Inhibition Of Protein Secretion Research Articles

A Novel Dual-Reporter System Reveals Distinct Characteristics of Exosome-Mediated Protein Secretion in Human Cells

BackgroundExosomes, a special subtype of extracellular vesicles derived from human cells, serve as vital mediators of intercellular communication by transporting diverse bioactive cargos, including proteins and enzymes. However, the underlying mechanisms governing exosome secretion and regulation remain poorly understood. In this study, we employed a dual-reporter system consisting of bioluminescent Gaussia luciferase and fluorescent proteins to investigate the dynamics and regulation of exosome secretion in cultured human cells.ResultsOur results demonstrated that the engineered dual-reporters effectively monitored both exosome-mediated and ER-Golgi-mediated secretory pathways in a specific and quantitative manner. Notably, we observed distinct characteristics of exosome-mediated protein secretion, including significantly lower capacity and different dynamics compared to the ER-Golgi pathway. This phenomenon was observed in human kidney 293T cells and liver HepG2 cells, emphasizing the conserved nature of exosome-mediated secretion across cell types. Furthermore, we investigated the impact of brefeldin A (BFA), an inhibitor of ER-to-Golgi membrane trafficking, on protein secretion. Interestingly, BFA inhibited protein secretion via the ER-Golgi pathway while stimulating exosome-mediated protein secretion under same experimental conditions.ConclusionsCollectively, our study highlights the utility of the dual-reporter system for real-time monitoring and quantitative analysis of protein secretion through conventional ER-Golgi and unconventional exosome pathways. Moreover, our findings unveil distinct features of exosome-mediated protein secretion, shedding light on its differential capacity, dynamics, and regulatory mechanisms compared to ER-Golgi-mediated proteins in human cells.

A Pyridyl-Furan Series Developed from the Open Global Health Library Block Red Blood Cell Invasion and Protein Trafficking in Plasmodium falciparum through Potential Inhibition of the Parasite's PI4KIIIB Enzyme.

With the resistance increasing to current antimalarial medicines, there is an urgent need to discover new drug targets and to develop new medicines against these targets. We therefore screened the Open Global Health Library of Merck KGaA, Darmstadt, Germany, of 250 compounds against the asexual blood stage of the deadliest malarial parasite Plasmodium falciparum, from which eight inhibitors with low micromolar potency were found. Due to its combined potencies against parasite growth and inhibition of red blood cell invasion, the pyridyl-furan compound OGHL250 was prioritized for further optimization. The potency of the series lead compound (WEHI-518) was improved 250-fold to low nanomolar levels against parasite blood-stage growth. Parasites selected for resistance to a related compound, MMV396797, were also resistant to WEHI-518 as well as KDU731, an inhibitor of the phosphatidylinositol kinase PfPI4KIIIB, suggesting that this kinase is the target of the pyridyl-furan series. Inhibition of PfPI4KIIIB blocks multiple stages of the parasite's life cycle and other potent inhibitors are currently under preclinical development. MMV396797-resistant parasites possess an E1316D mutation in PfPKI4IIIB that clusters with known resistance mutations of other inhibitors of the kinase. Building upon earlier studies that showed that PfPI4KIIIB inhibitors block the development of the invasive merozoite parasite stage, we show that members of the pyridyl-furan series also block invasion and/or the conversion of merozoites into ring-stage intracellular parasites through inhibition of protein secretion and export into red blood cells.

Identification of 2-Amino Benzothiazoles with Bactericidal Activity against Mycobacterium tuberculosis.

We identified an amino-benzothiazole scaffold from a whole-cell screen against recombinant Mycobacterium tuberculosis under expressing the essential signal peptidase LepB. The seed molecule had 2-fold higher activity against the LepB hypomorph. Through a combination of purchase and chemical synthesis, we explored the structure-activity relationship for this series; 34 analogs were tested for antitubercular activity and for cytotoxicity against eukaryotic cells. We identified molecules with improved potency and reduced cytotoxicity. However, molecules did not appear to target LepB directly and did not inhibit protein secretion. Key compounds showed good permeability, low protein binding, and lack of CYP inhibition, but metabolic stability was poor with short half-lives. The seed molecule showed good bactericidal activity against both replicating and nonreplicating bacteria, as well as potency against intracellular M. tuberculosis in murine macrophages. Overall, the microbiological properties of the series are attractive if metabolic stability can be improved, and identification of the target could assist in the development of this series. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis, is a serious global health problem requiring the development of new therapeutics. We previously ran a high-throughput screen and identified a series of compounds with antitubercular activity. In this paper, we test analogs of our hit molecules for activity against M. tuberculosis, as well as for activity against eukaryotic cells. We identified molecules with improved selectivity. Our molecules killed both replicating and nonreplicating bacteria but did not work by targeting protein secretion.

The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites.

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs—modifications which are both mediated by parasite‐derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host.

Abstract 323: Quantitative proteomic profiling of novel anti-cancer small molecule inhibitors of Sec61: Mechanistic investigation and biomarker discovery

Abstract Secreted and transmembrane (TM) proteins play key roles in malignant transformation and tumor growth. During translation, the majority of these proteins require translocation through the Sec61 protein complex into the endoplasmic reticulum (ER) for further processing. Thus, Sec61 represents a novel therapeutic target for cancer treatment through selective blockade of protein secretion. The structural analogs KZR-261 and KZR-834 were identified through a medicinal chemistry campaign and found to have broad in vitro anti-tumor activity and efficacy in mouse xenograft models. KZR-261 was nominated for clinical development and we describe here the proteomic assessment and in vitro pharmacodynamics of these novel anti-cancer agents. We utilized quantitative proteomic methods to study inhibition of protein secretion and global modulation of protein homeostasis across multiple tumor cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors. Cells were treated with KZR-834 or KZR-261 followed by subcellular fractionation of cytosolic and membrane/ER proteomes. The membrane/ER fraction was expected to be enriched for Sec61 client proteins. Subsequent proteomic profiling was performed with Tandem Mass Tag 6-plex (TMTsixplex™). Across tumor cell lines, a total of 2222 Sec61 client proteins were detected in the membrane/ER proteome, representing >30% of the annotated Sec61 clientome. Of these proteins, 5-17% were inhibited by >50% upon treatment, indicating that only a small portion of the total flux of secreted and TM proteins in cells was affected by our compounds. KZR-261 and KZR-834 preferentially targeted secreted and type 1 TM proteins relative to multi-pass TM proteins, which may be related to the smaller size of Sec61 targeting sequences in these proteins. Additionally, Ingenuity®Pathway Analysis (Qiagen) of the cytosolic proteome from tumor cell lines indicated that activation of the ER stress response was induced by KZR-834, which were further verified by gene expression and western blot analyses. In PBMCs treated with KZR-261, multiple type 1 TM proteins (IL-7 receptor, L-selectin, ICAM-1, HLA-DR) and the secreted protein beta-2-microglobulin were identified as sensitive cell surface targets. Flow cytometric analysis of PBMCs also showed a significant reduction in surface expression of those proteins in multiple cell populations. Our results highlight quantitative proteomic profiling as a valuable tool towards elucidation of the mechanism of pleiotropically-acting molecules. By assessing global proteomic changes in non-transformed cells, pharmacodynamic markers were identified that will be employed in the Phase 1 trial of KZR-261 in solid tumors. Citation Format: Yu Qian, Jennifer Whang, Janet Anderl, Henry W. Johnson, Christopher J. Kirk, Eric Lowe, Dustin McMinn, Beatriz Millare, Tony Muchamuel, Jinhai Wang. Quantitative proteomic profiling of novel anti-cancer small molecule inhibitors of Sec61: Mechanistic investigation and biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 323.

Cross-Kingdom Activation of Vibrio Toxins by ADP-Ribosylation Factor Family GTPases.

Pathogenic Vibrio species use many different approaches to subvert, attack, and undermine the host response. The toxins they produce are often responsible for the devastating effects associated with their diseases. These toxins target a variety of host proteins, which leads to deleterious effects, including dissolution of cell organelle integrity and inhibition of protein secretion. Becoming increasingly prevalent as cofactors for Vibrio toxins are proteins of the small GTPase families. ADP-ribosylation factor small GTPases (ARFs) in particular are emerging as a common host cofactor necessary for full activation of Vibrio toxins. While ARFs are not the direct target of Vibrio cholerae cholera toxin (CT), ARF binding is required for its optimal activity as an ADP-ribosyltransferase. The makes caterpillars floppy (MCF)-like and the domain X (DmX) effectors of the Vibrio vulnificus multifunctional autoprocessing repeats-in-toxin (MARTX) toxin also both require ARFs to initiate autoprocessing and activation as independent effectors. ARFs are ubiquitously expressed in eukaryotes and are key regulators of many cellular processes, and as such they are ideal cofactors for Vibrio pathogens that infect many host species. In this review, we cover in detail the known Vibrio toxins that use ARFs as cross-kingdom activators to both stimulate and optimize their activity. We further discuss how these contrast to toxins and effectors from other bacterial species that coactivate, stimulate, or directly modify host ARFs as their mechanisms of action.

Novel Antimicrobials from Uncultured Bacteria Acting against Mycobacterium tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis (TB), is estimated to infect one-third of the world's population. The overall burden and the emergence of drug-resistant strains of Mycobacterium tuberculosis underscore the need for new therapeutic options against this important human pathogen. Our recent work demonstrated the success of natural product discovery in identifying novel compounds with efficacy against Mycobacterium tuberculosis Here, we improve on these methods by combining improved isolation and Mycobacterium tuberculosis selective screening to identify three new anti-TB compounds: streptomycobactin, kitamycobactin, and amycobactin. We were unable to obtain mutants resistant to streptomycobactin, and its target remains to be elucidated. We identify the target of kitamycobactin to be the mycobacterial ClpP1P2C1 protease and confirm that kitamycobactin is an analog of the previously identified compound lassomycin. Further, we identify the target of amycobactin to be the essential protein secretion pore SecY. We show further that amycobactin inhibits protein secretion via the SecY translocon. Importantly, this inhibition is bactericidal to nonreplicating Mycobacterium tuberculosis This is the first compound, to our knowledge, that targets the Sec protein secretion machinery in Mycobacterium tuberculosis This work underscores the ability of natural product discovery to deliver not only new compounds with activity against Mycobacterium tuberculosis but also compounds with novel targets.IMPORTANCE Decreasing discovery rates and increasing resistance have underscored the need for novel therapeutic options to treat Mycobacterium tuberculosis infection. Here, we screen extracts from previously uncultured soil microbes for specific activity against Mycobacterium tuberculosis, identifying three novel compounds. We further define the mechanism of action of one compound, amycobactin, and demonstrate that it inhibits protein secretion through the Sec translocation machinery.

Protein Modifications Critical for Myonectin/Erythroferrone Secretion and Oligomer Assembly.

Myonectin/erythroferrone (also known as CTRP15) is a secreted hormone with metabolic function and a role in stress erythropoiesis. Despite its importance in physiologic processes, biochemical characterization of the protein is lacking. Here, we show that multiple protein modifications are critical for myonectin secretion and multimerization. Abolishing N-linked glycosylation by tunicamycin, glucosamine supplementation, or glutamine substitutions of all four potential Asn glycosylation sites blocked myonectin secretion. Mass spectrometry confirmed that Asn-229 and Asn-281 were glycosylated, and substituting both Asn sites with Gln prevented myonectin secretion. Although Asn-319 is not identified as glycosylated, Gln substitution caused protein misfolding and retention in the endoplasmic reticulum. Of the four conserved cysteines, Cys-273 and Cys-278 were required for proper protein folding; Ala substitution of either site inhibited protein secretion. In contrast, Ala substitutions of Cys-142, Cys-194, or both markedly enhanced protein secretion, suggesting endoplasmic reticulum retention that facilitates myonectin oligomer assembly. Secreted myonectin consists of trimers, hexamers, and high-molecular weight (HMW) oligomers. The formation of higher-order structures via intermolecular disulfide bonds depended on Cys-142 and Cys-194; while the C142A mutant formed almost exclusively trimers, the C194A mutant was impaired in HMW oligomer formation. Most Pro residues within the short collagen domain of myonectin were also hydroxylated, a modification that stabilized the collagen triple helix. Inhibiting Pro hydroxylation or deleting the collagen domain markedly reduced the rate of protein secretion. Together, our results reveal key determinants that are important for myonectin folding, secretion, and multimeric assembly and provide a basis for future structure-function studies.

Proteomic Profiling and Mechanistic Investigating of a Novel Anti-Cancer Small Molecule Inhibitor of Sec61

Secreted and transmembrane (TM) proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. During translation, the majority of such proteins require translocation through the Sec61 translocon into the Endoplasmic Reticulum (ER) for further processing. This process is negotiated by unique signal sequences of the translating protein. Therefore, Sec61 represents a novel therapeutic target for cancer treatment through selective blockade of protein secretion. We generated Sec61 inhibitors and assessed their potential against target proteins using HEK293 cell lines stably expressing secreted or TM proteins of interest fused to a luciferase reporter. Additionally, anti-tumor activity was determined across both solid and liquid tumor cell lines in vitro and in mouse models. KZR-8834, a lead candidate identified through a medicinal chemistry campaign, induced cell death in multiple tumor cell lines in vitro, including multiple myeloma (MM), and was effective in xenograft models at doses that did not induce significant body weight loss or clinical signs of toxicity. We utilized quantitative proteomic methods to study KZR-8834 for inhibition of protein secretion and global modulation of protein homeostasis in sensitive and resistant tumor cell lines. Multiple tumor cell types were tested at various doses and time courses followed by subcellular fractionation of cytosolic and membrane/ER proteomes. Subsequent proteomic profiling was performed with Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) and/or Tandem Mass Tag 6-plex (TMT-sixplex). Sensitive targets from both proteomes were further verified using downstream biochemical methods. Sec61 client proteins showed both time- and dose-dependent inhibition upon compound treatment and proteomic results were verified via western blot analysis. Approximately 20% of the total Sec61 clientome and 25% of total proteins detected in a sensitive multiple myeloma (MM) cell line, H929, were significantly down-regulated in response to KZR-8834 treatment at concentrations leading to cell death. IPA pathway analysis suggested that activation of the ER stress response gene ATF4 was induced by KZR-8834 treatment in H929 cells. In a resistant MM cell line, U266, only 13% of the total Sec61 clientome and 5% of total protein detected were significantly down-regulated in response to the same compound treatment. A distinct profile of down-regulated Sec61 clientome was noted with overlap in only 11 of 394 commonly expressed proteins across those two cell lines. Interestingly, in compound treated cells, 39 down-regulated Sec61 client proteins in H929 were either unchanged or upregulated in U266 cells. Conversely, 38 upregulated H929 Sec61 clients were either unchanged or down-regulated in U266 cells. We further explored the ER stress response induced by KZR-8834 via comparative proteomic analysis in H929 cells treated with known ER stress inducers, Tunicamycin and Thapsigargin. These agents, which exert ER stress upon inhibition of N-linked glycosylation and blockade of ER Ca2+ flux, respectively, showed distinct cytosolic proteomic profiles in H929 cells relative to KZR-8834 treatment. These data suggest that KZR-8834-induced blockade of Sec61 results in a unique form of proteotoxic stress in sensitive MM cells. Collectively our results highlight quantitative proteomic profiling as a valuable tool toward elucidating the mechanism of pleiotropic acting molecules like KZR-8834. These studies constitute important first steps toward clarifying the anti-tumor mechanism inhibiting Sec61, a novel pathway agent, for the potential treatment of hematologic tumors. Disclosures Qian: Kezar Life Sciences: Employment, Equity Ownership. Johnson:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Lowe:Kezar Life Sciences: Employment, Equity Ownership. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Millare:Kezar Life Sciences: Employment, Equity Ownership. Muchamuel:Kezar Life Sciences: Employment, Equity Ownership. Wang:Kezar Life Sciences: Employment, Equity Ownership.

Abstract 3087: KZR-8834: A novel, small molecule inhibitor of Sec61-dependent protein secretion with anti-tumor activity

Abstract The majority of secreted and transmembrane (TM) proteins, including growth factors and TM oncogenes, require translocation through Sec61 into the ER for further processing. Sec61, therefore, represents a unique drug discovery opportunity through blockade of typically “undruggable” targets and modulation of protein homeostasis. Multiple inhibitors of protein secretion have been described that directly target Sec61 and have anti-tumor activity but lack adequate pharmaceutical properties and/or tolerability to be considered clinical candidates. We aimed to discover novel, selective inhibitors of Sec61 with increased tolerability as a potential treatment for various malignancies. Sec61 inhibitors were identified using HEK293 cell lines stably expressing secreted or TM proteins of interest fused to a luciferase reporter. Selective anti-tumor activity was assessed via HEK293 reporter cell viability in comparison to tumor cell lines with known levels of sensitivity to published Sec61-modulating compounds. An initial screen resulted in KZR-4152 (4152), which blocked secretion of several target proteins but had no impact on tumor cell viability. Following a medicinal chemistry campaign, KZR-8834 (8834) was identified, which exhibited a 60-fold increase in potency against target proteins relative to 4152. On-target activity of 8834 was demonstrated via protein secretion assays in cells overexpressing a previously described Sec61 mutant with resistance to published compounds. In addition, 8834 did not affect the viability of HEK293 cells or endotoxin- or TCR-stimulated PBMCs. 8834 induced cell death in H929 multiple myeloma cells in vitro (IC50 = 98 nM) with rapid activation (≤8 hours) of caspase 3/7 via dual activation of caspases 8 and 9. When assessed in a panel of 346 human cancer cell lines across 25 different tumor types, head and neck squamous cell carcinoma, acute lymphocytic leukemia, non-Hodgkin’s lymphoma, melanoma, multiple myeloma, and prostate cancer were among the most sensitive tumor types. Weekly and twice-weekly administration of 8834 was effective in an H929 mouse xenograft model at doses that resulted in >90% tumor growth inhibition without significant body weight loss or clinical signs of toxicity. Anti-tumor activity was >50% at doses 1/3rd of the maximum tolerated dose, suggesting a broad therapeutic index. 8834 was also effective in the B16F0 melanoma model, showing tumor growth inhibition similar to that of anti-PD1 therapy. KZR-8834 is a novel, small molecule inhibitor of Sec61-dependent translocation of multiple Sec61 client proteins. 8834 exhibits a broad anti-tumor profile in vitro and therapeutic activity in multiple mouse tumor models, demonstrating a potential new therapy for the treatment of malignant diseases. Citation Format: Eric Lowe, Janet L. Anderl, Andrea R. Fan, Jing Jiang, Henry W. Johnson, Christopher J. Kirk, Dustin McMinn, Tony Muchamuel, Jack Taunton, Jennifer A. Whang. KZR-8834: A novel, small molecule inhibitor of Sec61-dependent protein secretion with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3087.

Abstract 3860: Discovery and characterization of novel anti-cancer small molecule inhibitors of Sec61

Abstract Secreted and membrane proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. The biogenesis of most secreted and transmembrane proteins involves cotranslational translocation of nascent polypeptides into the endoplasmic reticulum through the Sec61 translocon. Broad inhibition of protein secretion can be mediated by natural product-derived Sec61 inhibitors that also possess anti-tumor activity, suggesting that Sec61 represents an untapped therapeutic target for cancer treatment. Here we describe a process for discovering and evaluating novel Sec61 inhibitors with varying degrees of selectivity for therapeutically relevant targets in oncology. We used HEK293 reporter cell lines stably transfected to express secreted and transmembrane proteins of interest fused to luciferase to enable high-throughput screening of potential inhibitors of Sec61-dependent protein secretion. As expected, target selectivity was largely dependent on respective signal sequences. However, full-length protein constructs were found to be 5-10 fold less sensitive to inhibition compared to reporters containing only the signal sequence plus 10 amino acids, suggesting that additional interactions beyond the signal sequence are involved in promoting cotranslational translocation. To verify that novel compounds specifically targeted Sec61, we found that compounds were unable to inhibit the activity of luciferase localized to the cytosol and had reduced potency in reporter cells expressing a Sec61 mutant known to confer resistance to published inhibitors such as cotransin, mycolactone, and apratoxin. We assessed compound selectivity using a library of 37 transiently expressed Sec61-client proteins of therapeutic relevance, revealing 4 distinct chemical scaffolds differentiated by target selectivity profile. Consistent with data from published compounds, broad inhibitors of protein secretion had anti-tumor activity. Of particular interest were compounds able to block expression of multiple immune checkpoints including PD-1, PD-L1, CTLA-4, and CD96. Activity in primary cells, such as receptor expression on activated T-cells and cytokine release from stimulated PBMC, was assessed to verify specificity. Compounds with adequate in vitro metabolic stability were tested in mice for pharmacokinetics as well as inhibition of target protein secretion and global inhibition of Sec61 activity. Taken together, our data suggest that Sec61 is a novel therapeutic target which represents a unique opportunity to both selectively block expression of multiple proteins involved in tumor growth and globally modulate protein homeostasis, resulting in tumor cell death. Citation Format: Eric Lowe, Janet L. Anderl, Andrea R. Fan, Ying Fang, Jing Jiang, Henry W. Johnson, Christopher J. Kirk, Dustin McMinn, Tony Muchamuel, Meera Rao, Phillip P. Sharp, Jack Taunton, Jinhai Wang, Jennifer A. Whang. Discovery and characterization of novel anti-cancer small molecule inhibitors of Sec61 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3860.

A Protein Acyltransferase Complex Promotes Nonapoptotic Cell Death

Lethal small molecules are useful probes to discover and characterize novel cell death pathways and biochemical mechanisms. Here we report that the synthetic oxime caspase-independent lethal 56 (CIL56) induces an unconventional form of nonapoptotic cell death distinct from necroptosis, ferroptosis and classic necrosis. CIL56-induced cell death requires a protein acyltransferase complex comprising the enzyme ZDHHC5 and an accessory subunit, GOLGA7, that is mutually stabilizing and localizes to the plasma membrane. ZDHHC5 activity is not required for CIL56 uptake into the cell, but rather promotes cell death in the context of CIL56 treatment, which inhibits protein secretion from the trans-Golgi apparatus. These results define a novel PAT complex that regulates an unconventional form of nonapoptotic cell death in response to compound treatment.

Golgi stress mediates redox imbalance and ferroptosis in human cells

Cytotoxic activities of several Golgi-dispersing compounds including AMF-26/M-COPA, brefeldin A and golgicide A have previously been shown to induce autophagy or apoptosis. Here, we demonstrate that these Golgi disruptors also trigger ferroptosis, a non-apoptotic form of cell death characterized by iron-dependent oxidative degradation of lipids. Inhibitors of ferroptosis not only counteract cell death, but they also protect from Golgi dispersal and inhibition of protein secretion in response to several Golgi stress agents. Furthermore, the application of sublethal doses of ferroptosis-inducers such as erastin and sorafenib, low cystine growth conditions, or genetic knockdown of SLC7A11 and GPX4 all similarly protect cells from Golgi stress and lead to modulation of ACSL4, SLC7A5, SLC7A11 or GPX4 levels. Collectively, this study suggests a previously unrecognized function of the Golgi apparatus, which involves cellular redox control and prevents ferroptotic cell death.

Identification of pharmacological inhibitors of conventional protein secretion

The retention using selective hooks (RUSH) system allows to withhold a fluorescent biosensor such as green fluorescent protein (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the biosensor from its hook. We constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and streptavidin within the endoplasmic reticulum (ER) lumen and then used this system to screen a compound library for inhibitors of the biotin-induced release of ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. In sum, we demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay.

A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production.

Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.

Non-muscular myosin light chain kinase triggers intermittent hypoxia-induced interleukin-6 release, endothelial dysfunction and permeability

Obstructive sleep apnea is characterized by intermittent hypoxia (IH) which alters endothelial function, induces inflammation and accelerates atherosclerosis-induced cardiovascular diseases. The non-muscular myosin light chain kinase (nmMLCK) isoform contributes to endothelial cell-cell junction opening. Deletion of nmMLCK protects mice from death in septic shock models and prevents atherosclerosis in high-fat diet-fed mice. The aim of the study was to analyze the implication of nmMLCK in IH-induced vascular inflammation. Human aortic endothelial cells were exposed to 6 hours of IH in absence or presence of nmMLCK inhibitors, ML-7 (5 µM) or PIK (150 µM). IH increased reactive oxygen species (ROS) and nitric oxide (NO) production, p65-NFκB activation and IL-6 secretion. While nmMLCK inhibition did not prevent IH-induced ROS production and p65-NFκB activation, it decreased NO production and partially prevented IL-6 secretion. IH-induced IL-6 secretion and vesicle-associated membrane protein-associated vesicles re-organization were inhibited in presence of the inhibitor of protein secretion, brefeldin A, or ML-7. IH increased monocytes transendothelial migration that was partially prevented by ML-7. Finally, IH reduced endothelium-dependent relaxation to acetylcholine of aortas from wild-type but not those taken from nmMLCK-deficient mice. These results suggest that nmMLCK participates to IH-induced endothelial dysfunction resulting from cytokines secretion and endothelial permeability.

Salidroside Regulates Inflammatory Response in Raw 264.7 Macrophages via TLR4/TAK1 and Ameliorates Inflammation in Alcohol Binge Drinking-Induced Liver Injury.

The current study was designed to investigate the anti-inflammatory effect of salidroside (SDS) and the underlying mechanism by using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro and a mouse model of binge drinking-induced liver injury in vivo. SDS downregulated protein expression of toll-like receptor 4 (TLR4) and CD14. SDS inhibited LPS-triggered phosphorylation of LPS-activated kinase 1 (TAK1), p38, c-Jun terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Degradation of IκB-α and nuclear translocation of nuclear factor (NF)-κB were effectively blocked by SDS. SDS concentration-dependently suppressed LPS mediated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels, as well as their downstream products, NO. SDS significantly inhibited protein secretion and mRNA expression of of interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Additionally C57BL/6 mice were orally administrated SDS for continuous 5 days, followed by three gavages of ethanol every 30 min. Alcohol binge drinking caused the increasing of hepatic lipid accumulation and serum transaminases levels. SDS pretreatment significantly alleviated liver inflammatory changes and serum transaminases levels. Further investigation indicated that SDS markedly decreased protein level of IL-1β in serum. Taken together, these data implied that SDS inhibits liver inflammation both in vitro and in vivo, and may be a promising candidate for the treatment of inflammatory liver injury.

Secretion-Positive LGI1 Mutations Linked to Lateral Temporal Epilepsy Impair Binding to ADAM22 and ADAM23 Receptors.

Autosomal dominant lateral temporal epilepsy (ADTLE) is a focal epilepsy syndrome caused by mutations in the LGI1 gene, which encodes a secreted protein. Most ADLTE-causing mutations inhibit LGI1 protein secretion, and only a few secretion-positive missense mutations have been reported. Here we describe the effects of four disease-causing nonsynonymous LGI1 mutations, T380A, R407C, S473L, and R474Q, on protein secretion and extracellular interactions. Expression of LGI1 mutant proteins in cultured cells shows that these mutations do not inhibit protein secretion. This finding likely results from the lack of effects of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair interaction of LGI1 with the ADAM22 and ADAM23 receptors on the cell surface. These results support the existence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing LGI1 mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE.

Tunicamycin-induced inhibition of protein secretion into culture medium of Arabidopsis T87 suspension cells through mRNA degradation on the endoplasmic reticulum.

The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants.

Plasmodium falciparum transfected with ultra bright NanoLuc luciferase offers high sensitivity detection for the screening of growth and cellular trafficking inhibitors.

Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.