Regulation Of Protein Secretion Research Articles

Orthogonalized human protease control of secreted signals.

Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. While protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. Here, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins. We sourced a specific human protease and its FDA-approved inhibitor. We engineered cytokines (IL-2, IL-6, and IL-10) whose activities can be activated and abrogated by proteolytic cleavage. We utilized species specificity and re-localization strategies to orthogonalize the cytokines and protease from the human context that they would be deployed in. hDIRECT should enable local cytokine activation to support a variety of cell-based therapies such as muscle regeneration and cancer immunotherapy. Our work offers a proof of concept for the emerging appreciation of humanization in synthetic biology for human health.

Two distinct SNARE complexes mediate vesicle fusion with the plasma membrane to ensure effective development and pathogenesis of Fusarium oxysporum f. sp. cubense.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) facilitate docking and fusion of vesicles with their target membranes, playing a crucial role in vesicle trafficking and exocytosis. However, the spatial assembly and roles of plasma membrane (PM)-associated SNAREs in phytopathogen development and pathogenicity are not clearly understood. In this study, we analysed the roles and molecular mechanisms of PM-associated SNARE complexes in the banana Fusarium wilt fungus Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4). Our findings demonstrate that FocSso1 is important for the fungal growth, conidiation, host penetration and colonization. Mechanistically, FocSso1 regulates protein secretion by mediating vesicle docking and fusion with the PM and hyphal apex. Interestingly, a FocSso1-FocSec9-FocSnc1 complex was observed to assemble not only at the fungal PM but also on the growing hyphal apex, facilitating exocytosis. FocSso2, a paralogue of FocSso1, was also found to form a ternary SNARE complex with FocSec9 and FocSnc1, but it mainly localizes to the PM in old hyphae. The functional analysis of this protein demonstrated that it is dispensable for the fungal growth but necessary for host penetration and colonization. The other subunits, FocSec9 and FocSnc1, are involved in the fungal development and facilitate host penetration. Furthermore, FocSso1 and FocSnc1 are functionally interdependent, as loss of FocSso1 leads to mis-sorting and degradation of FocSnc1 in the vacuole and vice versa. Overall, this study provides insight into the formation of two spatially and functionally distinct PM SNARE complexes and their involvement in vesicle exocytosis to regulate development and pathogenicity of FocTR4.

Linear motifs regulating protein secretion, sorting and autophagy in Leishmania parasites are diverged with respect to their host equivalents.

The pathogenic, tropical Leishmania flagellates belong to an early-branching eukaryotic lineage (Kinetoplastida) with several unique features. Unfortunately, they are poorly understood from a molecular biology perspective, making development of mechanistically novel and selective drugs difficult. Here, we explore three functionally critical targeting short linear motif systems as well as their receptors in depth, using a combination of structural modeling, evolutionary sequence divergence and deep learning. Secretory signal peptides, endoplasmic reticulum (ER) retention motifs (KDEL motifs), and autophagy signals (motifs interacting with ATG8 family members) are ancient and essential components of cellular life. Although expected to be conserved amongst the kinetoplastids, we observe that all three systems show a varying degree of divergence from their better studied equivalents in animals, plants, or fungi. We not only describe their behaviour, but also build models that allow the prediction of localization and potential functions for several uncharacterized Leishmania proteins. The unusually Ala/Val-rich secretory signal peptides, endoplasmic reticulum resident proteins ending in Asp-Leu-COOH and atypical ATG8-like proteins are all unique molecular features of kinetoplastid parasites. Several of their critical protein-protein interactions could serve as targets of selective antimicrobial agents against Leishmaniasis due to their systematic divergence from the host.

Bioinformatic Analysis for Identifying Transcription Factors Involved in Protein Secretion and Vacuole Formation.

Protein secretion and vacuole formation are vital processes in plant cells, playing crucial roles in various aspects of plant development, growth, and stress responses. Multiple regulators have been uncovered to beinvolved in these processes. In animal cells, the transcription factor TFEB has been extensively studied and its role in lysosomal biogenesis is well understood. However, the transcription factors governing protein secretion and vacuole formation in plants remain largely unexplored. In recent years, an increasing number of bioinformatics databases and tools have been developed, facilitating computational prediction and analysis of the function of genes or proteins in specific cellular processes. Leveraging these resources, this chapter aims to provide practical guidance on how to effectively utilize these existing databases and tools for the analysis of key transcription factors involved in regulating protein secretion and vacuole formation in plants, with a particular focus on Arabidopsis and other higher plants. The findings from this analysis can serve as a valuable resource for future experimental investigations and the development of targeted strategies to manipulate protein secretion and vacuole formation in plants.

Exogenous abscisic acid treatment regulates protein secretion in sorghum cell suspension cultures

ABSTRACT Drought stress adversely affects plant growth, often leading to total crop failure. Upon sensing soil water deficits, plants switch on biosynthesis of abscisic acid (ABA), a stress hormone for drought adaptation. Here, we used exogenous ABA application to dark-grown sorghum cell suspension cultures as an experimental system to understand how a drought-tolerant crop responds to ABA. We evaluated intracellular and secreted proteins using isobaric tags for relative and absolute quantification. While the abundance of only ~ 7% (46 proteins) intracellular proteins changed in response to ABA, ~32% (82 proteins) of secreted proteins identified in this study were ABA responsive. This shows that the extracellular matrix is disproportionately targeted and suggests it plays a vital role in sorghum adaptation to drought. Extracellular proteins responsive to ABA were predominantly defense/detoxification and cell wall-modifying enzymes. We confirmed that sorghum plants exposed to drought stress activate genes encoding the same proteins identified in the in vitro cell culture system with ABA. Our results suggest that ABA activates defense and cell wall remodeling systems during stress response. This could underpin the success of sorghum adaptation to drought stress.

Timing of dense granule biogenesis in asexual malaria parasites.

Malaria is an important infectious disease that continues to claim hundreds of thousands of lives annually. The disease is caused by infection of host erythrocytes by apicomplexan parasites of the genus Plasmodium. The parasite contains three different apical organelles - micronemes, rhoptries and dense granules (DGs) - whose contents are secreted to mediate binding to and invasion of the host cell and the extensive remodelling of the host cell that occurs following invasion. Whereas the roles of micronemes and rhoptries in binding and invasion of the host erythrocyte have been studied in detail, the roles of DGs in Plasmodium parasites are poorly understood. They have been proposed to control host cell remodelling through regulated protein secretion after invasion, but many basic aspects of the biology of DGs remain unknown. Here we describe DG biogenesis timing for the first time, using RESA localization as a proxy for the timing of DG formation. We show that DG formation commences approximately 37 min prior to schizont egress, as measured by the recruitment of the DG marker RESA. Furthermore, using a bioinformatics approach, we aimed to predict additional cargo of the DGs and identified the J-dot protein HSP40 as a DG protein, further supporting the very early role of these organelles in the interaction of the parasite with the host cell.

Profiling the Pyrenophora tritici-repentis secretome: The Pf2 transcription factor regulates the secretion of the effector proteins ToxA and ToxB.

The global wheat disease tan spot is caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) which secretes necrotrophic effectors to facilitate host plant colonization. We previously reported a role of the Zn2 Cys6 binuclear cluster transcription factor Pf2 in the regulation of the Ptr effector ToxA. Here, we show that Pf2 is also a positive regulator of ToxB, via targeted deletion of PtrPf2 which resulted in reduced ToxB expression and defects in conidiation and pathogenicity. To further investigate the function of Ptr Pf2 in regulating protein secretion, the secretome profiles of two Δptrpf2 mutants of two Ptr races (races 1 and 5) were evaluated using a SWATH-mass spectrometry (MS) quantitative approach. Analysis of the secretomes of the Δptrpf2 mutants from in vitro culture filtrate identified more than 500 secreted proteins, with 25% unique to each race. Of the identified proteins, less than 6% were significantly differentially regulated by Ptr Pf2. Among the downregulated proteins were ToxA and ToxB, specific to race 1 and race 5 respectively, demonstrating the role of Ptr Pf2 as a positive regulator of both effectors. Significant motif sequences identified in both ToxA and ToxB putative promoter regions were further explored via GFP reporter assays.

Small GTPase FoSec4-Mediated Protein Secretion Is Important for Polarized Growth, Reproduction and Pathogenicity in the Banana Fusarium Wilt Fungus Fusarium odoratissimum.

Apical secretion at hyphal tips is important for the growth and development of filamentous fungi. In this study, we analyzed the role of the Rab GTPases FoSec4 involved in the secretion of the banana wilt fungal pathogen Fusarium odoratissimum. We found that the deletion of FoSEC4 affects the activity of extracellular hydrolases and protein secretion, indicating that FoSec4 plays an important role in the regulation of protein secretion in F. odoratissimum. As a typical Rab GTPase, Sec4 participates in the Rab cycle through the conversion between the active GTP-bound state and the inactive GDP-bound state, which is regulated by guanine nucleate exchange factors (GEFs) and GTPase-activating proteins (GAPs). We further found that FoSec2 can interact with dominant-negative FoSec4 (GDP-bound and nucleotide-free form, FoSec4DN), and that FoGyp5 can interact with dominant active FoSec4 (GTP-bound and constitutively active form, FoSec4CA). We evaluated the biofunctions of FoSec4, FoSec2 and FoGyp5, and found that FoSec4 is involved in the regulation of vegetative growth, reproduction, pathogenicity and the environmental stress response of F. odoratissimum, and that FocSec2 and FoGyp5 perform biofunctions consistent with FoSec4, indicating that FoSec2 and FoGyp5 may work as the GEF and the GAP, respectively, of FoSec4 in F. odoratissimum. We further found that the amino-terminal region and Sec2 domain are essential for the biological functions of FoSec2, while the carboxyl-terminal region and Tre-2/Bub2/Cdc16 (TBC) domain are essential for the biological functions of FoGyp5. In addition, FoSec4 mainly accumulated at the hyphal tips and partially colocalized with Spitzenkörper; however, FoGyp5 accumulated at the periphery of Spitzenkörper, suggesting that FoGyp5 may recognize and inactivate FoSec4 at a specific location in hyphal tips.

A Proteomic Approach to Elucidate the Changes in Saliva and Serum Proteins of Pigs with Septic and Non-Septic Inflammation

Sepsis is a systemic inflammatory response triggered by an infectious agent and is recognized by the World Health Organization as a global concern, since it is one of the major causes of severe illness in humans and animals. The study of the changes that can occur in saliva and serum in sepsis can contribute to a better understanding of the pathophysiological mechanisms involved in the process and also to discover potential biomarkers that can help in its diagnosis and monitoring. The objective of this study was to characterize the changes that occur in the salivary and serum proteome of pigs with experimentally-induced sepsis. The study included five pigs with sepsis induced by LPS administration and five pigs with non-septic inflammation induced by turpentine for comparative purposes. In saliva, there were eighteen salivary proteins differentially expressed in the sepsis condition and nine in non-septic inflammation. Among these, significant increments in aldolase A and serpin B12 only occurred in the sepsis model. Changes in aldolase A were validated in a larger population of pigs with sepsis due to Streptococcus suis infection. In serum, there were 30 proteins differentially expressed in sepsis group and 26 proteins in the non-septic group, and most of the proteins that changed in both groups were related to non-specific inflammation. In the saliva of the septic animals there were some specific pathways activated, such as the organonitrogen compound metabolic process and lipid transport, whereas, in the serum, one of the main activated pathways was the regulation of protein secretion. Overall, saliva and serum showed different proteome variations in response to septic inflammation and could provide complementary information about the pathophysiological mechanisms occurring in this condition. Additionally, salivary aldolase A could be a potential biomarker of sepsis in pigs that should be confirmed in a larger population.

Spinning the Matrix – Mechanisms of Basement Membrane Secretion and Assembly

Basement membranes (BMs) are sheet‐like extracellular matrices that line the basal surfaces of epithelial tissues and surround muscles, adipose tissue, and nerves. My lab studies how regulated changes to BM architecture contribute to tissue morphogenesis. In Drosophila, elongation of the initially spherical egg chamber coincides with the generation of a polarized network of fibrils in its surrounding BM. We found that BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber’s epithelial cells and undergo oriented insertion into the planar BM network by directed epithelial migration. We further found that a Rab10‐dependent secretion pathway ensures that a subset of new BM proteins exit the cell through a basal region of the lateral membrane for fibril formation. Rab10 recruits kinesin motors to BM secretory vesicles and transports them to this location on a polarized microtubule array. Finally, by manipulating Rab10 function, we showed for the first time that BM fibrils play an instructive role in egg chamber elongation. This work identifies a novel intracellular trafficking pathway for BM proteins and highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape.

Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Response of protein secretion based on transcriptional control is rather slow, as it requires transcription, translation and transport from the endoplasmic reticulum (ER) to the plasma membrane via the conventional protein secretion (CPS) pathway. An alternative regulation to provide faster response would be valuable. Here we present two genetically encoded orthogonal regulatory secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by a cytosolic protease) or inside the ER lumen (lumER, released by an ER-luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables the combination of signals into logic functions. Its application was demonstrated on a chemically regulated therapeutic protein secretion and regulated membrane translocation of a chimeric antigen receptor (CAR) targeting cancer antigen. Regulation of the ER escape represents a platform for the design of fast-responsive and tightly-controlled modular and scalable protein secretion system for mammalian cells.

Alternative splicing plays key roles in response to stress across different stages of fighting in the fish Betta splendens

BackgroundAggression is an evolutionarily conserved behavior critical for animal survival. In the fish Betta splendens, across different stages of fighting interactions, fighting opponents suffer from various stressors, especially from the great demand for oxygen. Using RNA sequencing, we profiled differential alternative splicing (DAS) events in the brains of fish collected before fighting, during fighting, and after fighting to study the involvement of alternative splicing (AS) in the response to stress during the fight.ResultsWe found that fighting interactions induced the greatest increase in AS in the ‘during-fighting’ fish, followed by that of the ‘after-fighting’ fish. Intron retention (IR) was the most enriched type among all the basic AS events. DAS genes were mainly associated with synapse assembly, ion transport, and regulation of protein secretion. We further observed that IR events significantly differentiated between winners and losers for 19 genes, which were associated with messenger RNA biogenesis, DNA repair, and transcription machinery. These genes share many common features, including shorter intron length and higher GC content.ConclusionsThis study is the first comprehensive view of AS induced by fighting interactions in a fish species across different stages of those interactions, especially with respect to IR events in winners and losers. Together, these findings facilitate future investigations into transcriptome complexity and AS regulation in response to stress under the context of aggression in vertebrates.

Microrna-142 Deficiency Promotes Chronic Myeloid Leukemia (CML) Transformation from Chronic Phase (CP) to Blast Crisis (BC)

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from the BCR-ABL1 fusion gene that encodes a constitutively activated tyrosine kinase (TK). Although TK inhibitors (TKIs) induce disease remission and prolonged survival in CML patients, a subset are resistant and progress from chronic phase (CP) to blast crisis (BC) with poor prognosis. Understanding the molecular mechanisms of transformation from CP to BC is necessary in the development of effective treatments. Here, we used the inducible SCLtTA/BCR-ABL transgenic CP CML model to study the molecular mechanism of disease evolution. Upon tetracycline withdrawal to induce BCR-ABL expression, both the SCLtTA/BCR-ABL homozygous (homo, i.e., SCLtTA+/+BCR-ABL+/+, hereafter called BCR-ABL) and heterozygous (het, i.e., SCLtTA+/-BCR-ABL+/-) mice developed and died of CP CML without developing BC CML, implying that BCR-ABL dosage is insufficient to induce transformation. MicroRNA (miR)-142 is highly expressed in hematopoietic cells with a critical role in normal hematopoiesis. In miR-142 knockout (KO)(miR-142−/−) mice, hematopoietic stem and progenitor cells expanded with a decrease of hematopoietic output. Loss of miR-142 function has been reported in lymphoma, acute lymphocytic leukemia and acute myeloid leukemia. Of note, we also observed lower levels of miR-142 in CD34+CD38- cells from patients with BC CML versus (vs) patients with CP CML. Thus, we hypothesized that miR-142 insufficiency may promote CML transformation from CP to BC. To test our hypothesis, we generated miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mice and observed increasing leukemic blasts over time after BCR-ABL induction in the blood and bone marrow (BM), but not in miR-142 wt (miR-142+/+)BCR-ABL controls even when the latter became moribund. MiR-142−/−BCR-ABL mice had larger spleens and significantly shorter survival [median: 26 vs 54 days (d); p<0.0001] than miR-142+/+BCR-ABL controls. Of note, while both homo (miR-142−/−) and het (miR-142+/−) miR-142 KO BCR-ABL mice eventually developed BC CML, the former had a significantly faster progression to BC and shorter survival (median: 26 vs 45 d; p=0.003) than the latter, suggesting miR-142 deficiency alone is sufficient to initiate BC transformation in the CP CML model in a dose-dependent manner. Importantly, all these features were recapitulated in congenic recipient mice transplanted with BM Lin-Sca-1+c-Kit+ cells (LSKs, 2000/mouse) from diseased miR-142−/−BCR-ABL mice, suggesting LSKs were enriched in leukemic stem cells and able to reproduce BC. Of note, in an RNA-seq analysis comparing LSKs from diseased miR-142−/−BCR-ABL (BC) and miR-142+/+ BCR-ABL(CP) mice, 504 genes were found differentially expressed. Gene set enrichment analysis (GSEA) showed only four pathways differentially expressed (upregulated); three [i.e., oxidative phosphorylation, glycolysis and adipogenesis] regulating cell metabolism and the fourth regulating protein secretion. Next, we developed a novel CpG-miR-142 mimic oligonucleotide, hereafter called CpG-M-miR-142, to restore miR-142 levels. Treatment with CpG-M-miR-142 (20mg/kg/day, iv, 4 weeks) on day 2 after BCR-ABL induction significantly prolonged survival of miR-142−/−BCR-ABL mice compared with CpG-scramble (SCR) (75% vs 33% survival rate at day 40 after BCR-ABL induction; median survival: not reached vs 25 d; p=0.03). Since we observed lower miR-142 levels in TKI-resistant vs TKI-sensitive CML patients (p=0.02), we selected LSKs from diseased miR-142−/−BCR-ABL and miR-142+/+BCR-ABL mice and exposed them to TKI nilotinib (NIL; 2µM) or vehicle for 72 hours to evaluate if downregulation of miR-142 was associated with TKI resistance. We observed lower apoptosis and higher cell growth in NIL-treated miR-142−/−BCR-ABL LSKs vs NIL-treated miR-142+/+BCR-ABL LSKs. The decreased sensitivity of miR-142−/−BCR-ABL LSKs to TKI was rescued by treatment with CpG-M-miR-142. CpG-M-miR-142 (2µM) plus NIL significantly increased apoptosis and reduced cell growth in miR-142−/−BCR-ABL LSKs compared with SCR+ NIL. We showed a key role of miR-142 deficiency in the transformation of CP CML to BC CML associated with deregulation of metabolic pathways. Restoring miR-142 expression in vivo with CpG-M-miR-142 significantly decreased the BC transformation rate, prolonged survival of miR-142−/−BCR-ABL mice and may increase sensitivity to TKIs. Disclosures Marcucci: Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Abbvie: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Other: Research Support (Investigation Initiated Clinical Trial); Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial).

Fam20C regulates protein secretion by Cab45 phosphorylation.

The TGN is a key compartment for the sorting and secretion of newly synthesized proteins. At the TGN, soluble proteins are sorted based on the instructions carried in their oligosaccharide backbones or by a Ca2+-mediated process that involves the cargo-sorting protein Cab45. Here, we show that Cab45 is phosphorylated by the Golgi-specific protein kinase Fam20C. Mimicking of phosphorylation translocates Cab45 into TGN-derived vesicles, which goes along with an increased export of LyzC, a Cab45 client. Our findings demonstrate that Fam20C plays a key role in the export of Cab45 clients by fine-tuning Cab45 oligomerization and thus impacts Cab45 retention in the TGN.

Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase.

Bacterial secretory preproteins are translocated across the inner membrane post-translationally by the SecYEG-SecA translocase. Mature domain features and signal peptides maintain preproteins in kinetically trapped, largely soluble, folding intermediates. Some aggregation-prone preproteins require chaperones, like trigger factor (TF) and SecB, for solubility and/or targeting. TF antagonizes the contribution of SecB to secretion by an unknown molecular mechanism. We reconstituted this interaction invitro and studied targeting and secretion of the model preprotein pro-OmpA. TF and SecB display distinct, unsuspected roles in secretion. Tightly associating TF:pro-OmpA targets the translocase at SecA, but TF prevents pro-OmpA secretion. In solution, SecB binds TF:pro-OmpA with high affinity. At the membrane, when bound to the SecA C-tail, SecB increases TF and TF:pro-OmpA affinities for the translocase and allows pro-OmpA to resume translocation. Our data reveal that TF, a main cytoplasmic folding pathway chaperone, is also a bona fide post-translational secretory chaperone that directly interacts with both SecB and the translocase to mediate regulated protein secretion. Thus, TF links the cytoplasmic folding and secretion chaperone networks.

Occludin protects secretory cells from ER stress by facilitating SNARE-dependent apical protein exocytosis.

Tight junctions (TJs) are fundamental features of both epithelium and endothelium and are indispensable for vertebrate organ formation and homeostasis. However, mice lacking Occludin (Ocln) develop relatively normally to term. Here we show that Ocln is essential for mammary gland physiology, as mutant mice fail to produce milk. Surprisingly, Ocln null mammary glands showed intact TJ function and normal epithelial morphogenesis, cell differentiation, and tissue polarity, suggesting that Ocln is not required for these processes. Using single-cell transcriptomics, we identified milk-producing cells (MPCs) and found they were progressively more prone to endoplasmic reticulum (ER) stress as protein production increased exponentially during late pregnancy and lactation. Importantly, Ocln loss in MPCs resulted in greatly heightened ER stress; this in turn led to increased apoptosis and acute shutdown of protein expression, ultimately leading to lactation failure in the mutant mice. We show that the increased ER stress was caused by a secretory failure of milk proteins in Ocln null cells. Consistent with an essential role in protein secretion, Occludin was seen to reside on secretory vesicles and to be bound to SNARE proteins. Taken together, our results demonstrate that Ocln protects MPCs from ER stress by facilitating SNARE-dependent protein secretion and raise the possibility that other TJ components may participate in functions similar to Ocln.

Autophagy Inhibition Induces the Secretion of Macrophage Migration Inhibitory Factor (MIF) with Autocrine and Paracrine Effects on the Promotion of Malignancy in Breast Cancer.

Breast cancer is the main cause of cancer-related death in women in the world. Because autophagy is a known survival pathway for cancer cells, its inhibition is currently being explored in clinical trials for treating several types of malignancies. In breast cancer, autophagy has been shown to be necessary for the survival of cancer cells from the triple negative subtype (TNBC), which has the worst prognosis among breast cancers and currently has limited therapeutic options. Autophagy has also been involved in the regulation of protein secretion and, of importance for this work, the inhibition of autophagy is known to promote the secretion of proinflammatory cytokines from distinct cell types. We found that the inhibition of autophagy in TNBC cell lines induced the secretion of the macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine involved in breast cancer invasion and immunomodulation. MIF secretion was dependent on an increase in reactive oxygen species (ROS) induced by the inhibition of autophagy. Importantly, MIF secreted from autophagy-deficient cells increased the migration of cells not treated with autophagy inhibitors, indicating that autophagy inhibition in cancer cells promoted malignancy in neighboring cells through the release of secreted factors, and that a combinatorial approach should be evaluated for cancer therapy.

Genome-wide CRISPR screening identifies new regulators of glycoprotein secretion

Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling invivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of β-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.

Autophagy and Its Relationship to Epithelial to Mesenchymal Transition: When Autophagy Inhibition for Cancer Therapy Turns Counterproductive.

The manipulation of autophagy for cancer therapy has gained recent interest in clinical settings. Although inhibition of autophagy is currently being used in clinical trials for the treatment of several malignancies, autophagy has been shown to have diverse implications for normal cell homeostasis, cancer cell survival, and signaling to cells in the tumor microenvironment. Among these implications and of relevance for cancer therapy, the autophagic process is known to be involved in the regulation of protein secretion, in tumor cell immunogenicity, and in the regulation of epithelial-to-mesenchymal transition (EMT), a critical step in the process of cancer cell invasion. In this work, we have reviewed recent evidence linking autophagy to the regulation of EMT in cancer and normal epithelial cells, and have discussed important implications for the manipulation of autophagy during cancer therapy.