway have historically been too low for commercial use. To improve the export efficiency of the Tat pathway, we identified the optimal Tat-dependent signal peptides and increased the abundance of the Tat translocases, the signal peptidase (SPase), and the intracellular chaperones. These strategic modifications significantly improved the Tat-dependent secretion of the cytoplasmic enzyme arginase into the culture medium using B. licheniformis. The extracellular enzymatic activity of arginase showed a 5.2-fold increase after these modifications. Moreover, compared to the start strain B. licheniformis 0F3, the production of extracellular GFP was improved by 3.8 times using the strategic modified strain B. licheniformis 0F13, and the extracellular enzymatic activity of SOX had a 1.3-fold increase using the strain B. licheniformis 0F14. This Tat-based production chassis has the potential for enhanced production of Sec-incompatible enzymes, therefore expanding the capability of B. licheniformis as an efficient cellular factory for the production of high-value proteins. KEY POINTS: • Systematic genetic modification of Tat-pathway in B. licheniformis. • Significant enhancement of the secretion capacity of Tat pathway for delivery the cytoplasmic enzyme arginase. • A new platform for efficient extracellular production of Sec-incompatible enzymes.

Identification of secretory pathway-related genes based on Random Forest algorithm to predict the prognosis and immunotherapy response of hepatocellular carcinoma.

BACKGROUND: The dysfunction of secretory pathways may represent biomarkers or therapeutic targets of cancer. The hepatocellular carcinoma (HCC) phenotype was studied in relation to the genes in the secretory pathway and to screen for a combination of genes that may be a viable therapeutic target for HCC and connected to the pathophysiological features of the tumor. METHODS: Using the HCC information from The Cancer Genome Atlas, somatic mutation and prognostic association analysis were performed on the secretory pathway genes. Based on prognostic genes in the secretory pathway, the samples were consensus clustered, and a Random Forest model was built. The clinical characteristics, tumor mutation burden, functional status and potential responses to immunotherapy and tumor suppressor medications of various subtypes and risk groups were discussed. RESULTS: Of the 84 genes for secretory pathway, 32 were prognostic genes related to HCC, which divided HCC into two categories: C1 and C2. By comparing the two types of HCC samples, it was found that the survival outcome of C1 was inferior, with stronger adaptive and innate immunity, but less sensitive to immunotherapy than C2. The constructed prognostic signature included seven of the 32 prognostic genes in the secretory pathway, which showed significant correlation with the prognosis, somatic mutation, biological pathway status, potential response to immunotherapy and sensitivity of 72 tumor suppressor drugs from different HCC cohorts, and had a feasible prognostic effect for 31 types of cancer and immunotherapy cohorts. CONCLUSIONS: In this study, HCC was divided into two molecular subtypes according to prognostic genes in the secretory pathway, and seven of them were combined into one signature, which produced significant results in evaluating the prognosis of different HCC cohorts, pan-cancer cohorts and immunotherapy cohorts, and had potential guiding significance for prophylactic immunotherapy in patients with HCC.

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.