Constitutive Secretion Research Articles

The secretory pathway in Tetrahymena is organized for efficient constitutive secretion at ciliary pockets

The secretory pathway in Tetrahymena is organized for efficient constitutive secretion at ciliary pockets

8464 A Unique SERM Reveals An Estrogen Receptor-Dependent Secretion Of DKK1 In Breast Cancer Cells

Abstract Disclosure: K.S. Young: None. G. Hancock: None. S. Kregel: None. S.W. Fanning: Grant Recipient; Self; Olema Oncology. Although Estrogen Receptor positive (ER+) breast cancer has been successfully targeted in primary tumors, the critical limitations of hormone therapies are ultimately revealed as many women often experience disease recurrence. A subset of these patients harbor a distinctive tyrosine to serine missense mutation at position 537 (Y537S) in ER leading to allele-specific transcriptional programs that enhance metastasis. ER is a ligand-dependent master transcriptional regulator. Probing unique ER structural features can reveal new transcriptional activities. We developed a structurally unconventional Selective Estrogen Receptor Modulator (SERM), T6I-29, to better understand these structure-transcriptional relationships. T6I-29 shows effective anti-tumoral activities in Y537S ESR1 breast cancer cells. To examine the anti-tumoral mechanism of T6I-29 action, we performed RNA-sequencing on ESR1 Y537S mutant breast cancer cells. Compared to other clinically relevant compounds, our drug uniquely and significantly downregulated DKK1, a tumor secreted protein known to modulate the Wnt/β-Catenin and PI3K/Akt pathways. Upregulation of DKK1 correlates with metastatic burden across multiple cancers but has not been evaluated in ESR1 mutant breast cancers. We find that DKK1 levels are significantly elevated in the plasma of 108 ER+ breast cancer patients compared to 100 matched healthy donors. Furthermore, in wild-type (WT) PIK3CA breast cancer cell lines, those with Y537S ESR1 mutation show constitutive expression and secretion of DKK1, while DKK1 expression and secretion is estrogen-dependent in WT breast cancer cells. Intriguingly, WT cells show significant surface bound DKK1, which is secreted into the media upon hormone stimulation and blocked by T6I-29. Understanding the ER-dependence and functional importance of DKK1 secretion offers a novel approach to potentially reduce breast cancer metastatic burden. Presentation: 6/3/2024

Influence of AMY1 gene copy number on salivary amylase activity changes induced by exercise in young adults.

Human salivary amylase secretion increases in response to stress; the activity has been reported to rise significantly with high-intensity exercise. The human salivary amylase gene (AMY1) has copy number variation, with the copy number correlating with salivary amylase activity. However, the relationship between individual AMY1 copy number and salivary amylase activity in response to exercise remains unclear. In this study, we investigated AMY1 copy number and fluctuations in amylase activity in 42 healthy university students (25 males and 17 females). Participants engaged in intermittent round-trip interval training on a basketball court. Saliva samples were collected pre- and post-exercise to measure amylase activity. DNA was extracted from the oral mucosa, and AMY1 copy number was quantified using RT-PCR. Results showed a significant increase in amylase activity postexercise. Additionally, amylase activity pre- and post-exercise was positively correlated with AMY1 copy number. The generalize linear model showed that the exercise-induced increase in amylase activity per AMY1 gene was negatively related to the AMY1 copy number and aerobic fitness. Gender has no effect on amylase activity. These results suggest a different mechanism for the constitutive and exercise-induced amylase secretion, while aerobic fitness may be independently involved in the secretion.

Creation of a spatially complex mucus bilayer on an in vitro colon model

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X–Y–Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air–liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 ± 180 μm thick) possessing an inner (149 ± 50 μm) and outer (435 ± 111 μm) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8–10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X–Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease.

Localized, highly efficient secretion of signaling proteins by migrasomes

Migrasomes, enriched with signaling molecules such as chemokines, cytokines and angiogenic factors, play a pivotal role in the spatially defined delivery of these molecules, influencing critical physiological processes including organ morphogenesis and angiogenesis. The mechanism governing the accumulation of signaling molecules in migrasomes has been elusive. In this study, we show that secretory proteins, including signaling proteins, are transported into migrasomes by secretory carriers via both the constitutive and regulated secretion pathways. During cell migration, a substantial portion of these carriers is redirected to the rear of the cell and actively transported into migrasomes, driven by the actin-dependent motor protein Myosin-5a. Once at the migrasomes, these carriers fuse with the migrasome membrane through SNARE-mediated mechanisms. Inhibiting migrasome formation significantly reduces secretion, suggesting migrasomes as a principal secretion route in migrating cells. Our findings reveal a specialized, highly localized secretion paradigm in migrating cells, conceptually paralleling the targeted neurotransmitter release observed in neuronal systems.

Neonatal and maternal upregulation of antileukoproteinase in horses.

The end of gestation, ensuing parturition, and the neonatal period represent highly dynamic phases for immunological changes in both mother and offspring. The regulation of innate immune cells at the maternal-fetal interface during late term pregnancy, after birth, and during microbial colonization of the neonatal gut and other mucosal surfaces, is crucial for controlling inflammation and maintaining homeostasis. Innate immune cells and mucosal epithelial cells express antileukoproteinase (SLPI), which has anti-inflammatory and anti-protease activity that can regulate cellular activation. Here, we developed and validated new monoclonal antibodies (mAbs) to characterize SLPI for the first time in horses. Peripheral blood and mucosal samples were collected from healthy adults horses and a cohort of mares and their foals directly following parturition to assess this crucial stage. First, we defined the cell types producing SLPI in peripheral blood by flow cytometry, highlighting the neutrophils and a subset of the CD14+ monocytes as SLPI secreting immune cells. A fluorescent bead-based assay was developed with the new SLPI mAbs and used to establish baseline concentrations for secreted SLPI in serum and secretion samples from mucosal surfaces, including saliva, nasal secretion, colostrum, and milk. This demonstrated constitutive secretion of SLPI in a variety of equine tissues, including high colostrum concentrations. Using immunofluorescence, we identified production of SLPI in mucosal tissue. Finally, longitudinal sampling of clinically healthy mares and foals allowed monitoring of serum SLPI concentrations. In neonates and postpartum mares, SLPI peaked on the day of parturition, with mares returning to the adult normal within a week and foals maintaining significantly higher SLPI secretion until three months of age. This demonstrated a physiological systemic change in SLPI in both mares and their foals, particularly at the time around birth, likely contributing to the regulation of innate immune responses during this critical period.

High-throughput assay for regulated secretion of neuropeptides in mouse and human neurons

Neuropeptides are the largest group of chemical signals in the brain. More than 100 different neuropeptides modulate various brain functions, and their dysregulation has been associated with neurological disorders. Neuropeptides are packed into dense core vesicles (DCVs), which fuse with the plasma membrane in a calcium-dependent manner. Here, we describe a novel high-throughput assay for DCV exocytosis using a chimera of Nanoluc luciferase and the DCV-cargo neuropeptide Y (NPY). The NPY-Nanoluc reporter colocalized with endogenous DCV markers in all neurons and little mis-localization to other cellular compartments. NPY-Nanoluc reported DCV exocytosis in both rodent and iPSC-derived human neurons, with similar depolarization-, Ca2+-, RAB3- and STXBP1/MUNC18-dependence as low throughput assays. Moreover, NPY-Nanoluc accurately reported modulation of DCV exocytosis by known modulators diacylglycerol-analog and Ca2+-channel blocker, and showed a higher assay sensitivity than a widely used single cell low throughput assay. Lastly, we showed that Nanoluc coupled to other secretory markers reports on constitutive secretion. In conclusion, the NPY-Nanoluc is a sensitive reporter of DCV exocytosis in mammalian neurons, suitable for pharmacological and genomic screening for DCV exocytosis genes and for mechanism-based treatments for CNS disorders.

HSP90β controls NLRP3 autoactivation.

Gain-of-function mutations in NLRP3 are linked to cryopyrin-associated periodic syndromes (CAPS). Although NLRP3 autoinflammasome assembly triggers inflammatory cytokine release, its activation mechanisms are not fully understood. Our study used a functional genetic approach to identify regulators of NLRP3 inflammasome formation. We identified the HSP90β-SGT1 chaperone complex as crucial for autoinflammasome activation in CAPS. A deficiency in HSP90β, but not in HSP90α, impaired the formation of ASC specks without affecting the priming and expression of inflammasome components. Conversely, activating NLRP3 with stimuli such as nigericin or alum bypassed the need for SGT1 and HSP90β, suggesting the existence of alternative inflammasome assembly pathways. The role of HSP90β was further demonstrated in PBMCs derived from CAPS patients. In these samples, the pathological constitutive secretion of IL-1β could be suppressed using a pharmacological inhibitor of HSP90β. This finding underscores the potential of SGT1-HSP90β modulation as a therapeutic strategy in CAPS while preserving NLRP3's physiological functions.

Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.

Selective and Concentrative Enteropancreatic Recirculation of Antibiotics by Pigs.

Antibiotics that are efficacious for infectious pancreatitis are present in pancreatic exocrine secretion (PES) after intravenous administration and above minimal inhibitory concentrations. We measured concentrations of four antibiotics by tandem liquid chromatography-mass spectroscopy in plasma and PES after enteral administration to juvenile pigs with jugular catheters and re-entrant pancreatic-duodenal catheters. Nystatin, which is not absorbed by the intestine nor used for infectious pancreatitis (negative control), was not detected in plasma or PES. Concentrations of amoxicillin increased in plasma after administration (p = 0.035), but not in PES (p = 0.51). Metronidazole and enrofloxacin that are used for infectious pancreatitis increased in plasma after enteral administration and even more so in PES, with concentrations in PES averaging 3.1 (±0.5)- and 2.3 (±0.6)-fold higher than in plasma, respectively (p's < 0.001). The increase in enrofloxacin in PES relative to plasma was lower after intramuscular administration (1.8 ± 0.5; p = 0.001). The present results demonstrate the presence of a selective and concentrative enteropancreatic pathway of secretion for some antibiotics. Unlike the regulated secretion of bile, the constitutive secretion of PES and intestinal reabsorption may provide a continuous exposure of pancreas tissue and the small intestine to recirculated antibiotics and potentially other therapeutic molecules. There is a need to better understand the enteropancreatic recirculation of antibiotics and the associated mechanisms.

Alteration of Immunoregulatory Patterns and Survival Advantage of Key Cell Types in Food Allergic Children.

All allergic responses to food indicate the failure of immunological tolerance, but it is unclear why cow's milk and egg (CME) allergies resolve more readily than reactivity to peanuts (PN). We sought to identify differences between PN and CME allergies through constitutive immune status and responses to cognate and non-cognate food antigens. Children with confirmed allergy to CME (n = 6) and PN (n = 18) and non-allergic (NA) (n = 8) controls were studied. Constitutive secretion of cytokines was tested in plasma and unstimulated mononuclear cell (PBMNC) cultures. Blood dendritic cell (DC) subsets were analyzed alongside changes in phenotypes and soluble molecules in allergen-stimulated MNC cultures with or without cytokine neutralization. We observed that in allergic children, constitutively high plasma levels IL-1β, IL-2, IL-4, IL-5 and IL-10 but less IL-12p70 than in non-allergic children was accompanied by the spontaneous secretion of sCD23, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IFN-γ and TNF-α in MNC cultures. Furthermore, blood DC subset counts differed in food allergy. Antigen-presenting cell phenotypic abnormalities were accompanied by higher B and T cell percentages with more Bcl-2 within CD69+ subsets. Cells were generally refractory to antigenic stimulation in vitro, but IL-4 neutralization led to CD152 downregulation by CD4+ T cells from PN allergic children responding to PN allergens. Canonical discriminant analyses segregated non-allergic and allergic children by their cytokine secretion patterns, revealing differences and areas of overlap between PN and CME allergies. Despite an absence of recent allergen exposure, indication of in vivo activation, in vitro responses independent of challenging antigen and the presence of unusual costimulatory molecules suggest dysregulated immunity in food allergy. Most importantly, higher Bcl-2 content within key effector cells implies survival advantage with the potential to mount abnormal responses that may give rise to the manifestations of allergy. Here, we put forward the hypothesis that the lack of apoptosis of key immune cell types might be central to the development of food allergic reactions.

Regulation of extracellular progranulin in medial prefrontal cortex

Progranulin is a secreted pro-protein that has anti-inflammatory and neurotrophic effects and is necessary for maintaining lysosomal function. Mutations in progranulin (GRN) are a major cause of frontotemporal dementia. Most pathogenic GRN mutations cause progranulin haploinsufficiency, so boosting progranulin levels is a promising therapeutic strategy. Progranulin is constitutively secreted, then taken up and trafficked to lysosomes. Before being taken up from the extracellular space, progranulin interacts with receptors that may mediate anti-inflammatory and growth factor-like effects. Modifying progranulin trafficking is a viable approach to boosting progranulin, but progranulin secretion and uptake by cells in the brain is poorly understood and may involve distinct mechanisms from other parts of the body. Understanding the cell types and processes that regulate extracellular progranulin in the brain could provide insight into progranulin's mechanism of action and inform design of progranulin-boosting therapies. To address this question we used microdialysis to measure progranulin in interstitial fluid (ISF) of mouse medial prefrontal cortex (mPFC). Grn+/− mice had approximately 50% lower ISF progranulin than wild-type mice, matching the reduction of progranulin in cortical tissue. Fluorescent in situ hybridization and immunofluorescence confirmed that microglia and neurons are the major progranulin-expressing cell types in the mPFC. Studies of conditional microglial (Mg-KO) and neuronal (N-KO) Grn knockout mice revealed that loss of progranulin from either cell type results in approximately 50% reduction in ISF progranulin. LPS injection (i.p.) produced an acute increase in ISF progranulin in mPFC. Depolarizing cells with KCl increased ISF progranulin, but this response was not altered in N-KO mice, indicating progranulin secretion by non-neuronal cells. Increasing neuronal activity with picrotoxin did not increase ISF progranulin. These data indicate that microglia and neurons are the source of most ISF progranulin in mPFC, with microglia likely secreting more progranulin per cell than neurons. The acute increase in ISF progranulin after LPS treatment is consistent with a role for extracellular progranulin in regulating inflammation, and may have been driven by microglia or peripheral immune cells. Finally, these data indicate that mPFC neurons engage in constitutive progranulin secretion that is not acutely changed by neuronal activity.

Cell-specific secretory granule sorting mechanisms: the role of MAGEL2 and retromer in hypothalamic regulated secretion.

Intracellular protein trafficking and sorting are extremely arduous in endocrine and neuroendocrine cells, which synthesize and secrete on-demand substantial quantities of proteins. To ensure that neuroendocrine secretion operates correctly, each step in the secretion pathways is tightly regulated and coordinated both spatially and temporally. At the trans-Golgi network (TGN), intrinsic structural features of proteins and several sorting mechanisms and distinct signals direct newly synthesized proteins into proper membrane vesicles that enter either constitutive or regulated secretion pathways. Furthermore, this anterograde transport is counterbalanced by retrograde transport, which not only maintains membrane homeostasis but also recycles various proteins that function in the sorting of secretory cargo, formation of transport intermediates, or retrieval of resident proteins of secretory organelles. The retromer complex recycles proteins from the endocytic pathway back to the plasma membrane or TGN and was recently identified as a critical player in regulated secretion in the hypothalamus. Furthermore, melanoma antigen protein L2 (MAGEL2) was discovered to act as a tissue-specific regulator of the retromer-dependent endosomal protein recycling pathway and, by doing so, ensures proper secretory granule formation and maturation. MAGEL2 is a mammalian-specific and maternally imprinted gene implicated in Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. In this review, we will briefly discuss the current understanding of the regulated secretion pathway, encompassing anterograde and retrograde traffic. Although our understanding of the retrograde trafficking and sorting in regulated secretion is not yet complete, we will review recent insights into the molecular role of MAGEL2 in hypothalamic neuroendocrine secretion and how its dysregulation contributes to the symptoms of Prader-Willi and Schaaf-Yang patients. Given that the activation of many secreted proteins occurs after they enter secretory granules, modulation of the sorting efficiency in a tissue-specific manner may represent an evolutionary adaptation to environmental cues.

A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells.

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Live-imaging of temporally controlled sFLT1 release from the endoplasmic reticulum showed clathrin-dependent sFLT1 trafficking at the Golgi into secretory vesicles that then trafficked to the plasma membrane. Depletion of STX6 altered vessel sprouting in 3D, suggesting that endothelial cell sFLT1 secretion influences proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.

200-OR: Role of Insulin-Degrading Enzyme (IDE) in the Tubulin–Primary Cilium Axis to Regulate Glucagon Secretion

Hyperglucagonemia is a hallmark of T2D, although molecular mechanisms underlying glucagon secretion dysregulation are poorly understood. We have recently reported that genetic ablation of IDE in α-cells (A-IDE-KO mouse) leads to constitutive glucagon secretion, hypertrophy, hyperplasia and increased proliferation. αTC1.9-KD-IDE cells (~30% IDE-knock-down) displayed reduced tubulin and acetylated-α-tubulin protein levels, impaired primary cilium and increased proliferation. Thus, we aimed to elucidate the role of IDE in α-cells tubulin network and ciliogenesis, and how this axis may be part of the glucagon secretion machinery. We have studied by western-blot IDE, tubulin, acetylated-α-tubulin and Arl13b (primary cilium marker) levels in αTC1.9 cells under low- and high-glucose conditions. We have found that high-glucose lowers IDE protein levels by 50% compared to low-glucose conditions, leading to a similar decrease in tubulin, acetylated-α-tubulin and Arl13b protein levels. These findings agree with our previous results using αTC1.9-KD-IDE cells, suggesting a relevant role of IDE in microtubule dynamics to control glucagon granules exocytosis. On the other hand, αTC1.9-KD-Arl13b cells (~70% Arl13b-knock-down) were used as a model of impaired ciliogenesis, showing 50% decrease in tubulin, and dysregulated glucagon secretion, mimicking αTC1.9-KD-IDE cells phenotype. In parallel, insulin-mediated inhibition of glucagon secretion is lost and insulin receptor protein levels were decreased by 40% in both, αTC1.9-KD-IDE and αTC1.9-KD-Arl13b cells, showing the relevance of IDE and primary cilium in the paracrine regulation of α-cells. Furthermore, these results have been reproduced in A-IDE-KO mice isolated islets. In conclusion, our results show that IDE-tubulin-primary cilium axis is regulated during glucose-stimulated glucagon secretion, this axis is also required for the paracrine action of insulin on α-cells to inhibit glucagon secretion. Disclosure E.Casanueva-alvarez: None. A.Sanz-gonzález: None. B.Merino: None. G.Perdomo: None. I.Cozar-castellano: None. Funding Ministerio de Ciencia e Innovación-Spain (PID2019-110496RB-C21)

Constitutive secretion of pro-IL-18 allows keratinocytes to initiate inflammation during bacterial infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases. It is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-γ. Under these conditions, isolates of several other major bacterial pathogens and microbiota of the skin were found to not have significant IL-18-maturing ability. These results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota.

Abstract 4089: PanCAR-specific antibody-cytokine fusion proteins

Abstract CAR-T cells are a potent immunotherapy that redirect T cell specificity towards cell surface tumor-associated antigens. While this therapy has been effective in hematological malignancies, therapies against solid tumors not achieved the same efficacy due to a multitude of additional challenges, including a lack of tumor specific antigens and an immunosuppressive tumor microenvironment. One strategy to increase T cell potency is to use fourth generation CAR-T cells that also secrete an immunostimulatory factor, such as a cytokine. We have created CAR-T cells targeting Tn-MUC1 utilizing a scFv from the 5E5 antibody that also secrete interleukin-12 (IL-12), interleukin-18 (IL-18), and interleukin-23 (IL-23). These cytokine-secreting CAR-T cells have shown an increase in efficacy against multiple epithelial based tumors in impedance-based killing assays. In a xenograft model of human breast cancer, CAR-T cells secreting IL-12 and IL-23 were able to significantly delay tumor growth compared to non-transduced T cells, CAR-T cells alone, and CAR-T cells secreting IL-18. While these cells showed good efficacy in vivo, clinical translation is difficult due to concerns over cytokine storm due to the constitutive secretion of cytokines. To overcome this, we created a fusion protein utilizing an antibody specific to an invariant region found in majority of CAR molecules, including those utilized commercially available therapies and under pre-clinical development, fused to the three immunostimulatory cytokines. In impedance-based killing assays, antibody-cytokine fusions stimulated non-cytokine secreting CAR-T cells to completely clear the tumor at an E:T ratio in which the CAR-T cells incubated with only the antibody alone were unable to control the tumor growth. Interestingly, combination therapy of CAR-T cells and antibody-cytokine fusions outperformed CAR-T cells constitutively secreting the same cytokines in in vitro assays. This work highlights the use of a single reagent capable of increasing the efficacy of the majority of CAR-T cell therapies and may be an important contribution towards treating solid tumor malignancies with CAR-T cell therapies. Citation Format: John T. Keane, Avery D. Posey. PanCAR-specific antibody-cytokine fusion proteins. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4089.

The exocyst complex is an essential component of the mammalian constitutive secretory pathway.

Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR-KO to study biosynthetic sorting from the Golgi to the plasma membrane. Depletion of all canonical exocyst subunits causes cargo accumulation in post-Golgi carriers. Exocyst subunits are recruited to and co-localize with carriers. Exocyst abrogation followed by kinetic trafficking assays of soluble cargoes results in intracellular cargo accumulation. Unbiased secretomics reveals impairment of soluble protein secretion after exocyst subunit knockout. Importantly, in specialized cell types, the loss of exocyst prevents constitutive secretion of antibodies in lymphocytes and of leptin in adipocytes. These data identify exocyst as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.

Do egg cell‐secreted aspartic proteases promote gamete attachment?

During the double fertilization process of flowering plants (angiosperms), pollen tubes transport a pair of immobile sperm cells towards and inside the embryo sac for fusion with egg and central cell, respectively. Male-female gamete interactions during fertilization are critical for reproductive success and seed development, and thus have interested biologists since decades. Compared to animals, little is known about gamete interaction mechanisms in angiosperms. This article is protected by copyright. All rights reserved.

TRAIL receptors promote constitutive and inducible IL-8 secretion in non-small cell lung carcinoma

Interleukin-8 (IL-8/CXCL8) is a pro-angiogenic and pro-inflammatory chemokine that plays a role in cancer development. Non-small cell lung carcinoma (NSCLC) produces high amounts of IL-8, which is associated with poor prognosis and resistance to chemo-radio and immunotherapy. However, the signaling pathways that lead to IL-8 production in NSCLC are unresolved. Here, we show that expression and release of IL-8 are regulated autonomously by TRAIL death receptors in several squamous and adenocarcinoma NSCLC cell lines. NSCLC constitutively secrete IL-8, which could be further enhanced by glucose withdrawal or by treatment with TRAIL or TNFα. In A549 cells, constitutive and inducible IL-8 production was dependent on NF-κB and MEK/ERK MAP Kinases. DR4 and DR5, known regulators of these signaling pathways, participated in constitutive and glucose deprivation-induced IL-8 secretion. These receptors were mainly located intracellularly. While DR4 signaled through the NF-κB pathway, DR4 and DR5 both regulated the ERK-MAPK and Akt pathways. FADD, caspase-8, RIPK1, and TRADD also regulated IL-8. Analysis of mRNA expression data from patients indicated that IL-8 transcripts correlated with TRAIL, DR4, and DR5 expression levels. Furthermore, TRAIL receptor expression levels also correlated with markers of angiogenesis and neutrophil infiltration in lung squamous carcinoma and adenocarcinoma. Collectively, these data suggest that TRAIL receptor signaling contributes to a pro-tumorigenic inflammatory signature associated with NSCLC.