Cargo Proteins Research Articles

Sortilin inhibition in microglial cells cannot alleviate ischemia and hypoxia-induced neuronal injury in co-culture.

Sortilin is a single-pass type I transmembrane protein which can bind to various cargo proteins, regulating their surface location, secretion, or degradation in lysosomes. In our previous study, we found that sortilin can regulate progranulin expression by transporting it to lysosomes and reduce neuronal cell injury in hypoxia-ischemia, but the expression and function of sortilin in microglial cells during hypoxia-ischemia are unknown. The purpose of this study was to further investigate the function of sortilin in microglial cells and its effect on neuron cells. In rat BV2 microglial cells, sortilin was knocked down by lentivirus. After oxygen-glucose deprivation/reperfusion (OGD/R), expression of sortilin, progranulin (PGRN) and JNK pathway was detected by western blot, immunofluorescence was used to show the localization of PGRN, secretion of TNFα/IL-6 was measured by Elisa. Then co-culture microglial cells with neuron cells during hypoxia-ischemia and detected the neuron injury by CCK-8 and TUNEL. The expression of sortilin, mature and cleaved PGRN were all increased after OGD/R in microglial cells. Furthermore, sortilin inhibition accompany with less PGRN localization in lysosomes and more mature and less cleaved PGRN expression in microglial cells. Sortilin inhibition also can reduce the inflammatory response in microglial cells, but it does not alleviate neuronal injury in co-culture. This study demonstrated that sortilin can regulate the expression of PGRN and reduce the inflammatory response in microglial cells. However, only inhibiting sortilin in microglial cells did not have an impact on the survival of neurons during ischemia-hypoxia.

Modulating material properties of cargo protein to probe exopher biology

Modulating material properties of cargo protein to probe exopher biology

The Reelin receptor ApoER2 is a cargo for the adaptor protein complex AP-4: Implications for Hereditary Spastic Paraplegia

Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.

Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma.

Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) - an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

Involvement of Extracellular Vesicles in the Proinflammatory Response to Clozapine: Implications for Clozapine-Induced Agranulocytosis.

Most idiosyncratic drug reactions (IDRs) appear to be immune-mediated, but mechanistic events preceding severe reaction onset remain poorly defined. Damage-associated molecular patterns (DAMPs) may contribute to both innate and adaptive immune phases of IDRs, and changes in extracellular vesicle (EV) cargo have been detected post-exposure to several IDR-associated drugs. To explore the hypothesis that EVs are also a source of DAMPs in the induction of the immune response preceding drug-induced agranulocytosis, the proteome and immunogenicity of clozapine- (agranulocytosis-associated drug) and olanzapine- (non-agranulocytosis-associated drug) exposed EVs were compared in two preclinical models: THP-1 macrophages and Sprague-Dawley rats. Compared with olanzapine, clozapine induced a greater increase in the concentration of EVs enriched from both cell culture media and rat serum. Moreover, treatment of drug-naïve THP-1 cells with clozapine-exposed EVs induced an inflammasome-dependent response, supporting a potential role for EVs in immune activation. Proteomic and bioinformatic analyses demonstrated an increased number of differentially expressed proteins with clozapine that were enriched in pathways related to inflammation, myeloid cell chemotaxis, wounding, transforming growth factor-β signaling, and negative regulation of stimuli response. These data indicate that, although clozapine and olanzapine exposure both alter the protein cargo of EVs, clozapine-exposed EVs carry mediators that exhibit significantly greater immunogenicity. Ultimately, this supports the working hypothesis that drugs associated with a risk of IDRs induce cell stress, release of proinflammatory mediators, and early immune activation that precedes severe reaction onset. Further studies characterizing EVs may elucidate biomarkers that predict IDR risk during development of drug candidates. SIGNIFICANCE STATEMENT: This work demonstrates that clozapine, an idiosyncratic drug-induced agranulocytosis (IDIAG)-associated drug, but not olanzapine, a safer structural analogue, induces an acute proinflammatory response and increases extracellular vesicle (EV) release in two preclinical models. Moreover, clozapine-exposed EVs are more immunogenic, as measured by their ability to activate inflammasomes, and contain more differentially expressed proteins, highlighting a novel role for EVs during the early immune response to clozapine and enhancing our mechanistic understanding of IDIAG and other idiosyncratic reactions.

Exosomes Reshape the Osteoarthritic Defect: Emerging Potential in Regenerative Medicine-A Review.

Osteoarthritis (OA) is a joint disorder caused by wear and tear of the cartilage that cushions the joints. It is a progressive condition that can cause significant pain and disability. Currently, there is no cure for OA, though there are treatments available to manage symptoms and slow the progression of the disease. A chondral defect is a common and devastating lesion that is challenging to treat due to its avascular and aneural nature. However, there are conventional therapies available, ranging from microfracture to cell-based therapy. Anyhow, its efficiency in cartilage defects is limited due to unclear cell viability. Exosomes have emerged as a potent therapeutic tool for chondral defects because they are a complicated complex containing cargo of proteins, DNA, and RNA as well as the ability to target cells due to their phospholipidic composition and the altering exosomal contents that boost regeneration potential. Exosomes are used in a variety of applications, including tissue healing and anti-inflammatory therapy. As in recent years, biomaterials-based bio fabrication has gained popularity among the many printable polymer-based hydrogels, tissue-specific decellularized extracellular matrix might boost the effects rather than an extracellular matrix imitating environment, a short note has been discussed. Exosomes are believed to be the greatest alternative option for current cell-based therapy, and future progress in exosome-based therapy could have a greater influence in the field of orthopaedics. The review focuses extensively on the insights of exosome use and scientific breakthroughs centered OA.

Nuclear-import receptors as gatekeepers of pathological phase transitions in ALS/FTD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders on a disease spectrum that are characterized by the cytoplasmic mislocalization and aberrant phase transitions of prion-like RNA-binding proteins (RBPs). The common accumulation of TAR DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), and other nuclear RBPs in detergent-insoluble aggregates in the cytoplasm of degenerating neurons in ALS/FTD is connected to nuclear pore dysfunction and other defects in the nucleocytoplasmic transport machinery. Recent advances suggest that beyond their canonical role in the nuclear import of protein cargoes, nuclear-import receptors (NIRs) can prevent and reverse aberrant phase transitions of TDP-43, FUS, and related prion-like RBPs and restore their nuclear localization and function. Here, we showcase the NIR family and how they recognize cargo, drive nuclear import, and chaperone prion-like RBPs linked to ALS/FTD. We also discuss the promise of enhancing NIR levels and developing potentiated NIR variants as therapeutic strategies for ALS/FTD and related neurodegenerative proteinopathies.

Acute exercise-induced release of innate immune proteins via small extracellular vesicles changes with aerobic fitness and age.

Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation in humans, enabling signalling crosstalk between tissues. Exercise-derived EVs and their cargo have been proposed to mediate adaptations to exercise; however, our understanding of how exercise-derived EV protein cargo is modulated by factors such as aerobic fitness and age of an individual is currently unknown. Here, we examined the circulating sEV proteome following aerobic exercise in healthy males of different ages and aerobic fitness to understand exercise-induced EV response during the aging process. Twenty-eight healthy men completed a bout of 20-min cycling exercise at 70% estimated VO2peak . Small EVs were isolated from blood samples collected before and immediately after exercise, and then quantified using particle analysis and Western blotting. Small EV proteome was examined using quantitative proteomic analysis. We identified a significant increase in 13 proteins in small plasma EVs following moderate-to-vigorous intensity exercise. We observed distinct changes in sEV proteome after exercise in young, mature, unfit, and fit individuals, highlighting the impact of aerobic fitness and age on sEV protein secretion. Functional enrichment and pathway analysis identified that the majority of the significantly altered sEV proteins are associated with the innate immune system, including proteins known to be damage-associated molecular patterns (DAMPs). Together, our findings suggest that exercise-evoked acute stress can positively challenge the innate immune system through the release of signalling molecules such as DAMPs in sEVs, proposing a novel EV-based mechanism for moderate-to-vigorous intensity exercise in immune surveillance pathways.

Targeting of Specialized Metabolites Biosynthetic Enzymes to Membranes and Vesicles by Posttranslational Palmitoylation: A Mechanism of Non-Conventional Traffic and Secretion of Fungal Metabolites.

In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi.

Abstract B052: Defining the lysosome proteome during tumor evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.

Abstract B088: The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle

Abstract As an integral aspect of the metabolic reprogramming that occurs in cancer, oncogenic KRAS mutations drive the stimulation of macropinocytosis, a type of endocytosis that mediates nonselective fluid-phase uptake. Using KRAS-driven models of pancreatic ductal adenocarcinoma (PDAC), we were the first to demonstrate that macropinocytosis functions in tumor cells as a nutrient acquisition pathway. Macropinocytosis triggers the internalization of extracellular proteins via discrete endocytic vesicles called macropinosomes. The incoming protein cargo is targeted for lysosome-dependent degradation, causing the intracellular release of amino acids. These protein-derived amino acids support metabolic fitness by contributing to the intracellular amino acid pools, as well as to the biosynthesis of central carbon metabolites. In this way, macropinocytosis represents a novel amino acid supply route that tumor cells use to survive the nutrient-poor conditions of the tumor microenvironment. While the tenets of the signal transduction events that drive macropinocytosis in cancer have emerged, a detailed picture of the macropinosome itself is not yet available. With the aid of nanotechnology, we have developed a methodology to isolate a pure fraction of macropinosomes from PDAC cells. Using a large-scale proteomic approach, we have been able to generate a list of proteins that reside in macropinosomes. The specificity of this approach was confirmed through the identification and validation of proteins known to specifically localize to these organelles in different contexts, such as Rab5 or SDC1. Thanks to this innovative method, we have demonstrated the involvement of two calcium channels, ATP2B1 and ATP2B4, in the formation of macropinosomes. By elucidating the molecular anatomy of KRAS-driven macropinosomes, we will gain further insight into the regulation and function of macropinocytosis in cancer. Moreover, having a clearer picture of the macropinosome will make us better positioned to exploit this pathway therapeutically and deliver breakthroughs to patients. Citation Format: Ambroise Manceau, Koen M.O. Galenkamp, Cheska M. Galapate, Cosimo Commisso. The macropinosome: Uncovering the molecular anatomy of an oncogene-driven organelle [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B088.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Selective degradation of PL2L60 by metabolic stresses‑induced autophagy suppresses multi‑cancer growth.

It has been reported that PL2L60 proteins, a product of PIWIL2 gene which might be activated by an intragenic promoter, could mediate a common pathway specifically for tumorigenesis. In the present study, it was further identified by using western blot assay that the PL2L60 proteins could be degraded in cancer cells through a mechanism of selective autophagy in response to oxidative stress. The PL2L60 was downregulated in various types of cancer cells under the hypoxic condition independently of HIF‑1α, resulting in apoptosis of cancer cells. Inhibition of autophagy by small interfering RNA targeting of either Beclin‑1 (BECN1) or Atg5 resulted in restoration of PL2L60 expression in hypoxic cancer cell. The hypoxic degradation of PL2L60 was also blocked by the attenuation of the autophagosome membrane protein Atg8/microtubule‑associated protein 1 light chain 3 (LC3) or autophagy cargo protein p62 expression. Surprisingly, Immunofluorescence analysis demonstrated that LC3 could be directly bound to PL2L60 and was required for the transport of PL2L60 from the nucleus to the cytoplasm for lysosomal flux under basal or activated autophagy in cancer cells. Moreover, flow cytometric analysis displayed that knocking down of PL2L60 mRNA but not PIWIL2 mRNA effectively inhibited cancer cell proliferation and promoted apoptosis of cancer cells. The similar results were obtained from invivo tumorigenic experiment, in which PL2L60 downregulation in necroptosis areas was confirmed by immunohistochemistry. These results suggested that various cancer could be suppressed by promoting autophagy. The present study revealed a key role of autophagic degradation of PL2L60 in hypoxia‑induced cancer cell death, which could be used as a novel therapeutic target of cancer.

Characterization of a Novel Mutation in the COPI Vesicle on Binding to Dilysine Motifs

Background/Objective: The heptameric COPI coatomer complex is involved in the formation of vesicles and the intracellular trafficking of proteins between the Golgi and Endoplasmic Reticulum as well as throughout the cytoplasm. Members of the COPI complex bind dilysine motifs found in the C-terminal domain of the cargo protein, particularly KKxx or KxKxx. We generated a point mutation in the WD40 domain of the COPI alpha subunit (⍺-COP). We hypothesized that the E269V mutant ⍺-COP would not co-immunoprecipitate (co-IP) COPI cargo proteins terminating with a dilysine domain of KxKxx (Nucleolin and Stasimon/Tmem41b), but would bind cargo proteins terminating in KKxx (FLAGSyntaxin17). We predicted that a mutation in the ⍺-COP C-terminus, which impairs interaction with ε–COP, would not affect its ability to co-IP dilysine-containing cargo.
 Experimental Design or Project Methods: HEK-293TT cells were transfected with Myctagged wild-type, E269V, and triple mutant (3X) ⍺-COP. The E269V ⍺-COP mutant has an amino acid change at position 269 from glutamic acid to valine. The triple ⍺-COP mutant has three amino acid changes that eliminate binding with the ε–COP COPI subunit. Transfected ⍺-COP was immunoprecipitated using magnetic anti-Myc beads. Endogenous Nucleolin was immunoprecipitated using magnetic Protein A beads conjugated to rabbit polyclonal anti- Nucleolin antibody. Western blots of inputs and immunoprecipitates of each experiment were conducted to determine the ability of ⍺-COP to co-IP C-terminal dilysine-containing proteins.
 Results: Endogenous Nucleolin and Stasimon co-immunoprecipitated with WT and 3X ⍺-COP, but not E269V ⍺-COP.
 Conclusions and Potential Impact: The inability of mutation E269V to co-IP dilysine proteins implies that the WD40 domain of the COPI ⍺-COP protein is required for binding to KxKxxterminating proteins, as typified by Nucleolin and Stasimon.The C-terminal 3X mutation shows that ε-COP is not necessary for dilysine recognition and implies that ⍺-COP directly binds to this KxKxx motif.

Extracellular vesicles released by cancer-associated fibroblast-induced myeloid-derived suppressor cells inhibit T-cell function

ABSTRACT Myeloid cells are known to play a crucial role in creating a tumor-promoting and immune suppressive microenvironment. Our previous study demonstrated that primary human monocytes can be polarized into immunosuppressive myeloid-derived suppressor cells (MDSCs) by cancer-associated fibroblasts (CAFs) in a 3D co-culture system. However, the molecular mechanisms underlying the immunosuppressive function of MDSCs, especially CAF-induced MDSCs, remain poorly understood. Using mass spectrometry-based proteomics, we compared cell surface protein changes among monocytes, in vitro differentiated CAF-induced MDSCs, M1/M2 macrophages, and dendritic cells, and identified an extracellular vesicle (EV)-mediated secretory phenotype of MDSCs. Functional assays using an MDSC/T-cell co-culture system revealed that blocking EV generation in CAF-induced MDSCs reversed their ability to suppress T-cell proliferation, while EVs isolated from CAF-induced MDSCs directly inhibited T-cell function. Furthermore, we identified fructose bisphosphatase 1 (FBP1) as a cargo protein that is highly enriched in EVs isolated from CAF-induced MDSCs, and pharmacological inhibition of FBP1 partially reversed the suppressive phenotype of MDSCs. Our findings provide valuable insights into the cell surface proteome of different monocyte-derived myeloid subsets and uncover a novel mechanism underlying the interplay between CAFs and myeloid cells in shaping a tumor-permissive microenvironment.

Significant Enhancement of Fibroblast Migration, Invasion, and Proliferation by Exosomes Loaded with Human Fibroblast Growth Factor 1.

Exosomes possess several inherent properties that make them ideal for biomedical applications, including robust stability, biocompatibility, minimal immunogenicity, and the ability to cross biological barriers. These natural nanoparticles have recently been developed as drug delivery vesicles. To do so, therapeutic molecules must be efficiently loaded into exosomes first. Very recently, we developed a cell-penetrating peptide (CPP)-based platform for loading of nucleic acids and small molecules into exosomes by taking advantage of the membrane-penetration power of CPPs. Here, we extended this simple but effective platform by loading a protein cargo into exosomes isolated from either mesenchymal stem cells from three different sources or two different cancer cell lines. The protein cargo is a fusion protein YARA-FGF1-GFP through the covalent conjugation of a model CPP called YARA to human fibroblast growth factor 1 (FGF1) and green fluorescence protein (GFP). Loading of YARA-FGF1-GFP into exosomes was time-dependent and reached a maximum of about 1600 YARA-FGF1-GFP molecules in each exosome after 16 h. The ladened exosomes were effectively internalized by mammalian cells, and subsequently, the loaded protein cargo YARA-FGF1-GFP was delivered intracellularly. In comparison to YARA, YARA-FGF1-GFP, the unloaded exosomes, and the exosomes loaded with YARA, the exosomes loaded with YARA-FGF1-GFP substantially promoted the migration, proliferation, and invasion capabilities of mouse and human fibroblasts, which are important factors for wound repair. The work extended our CPP-based exosomal cargo loading platform and established a foundation for developing novel wound-healing therapies using exosomes loaded with FGF1 and other growth factors.

Directional Endothelial Communication by Polarized Extracellular Vesicle Release.

Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1β activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.

Toxoplasma protein export and effector function.

Toxoplasma gondii is a single-celled eukaryotic parasite with a considerable host range that must invade the cells of warm-blooded hosts to survive and replicate. The challenges and opportunities that such a strategy represent have been met by the evolution of effectors that are delivered into host cells, counter host defences and co-opt host cell functions for their own purposes. These effectors are delivered in two waves using distinct machinery for each. In this Review, we focus on understanding the architecture of these protein-export systems and how their protein cargo is recognized and selected. We discuss the recent findings on the role that host manipulation has in latent Toxoplasma infections. We also discuss how these recent findings compare to protein export in the related Plasmodium spp. (the causative agent of malaria) and how this can inform our understanding of host manipulation in the larger Apicomplexa phylum and its evolution.

Cleavage of Hsp70.1 causes lysosomal cell death under stress conditions.

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, β-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated μ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.

Dysregulation of extracellular vesicle protein cargo in female myalgic encephalomyelitis/chronic fatigue syndrome cases and sedentary controls in response to maximal exercise.

In healthy individuals, physical exercise improves cardiovascular health and muscle strength, alleviates fatigue and reduces the risk of chronic diseases. Although exercise is suggested as a lifestyle intervention to manage various chronic illnesses, it negatively affects people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), who suffer from exercise intolerance. We hypothesized that altered extracellular vesicle (EV) signalling in ME/CFS patients after an exercise challenge may contribute to their prolonged and exacerbated negative response to exertion (post-exertional malaise). EVs were isolated by size exclusion chromatography from the plasma of 18 female ME/CFS patients and 17 age- and BMI-matched female sedentary controls at three time points: before, 15 min, and 24h after a maximal cardiopulmonary exercise test. EVs were characterized using nanoparticle tracking analysis and their protein cargo was quantified using Tandem Mass Tag-based (TMT) proteomics. The results show that exercise affects the EV proteome in ME/CFS patients differently than in healthy individuals and that changes in EV proteins after exercise are strongly correlated with symptom severity in ME/CFS. Differentially abundant proteins in ME/CFS patients versus controls were involved in many pathways and systems, including coagulation processes, muscle contraction (both smooth and skeletal muscle), cytoskeletal proteins, the immune system and brain signalling.