ESCRT Complex Research Articles

Abstract 2139: Galectin-3 Plays A Critical Intracellular Role In Macrophages By Promoting The Repair, Removal, And Replacement Of Damaged Lysosomes And Protecting Against Atherosclerosis

Galectin-3 (Gal3) is a β-galactoside-binding lectin predominantly known as a secreted inflammatory biomarker in cardiovascular disease, with significantly elevated circulating levels in patients with atherosclerosis and myocardial infarction. However, the molecular mechanisms of Galectin-3 action and whether its intracellular role contributes to atherogenesis remain unclear. Using several databases of bulk and single cell RNAseq, we first show that Gal3 transcripts are upregulated during plaque progression with particular abundance in the myeloid/macrophage lineage and foamy macrophages. Furthermore, Gal3 transcripts correlate significantly with increases in a network of lysosomal genes, suggesting a possible link between macrophage Gal3 and lysosomal function. Indeed, instigation of lysosome membrane damage in primary macrophages via cholesterol crystals and LLOMe triggers robust recruitment of Gal3 to lysosomes by sensing exposed carbohydrate moieties of proteins including Lamp1. Gal3 recruitment initiates a two-pronged lysosomal recovery program composed of either lysosomal repair involving the ESCRT complex or lysosomal removal and replacement involving autophagy/lysophagy and TFEB-mediated lysosomal biogenesis. Gal3-KO macrophages corroborate these findings displaying blunted ESCRT recruitment, diminished autophagy and TFEB activation, and resultant accumulation of damaged lysosomes. Gal3-deficiency also results in enhanced apoptosis and inflammasome/IL-1β activation upon triggering of lysosomal stress in macrophages. These findings are recapitulated in vivo, where Gal3-null bone marrow transplanted in atherogenic LDLR-null mice yields increased lesion size as well as altered plaque composition with macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of the advanced plaque. Taken together, our data implicate unique and previously unrecognized protective function for intracellular Galectin-3 in macrophages and atherosclerosis which are distinct from its traditional role as an inflammatory biomarker in cardiovascular disease.

The remnant of the midbody as a cellular signaling mechanism

Cell signaling mechanisms are the basis for intercellular integration and regulation of proliferation and differentiation processes at the systemic level. One of the most plausible ways to control cell-to-cell interaction and targeted distribution of genetic information is for cells to use their own structures that are formed during mitosis and carry RNA-dependent signaling molecules that affect the mechanisms of control of intercellular interaction, cell proliferation and differentiation. The midbody remnant is a microtubule-rich structure that forms between dividing cells in the last stages of cytokinesis. Previously, it was thought to be only a temporary structure of the intercellular bridge during cytokinesis, which served to connect two future daughter cells. This structure is a key regulator of abscission and functions as a signaling platform that coordinates the cytoskeleton and endosomal dynamics during the terminal stages of cell division. The midbody is a subcellular structure that is formed during cell division, during penetration into the cleavage sulcus, when the microtubules of the central spindle are compacted and cross-linked by a thin intracellular bridge connecting the two daughter cells. The midbody plays a key role in organizing cytokinesis by recruiting a variety of mitotic kinases such as Aurora B and Plk1, as well as sulcus endosomes containing Rab11/FIP3, the membrane-rupturing ESCRT complex and the microtubule-rupturing enzyme spastin, all of which are responsible for mediated rupture during the later stages of cytokinesis. The midbodies can serve as extracellular and intracellular polarity signals during early embryogenesis, as well as during epithelialization and polarization of neurons. The molecular mechanism that governs the positioning of the middle body and how it transmits signals to neurons during differentiation or epithelium remains unknown. Importantly, the remains of the middle bodies can also function as intracellular signaling scaffolds that regulate proliferation and fate postmitotic cells. Since these structures can be released outside cells and taken up by other non-mitotic cells, it is suggested that they may function as vehicles for alternative transmission of complex sets of signaling molecules and/or receptors between cells, thus profoundly affecting signaling in general.

Emerging roles of lysosome homeostasis (repair, lysophagy and biogenesis) in cancer progression and therapy

In the era of personalized therapy, precise targeting of subcellular organelles holds great promise for cancer modality. Taking into consideration that lysosome represents the intersection site in numerous endosomal trafficking pathways and their modulation in cancer growth, progression, and resistance against cancer therapies, the lysosome is proposed as an attractive therapeutic target for cancer treatment. Based on the recent advances, the current review provides a comprehensive understanding of molecular mechanisms of lysosome homeostasis under 3R responses: Repair, Removal (lysophagy) and Regeneration of lysosomes. These arms of 3R responses have distinct role in lysosome homeostasis although their interdependency along with switching between the pathways still remain elusive. Recent advances underpinning the crucial role of (1) ESCRT complex dependent/independent repair of lysosome, (2) various Galectins-based sensing and ubiquitination in lysophagy and (3) TFEB/TFE proteins in lysosome regeneration/biogenesis of lysosome are outlined. Later, we also emphasised how these recent advancements may aid in development of phytochemicals and pharmacological agents for targeting lysosomes for efficient cancer therapy. Some of these lysosome targeting agents, which are now at various stages of clinical trials and patents, are also highlighted in this review.

PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target

Aberrant overexpression or activation of EGFR drives the development of non-small cell lung cancer (NSCLC) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) by secondary EGFR mutations or c-MET amplification/activation remains as a major hurdle for NSCLC treatment. We previously identified WDR4 as a substrate adaptor of Cullin 4 ubiquitin ligase and an association of WDR4 high expression with poor prognosis of lung cancer. Here, using an unbiased ubiquitylome analysis, we uncover PTPN23, a component of the ESCRT complex, as a substrate of WDR4-based ubiquitin ligase. WDR4-mediated PTPN23 ubiquitination leads to its proteasomal degradation, thereby suppressing lysosome trafficking and degradation of wild type EGFR, EGFR mutant, and c-MET. Through this mechanism, WDR4 sustains EGFR and c-MET signaling to promote NSCLC proliferation, migration, invasion, stemness, and metastasis. Clinically, PTPN23 is downregulated in lung cancer and its low expression correlates with WDR4 high expression and poor prognosis. Targeting WDR4-mediated PTPN23 ubiquitination by a peptide that competes with PTPN23 for binding WDR4 promotes EGFR and c-MET degradation to block the growth and progression of EGFR TKI-resistant NSCLC. These findings identify a central role of WDR4/PTPN23 axis in EGFR and c-MET trafficking and a potential therapeutic target for treating EGFR TKI-resistant NSCLC.

Birth, life and death of the Arabidopsis IRT1 iron transporter: the role of close friends and foes.

IRT1 intracellular dynamics and function are finely controlled through protein-protein interactions. In plants, iron uptake from the soil is tightly regulated to allow optimal growth and development. Iron acquisition in Arabidopsis root epidermal cells requires the IRT1 transporter, which also mediates the entry of non-iron metals. In this mini-review, we describe how protein-protein interactions regulate IRT1 intracellular dynamics and IRT1-mediated metal uptake to maintain iron homeostasis. Recent interactomic data provided interesting clues on IRT1 secretion and the putative involvement of COPI- and COPII-mediated pathways. Once delivered to the plasma membrane, IRT1 can interact with other components of the iron uptake machinery to form an iron acquisition complex that likely optimizes iron entrance in root epidermal cells. Then, IRT1 may be internalized from the plasma membrane. In the past decade, IRT1 endocytosis emerged as an essential mechanism to control IRT1 subcellular localization and thus to tune iron uptake. Interestingly, IRT1 endocytosis and degradation are regulated by its non-iron metal substrates in an ubiquitin-dependent manner, which requires a set of interacting-proteins including kinases, E3 ubiquitin ligases and ESCRT complex subunits. This mechanism is essential to avoid non-iron metal overload in Arabidopsis when the iron is scarce.

Abstract 335: Hsf1 Regulates Exosome Biogenesis And Cargo Enrichment Of These Exosomes In Stem Cells Thereby Enhancing Their Potential Of Ischemic Heart Regeneration.

Introduction: Paracrine mechanism of stem cell-based therapy holds importance. However, genes regulating the paracrine factors have not been elucidated in detail. Here in this study, we evaluated the role of HSF1 in exosome biogenesis and their composition in neonatal cardiac mesenchymal stem cells (nMSCs) in concert with ischemic heart generation. Hypothesis: HSF1 in nMSCs promotes production of exosomes, functional exosomal miRs cargo, and exosome acquisition by recipient cells. Methodology: nMSCs and adult MSCs (aMSCs) were respectively isolated from right atrial appendage of neonate (<1month) and adult ( > 40 years) patients. nMSCs were knocked down (KD) for HSF1 gene using CRISPR-CAS9 technique while HSF1 gene was overexpressed (OE) in aMSCs using HSF1 overexpressing lentiviral constructs. Exosome biogenesis was evaluated using nano site technique. We next evaluated effect of HSF1 KD and OE on miRs cargo in nMSCs and aMSCs respectively. Exosome acquisition by target cells was studied by mCherry labeling technique. The potential of nMSCs and aMSCs and their corresponding exosomes to repair rat ischemic heart was evaluated under the effect of HSF1 KD and OE. Results: HSF1-KD in nMSCs resulted in significant reduction in production of exosomes, downregulation of ESCRT complex genes and therapeutic miRs viz, miR-199a, mi590-3P and miR-146. In contrast, HSF-1 over expression in aMSCs statistically increased production of exosomes, ESCRT complex genes and of therapeutic miRs. Moreover, HSF-1 OE in aMSCs resulted in statistical downregulation of miR-122, known to induce apoptosis, and downregulation of p27, a known target of miR-590. We identified that potential of nMSCs to repair ischemic heart decreased statistically after HSF1-KD while that of aMSCs increased significantly after HSF1-OE. Finally, we identified exosomes from these stem cells were preferentially taken up by cells in injured myocardium and not by cells in non-injured one and that HSF1-OE increased the uptake by recipient cells while HSF1-KD had opposite effect. Conclusion: Together, our results demonstrate that HSF-1 plays an important role in exosome biogenesis, miRs cargo enrichment and uptake of exosomes by recipient cells.

A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes

Exosomes are small extracellular vesicles of ∼30 to 150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes. We show that exosome cargo proteins bud from cells (i) in exosome-sized vesicles regardless of whether they are localized to plasma or endosome membranes, (ii) ∼5-fold more efficiently when localized to the plasma membrane, (iii) ∼5-fold less efficiently when targeted to the endosome membrane, (iv) by a stochastic process that leads to ∼100-fold differences in their abundance from one exosome to another, and (v) independently of small GTPase Rab27a, the ESCRT complex–associated protein Alix, or the cargo protein CD63. Taken together, our results demonstrate that cells use a shared, stochastic mechanism to bud exosome cargoes along the spectrum of plasma and endosome membranes and far more efficiently from the plasma membrane than the endosome. Our observations also indicate that the pronounced variation in content between different exosome-sized vesicles is an inevitable consequence of a stochastic mechanism of small vesicle biogenesis, that the origin membrane of exosome-sized extracellular vesicles simply cannot be determined, and that most of what we currently know about exosomes has likely come from studies of plasma membrane-derived vesicles.

HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8+ T-cell infiltration into tumors

The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.

Abstract 522: Macrophage Galectin 3 Protects Against Atherosclerosis By Promoting Repair, Removal And Replacement Of Damaged Lysosomes

Understanding the derangements in cellular processes that underlie macrophage dysfunction is critical to the development of future therapeutics. The autophagy-lysosome system in macrophages plays critical role in the degradation and recycling of long-lived/damaged intracellular material. Atherosclerotic plaque macrophages develop an impairment in autophagy and lysosomes which is a contributing factor to plaque progression. Thus, one can surmise that harnessing programs of lysosomal repair and lysosomal biogenesis in macrophages can have atheroprotective effects. A class of important regulators of the autophagy-lysosomal system are cytosolic lectins. When the lysosome membrane becomes damaged, Galectin-3 (Gal3) recognizes the damage and recruits autophagic receptors and delivers the cargo to autophagosomes. Additionally, Gal3 may recruit the ESCRT complex and facilitate the repair and restoration of lysosomes. We hypothesized that Gal3 provides a protective role in atherogenic macrophages, by repairing or removing damaged lysosomes, thus preventing the relevant deleterious signaling pathways such as inflammasome activation and apoptosis. We utilized Gal3-KO macrophages and mice to study the role of Gal3 in atherosclerosis. First, we show that Gal3-KO macrophages are highly sensitive to lysosome membrane damaging agents including atherogenic oxLDL and cholesterol crystals, exhibiting decreased autophagy and protein aggregate clearance compared to the WT controls. Furthermore, the recruitment of lysosome repair mechanisms involving the ESCRT complex are blunted. Such blunting of critical repair mechanisms results in increased macrophage apoptosis and inflammasome/IL-1β activation. These findings are also recapitulated in vivo, where bone marrow transplants of atherogenic LDLR-null mice with Gal3-deficient bone marrow results in increased plaque size and altered plaque composition with significant macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of advanced plaques. Overall, our data demonstrate important roles for Gal3 in macrophage lysosome repair mechanisms which are a critical response against atherosclerosis.

Origin of eukaryotes: What can be learned from the first successfully isolated Asgard archaeon.

The origin of cellular complexity characterizing eukaryotic cells remains a central unresolved issue in the study of diversification of cellular life on Earth. The isolation by Imachi et al.1 of a member of the Asgard archaea2 - a contemporary relative of organisms thought to have given rise to eukaryotic cells about 2 billion years ago - now promises new insight. The complete genome sequence of the isolated Lokiarchaeum strain confirms that the eukaryotic signature proteins (ESPs) previously identified in the Lokiarchaeota3 and other Asgard archaea2 are indeed encoded by these archaeal genomes and do not represent contamination from eukaryotes. These ESPs encode homologs of eukaryotic actins, small GTPases and the ESCRT complex proteins and are required for the functioning of complex eukaryotic cells. The new, slowly growing, anaerobic laboratory strain allows a first direct look at these organisms and provides key insights into the morphology and metabolism of an Asgard archaeal organism. The work has provided valuable information for other laboratories that aim to isolate and characterize related organisms from other environments.

Proteomic and metabolomic profiling of urine uncovers immune responses in patients with COVID-19.

The utility of the urinary proteome in infectious diseases remains unclear. Here, we analyzed the proteome and metabolome of urine and serum samples from patients with COVID-19 and healthy controls. Our data show that urinary proteins effectively classify COVID-19 by severity. We detect 197 cytokines and their receptors in urine, but only 124 in serum using TMT-based proteomics. The decrease in urinary ESCRT complex proteins correlates with active SARS-CoV-2 replication. The downregulation of urinary CXCL14 in severe COVID-19 cases positively correlates with blood lymphocyte counts. Integrative multiomics analysis suggests that innate immune activation and inflammation triggered renal injuries in patients with COVID-19. COVID-19-associated modulation of the urinary proteome offers unique insights into the pathogenesis of this disease. This study demonstrates the added value of including the urinary proteome in a suite of multiomics analytes in evaluating the immune pathobiology and clinical course of COVID-19 and, potentially, other infectious diseases.

Platelet-derived extracellular vesicles express NADPH oxidase-1 (Nox-1), generate superoxide and modulate platelet function

Rapid repair of plasma membrane wounds is critical for cellular survival. Exocytic patches, membrane tension reduction and endocytosis were previously proposed to mediate resealing. A recent study implicating the ESCRT complex adds to the growing evidence that repair involves removal of damaged plasma membrane, and not simply patching the wound.

Abstract P478: Farnesyl Transferase Inhibitor Tipifarnib Reduces Circulating Exosomes And Protects Against Pressure Overload-mediated Cardiac Hypertrophy

Clinically, Hypertrophic cardiomyopathy (HCM) in response to pathophysiological stress is one of the major initiating factors for the onset of cardiac remodeling leading to heart failure. Studies have revealed that HCM characterized by left ventricular hypertrophy, hypercontractility, and impaired relaxation is mainly driven by an intricate crosstalk among the multiple cellular and molecular mechanisms, which leads to heart failure. In agreement with this observation, we investigated if the Tipifarnib-mediated reduction/alteration of circulating exosomes mediates cardiac cell communication during HCM. Several studies have shown Tipifarnib as a potential Farnesyl transferase inhibitor. However, in recent past Tipifarnib has been shown to target exosomes biogenesis by several mechanisms such as inhibiting Ras pathway, ESCRT complex etc. Tipifarnib treatment in mice significantly reduced the number of circulating plasma exosomes. We examined the response of Tipifarnib treatment (10 mg/kg body weight) in C57BL6J male mice subjected to transverse aortic constriction (TAC) surgery. Untreated TAC mice had worsening of systolic Left Ventricular function at 4 weeks that further deteriorated at 8 weeks, while the treatment with Tipifarnib substantially improved cardiac functions by reducing cardiac hypertrophy and fibrosis. Exosomes isolated from the serum of sham and TAC mice with or without tipifarnib were used for in vitro cell based analyses. We observed that the exosomes isolated from Tipifarnib treated TAC mice reduced isoproterenol (ISO)-induced cardiomyoblast hypertrophy and fibrosis-associated genes in adult cardiac fibroblasts. Taken together, our studies suggest Tipifarnib protects against pressure overload induced cardiac remodeling and dysfunction by altering hypertrophic and fibrotic gene expression, by potentially reducing circulating exosomes or by altering exosome contents. Ongoing studies will clarify the molecular mechanisms of these observations.

The ESCRT-III complex contributes to macromitophagy in yeast.

Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, glutathione-S-transferase (GST) pulldown and bimolecular fluorescence complementation (BiFC) assay, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32-marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.

SNX-3 mediates retromer-independent tubular endosomal recycling by opposing EEA-1-facilitated trafficking.

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.

Loss of HCRP1 leads to upregulation of PD-L1 via STAT3 activation and is of prognostic significance in EGFR-dependent cancer

Loss of hepatocellular carcinoma-related protein 1 (HCRP1) (alias VPS37A) plays a role in endocytosis of receptor tyrosine kinases as a member of the ESCRT complex and has been linked to poor patient outcome in various types of epithelial cancer. To this date, the molecular and biological mechanisms explaining how its absence would contribute to tumor progression remain unknown. Using genomic editing with CRISPR-Cas9, we generated ovarian and breast cancer cell lines with loss-of-function mutations of HCRP1. We hypothesized that pathways downstream of receptor tyrosine kinases such as epidermal growth factor receptor are affected by HCRP1 loss and looked for deregulated signaling using immunoblotting and classical cancer biology assays. In our study, we show that endogenous deletion of HCRP1 leads to elevated phosphorylation of the transcription factor Signal transducer and activator of transcription 3 (STAT3) and induces upregulation of PD-L1, an important regulator of immune checkpoint inhibition. HCRP1 loss further leads to a mesenchymal phenotype switch in cancer cells, leading to increased proliferation and migration. Concludingly, our data emphasize the role of the tumor microenvironment in tumors with low or absent HCRP1 expression and suggest HCRP1 loss as a potential marker for metastatic potential and immunogenicity of epidermal growth factor receptor-driven cancer.

The ambiguity of the basic terms related to eukaryotes and the more consistent etymology based on eukaryotic signatures in Asgard archaea

The endosymbiosis theory most widely accepted variant surmises the engulfment of a bacterial cell by an archaeal cell. For decades, this scenario was reputed to be an unconfirmed hypothesis, and only recently it has obtained an indirect proof in Asgard archaea environmental DNA encoding eukaryotic signatures − actin cytoskeleton, small GTPases, and ESCRT complex. In view of growing interest to this aspect of the endosymbiosis theory, it seemed timely to revisit the basic terms eukaryotic cell/eukaryotes/nucleated organisms. The article highlights the ambiguous applications of these terms, and seeks for their consistency with regard to phylogeny and taxonomy. Additionally, new name Caryosignifera is proposed for the phylum represented by: (1) the underexplored Asgard archaea manifested by above-mentioned environmental DNA; (2) cultured species of engulfing Asgard archaea; (3) eukaryotic host cells in nucleated organisms (protists, algae, plants, fungi, and animals).

ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC–MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

Endocytic sorting and downregulation of the M2 acetylcholine receptor is regulated by ubiquitin and the ESCRT complex

Cholinergic dysfunction plays a critical role in a number of disease states, and the loss of functional muscarinic acetylcholine receptors plays a key role in disease pathogenesis. Therefore, preventing receptor downregulation would maintain functional receptor number, and be predicted to alleviate symptoms. However, the molecular mechanism(s) underlying muscarinic receptor downregulation are currently unknown. Here we demonstrate that the M2 muscarinic receptor undergoes rapid lysosomal proteolysis, and this lysosomal trafficking is facilitated by ubiquitination of the receptor. Importantly, we show that this trafficking is driven specifically by ESCRT mediated involution. Critically, we provide evidence that disruption of this process leads to a re-routing of the trafficking of the M2 receptor away from the lysosome and into recycling pathway, and eventually back to the plasma membrane. This study is the first to identify the process by which the M2 muscarinic acetylcholine receptor undergoes endocytic sorting, and critically reveals a regulatory checkpoint that represents a target to pharmacologically increase the number of functional muscarinic receptors within the central nervous system.

Abstract 1897: Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3

Abstract Introduction: Tumors are complex systems where cell-cell communication between intra-tumoral and extra-tumoral cells play a crucial role. Extracellular vesicles (EVs), membrane-limited vesicles secreted by normal and malignant cells, have been proposed to act as mediators of intercellular communication in physiological and pathological scenarios. It is now well established that integrins are constantly endocytosed and recycled back to the plasma membrane through multiple routes. Accumulating evidence clearly indicates that endocytosis and recycling of integrins plays a crucial role during cancer progression, invasion, and metastasis. Here we describe a fundamental link between endocytosis mediated integrin trafficking and the integrin-mediated uptake of exosomes. Objectives: As the integrins are crucial for the role of exosomes in cancer progression and particularly in metastasis we were aiming at delineating the role of ITGB3 in exosome-mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231. Methodology: Using stable shRNA of ITGB3 (shITGB3) in the triple-negative breast cancer cell line MDA.MB.231, EVs were isolated by ultracentrifugation and characterized by Western blot analysis to determine the protein composition and by NTA and CryoEM to measure the amount of EVs. An LC-MS/MS-based survey was performed, to determine the differences in the protein composition of EVs after knock-down of ITGB3. Uptake of fluorescently labeled exosomes into recipient cells was measured by flow cytometry. The colony forming capacity induced by conditioned medium was used to assess the functional impact on the role of ITGB3 in intercellular communication. Results: Analysis of the EVs fraction of shITGB3 cell revealed a striking decrease of classical markers of exosomes as judged by western blot analysis of proteins such as TSG101, CD81, but not others like Flotillin-1. LC-MS/MS analysis of shITGB3-MDA.MB.231 derived-exosomes furthermore revealed clear defects in protein complexes required for exosome formation, namely the ESCRT complex and the Syndecan-syntenin-ALIX complex. Both complexes are required for the formation of endosomal intraluminal vesicles that get released as exosomes. On the other hand, NTA analysis and CryoEM showed the overall amount of EVs was increased. In line with this, the uptake of fluorescently labeled EVs was strongly impaired in shITGB3 cells. By the use of small molecule inhibitors, we can furthermore demonstrate that this defect linked to endocytosis of EVs. Conclusions: Here we demonstrate that ITGB3 plays a crucial role in controlling the composition of different vesicle populations in the extracellular space, linked to the endocytosis of integrins and interference with this mechanism results in defects in the uptake of EVs and impedes the generation of exosomes. The functional effects are discussed Citation Format: Pedro Fuentes Varela, Marta Sesé, Pedro Jesús Guijarro, Marta Emperador, Héctor Peinado, Stefan Hümmer, Santiago Ramon y Cajal. Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3 [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 1897.