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1.
EMBO J ; 43(6): 904-930, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38337057

ABSTRACT

Mitochondrial outer membrane permeabilisation (MOMP) is often essential for apoptosis, by enabling cytochrome c release that leads to caspase activation and rapid cell death. Recently, MOMP has been shown to be inherently pro-inflammatory with emerging cellular roles, including its ability to elicit anti-tumour immunity. Nonetheless, how MOMP triggers inflammation and how the cell regulates this remains poorly defined. We find that upon MOMP, many proteins localised either to inner or outer mitochondrial membranes are ubiquitylated in a promiscuous manner. This extensive ubiquitylation serves to recruit the essential adaptor molecule NEMO, leading to the activation of pro-inflammatory NF-κB signalling. We show that disruption of mitochondrial outer membrane integrity through different means leads to the engagement of a similar pro-inflammatory signalling platform. Therefore, mitochondrial integrity directly controls inflammation, such that permeabilised mitochondria initiate NF-κB signalling.


Subject(s)
NF-kappa B , Ubiquitin , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , Ubiquitin/metabolism , Mitochondrial Membranes/metabolism , Mitochondria/metabolism , Apoptosis/physiology , Inflammation/metabolism
2.
EMBO J ; 42(18): e113987, 2023 09 18.
Article in English | MEDLINE | ID: mdl-37577760

ABSTRACT

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.


Subject(s)
Monomeric GTP-Binding Proteins , Ovarian Neoplasms , Humans , Female , Integrins/metabolism , Proteomics , Phosphatidylinositol 3-Kinases/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Monomeric GTP-Binding Proteins/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism
3.
Nucleic Acids Res ; 52(5): 2389-2415, 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38224453

ABSTRACT

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.


Subject(s)
Cell Nucleus , DNA Damage , Stromal Interaction Molecule 1 , Chromatin/genetics , DNA Repair , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Mitomycin/pharmacology , Proteomics , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/metabolism , Humans , Cell Nucleus/metabolism , Neoplasm Proteins/metabolism
4.
Proc Natl Acad Sci U S A ; 119(26): e2111506119, 2022 06 28.
Article in English | MEDLINE | ID: mdl-35737835

ABSTRACT

Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.


Subject(s)
Fucosidosis , Macroautophagy , Polysaccharides , Animals , Fucosidosis/genetics , Fucosidosis/metabolism , Lysosomes/metabolism , Macroautophagy/physiology , Mice , Polysaccharides/metabolism , alpha-L-Fucosidase/genetics , alpha-L-Fucosidase/metabolism
5.
PLoS Biol ; 18(8): e3000774, 2020 08.
Article in English | MEDLINE | ID: mdl-32745097

ABSTRACT

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.


Subject(s)
Dictyostelium/genetics , MAP Kinase Kinase Kinase 3/genetics , Protozoan Proteins/genetics , Pseudopodia/metabolism , Wiskott-Aldrich Syndrome Protein Family/genetics , Animals , CRISPR-Cas Systems , Cell Adhesion , Cell Line, Tumor , Chemotaxis/genetics , Dictyostelium/metabolism , Dictyostelium/ultrastructure , Gene Editing/methods , Gene Expression Regulation , MAP Kinase Kinase Kinase 3/metabolism , Melanocytes/metabolism , Melanocytes/ultrastructure , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mutation , NIH 3T3 Cells , Phenotype , Phosphorylation , Ploidies , Protozoan Proteins/metabolism , Pseudopodia/genetics , Pseudopodia/ultrastructure , Wiskott-Aldrich Syndrome Protein Family/metabolism
6.
EMBO J ; 37(13)2018 07 02.
Article in English | MEDLINE | ID: mdl-29844016

ABSTRACT

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.


Subject(s)
Centrosome/metabolism , Heat-Shock Proteins/metabolism , Microfilament Proteins/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Humans , Models, Molecular , Prognosis
7.
J Cell Sci ; 133(8)2020 04 30.
Article in English | MEDLINE | ID: mdl-32184267

ABSTRACT

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is crucial for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the crucial role of the cytosol in regulating ER redox homeostasis, ensuring correct protein folding and facilitating the degradation of misfolded ER proteins.


Subject(s)
Membrane Proteins , Thioredoxin-Disulfide Reductase , Animals , Cytosol , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Oxidation-Reduction , Protein Folding , Thioredoxin-Disulfide Reductase/genetics , Thioredoxin-Disulfide Reductase/metabolism
8.
J Cell Sci ; 128(2): 251-65, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25413351

ABSTRACT

Podosomes are integrin-containing adhesion structures commonly found in migrating leukocytes of the monocytic lineage. The actin cytoskeletal organisation of podosomes is based on a WASP- and Arp2/3-mediated mechanism. WASP also associates with a second protein, WIP (also known as WIPF1), and they co-localise in podosome cores. Here, we report for the first time that WIP can be phosphorylated on tyrosine residues and that tyrosine phosphorylation of WIP is a trigger for release of WASP from the WIP-WASP complex. Using a knockdown approach together with expression of WIP phosphomimics, we show that in the absence of WIP-WASP binding, cellular WASP is rapidly degraded, leading to disruption of podosomes and a failure of cells to degrade an underlying matrix. In the absence of tyrosine phosphorylation, the WIP-WASP complex remains intact and podosome lifetimes are extended. A screen of candidate kinases and inhibitor-based assays identified Bruton's tyrosine kinase (Btk) as a regulator of WIP tyrosine phosphorylation. We conclude that tyrosine phosphorylation of WIP is a crucial regulator of WASP stability and function as an actin-nucleation-promoting factor.


Subject(s)
Cytoskeletal Proteins/metabolism , Extracellular Matrix/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Wiskott-Aldrich Syndrome Protein/metabolism , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Agammaglobulinaemia Tyrosine Kinase , Animals , Cytoskeletal Proteins/genetics , Extracellular Matrix/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Macrophages/metabolism , Phosphorylation/genetics , Podosomes/metabolism , Protein Binding , Protein-Tyrosine Kinases/genetics , Tyrosine/metabolism , Wiskott-Aldrich Syndrome Protein/genetics
9.
Mol Cell Proteomics ; 11(6): M111.015131, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22223895

ABSTRACT

N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.


Subject(s)
Arabidopsis Proteins/metabolism , Protein Processing, Post-Translational , Proteome/metabolism , Acetylation , Acetyltransferases/chemistry , Acetyltransferases/metabolism , Arabidopsis Proteins/chemistry , Cell Line , Chromatography, Ion Exchange , Computer Simulation , Humans , Models, Biological , Peptide Fragments/chemistry , Phylogeny , Protein Structure, Tertiary , Protein Transport , Proteome/chemistry , Tandem Mass Spectrometry
10.
Curr Biol ; 34(15): 3564-3581.e6, 2024 Aug 05.
Article in English | MEDLINE | ID: mdl-39059394

ABSTRACT

Hermansky-Pudlak syndrome (HPS) is an inherited disorder of intracellular vesicle trafficking affecting the function of lysosome-related organelles (LROs). At least 11 genes underlie the disease, encoding four protein complexes, of which biogenesis of lysosome-related organelles complex-2 (BLOC-2) is the last whose molecular action is unknown. We find that the unicellular eukaryote Dictyostelium unexpectedly contains a complete BLOC-2, comprising orthologs of the mammalian subunits HPS3, -5, and -6, and a fourth subunit, an ortholog of the Drosophila LRO-biogenesis gene, Claret. Lysosomes from Dictyostelium BLOC-2 mutants fail to mature, similar to LROs from HPS patients, but for all endolysosomes rather than a specialized subset. They also strongly resemble lysosomes from WASH mutants. Dictyostelium BLOC-2 localizes to the same compartments as WASH, and in BLOC-2 mutants, WASH is inefficiently recruited, accounting for their impaired lysosomal maturation. BLOC-2 is recruited to endolysosomes via its HPS3 subunit. Structural modeling suggests that all four subunits are proto-coatomer proteins, with important implications for BLOC-2's molecular function. The discovery of Dictyostelium BLOC-2 permits identification of orthologs throughout eukaryotes. BLOC-2 and lysosome-related organelles, therefore, pre-date the evolution of Metazoa and have broader and more conserved functions than previously thought.


Subject(s)
Dictyostelium , Lysosomes , Protozoan Proteins , Dictyostelium/genetics , Dictyostelium/metabolism , Lysosomes/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Animals , Evolution, Molecular , Coatomer Protein/genetics , Coatomer Protein/metabolism , Hermanski-Pudlak Syndrome/genetics , Hermanski-Pudlak Syndrome/metabolism
11.
Sci Signal ; 17(827): eade0580, 2024 Mar 12.
Article in English | MEDLINE | ID: mdl-38470957

ABSTRACT

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.


Subject(s)
Cancer-Associated Fibroblasts , Neoplasms , Humans , Cancer-Associated Fibroblasts/metabolism , Endothelial Cells , Neoplasms/metabolism , Cell Membrane , Cell Line , Fibroblasts/metabolism , Tumor Microenvironment , Cell Line, Tumor
12.
Nat Cancer ; 5(4): 659-672, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38286828

ABSTRACT

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification.


Subject(s)
DNA, Mitochondrial , Glycolysis , Immune Checkpoint Inhibitors , Melanoma , Mutation , DNA, Mitochondrial/genetics , Animals , Melanoma/genetics , Melanoma/drug therapy , Mice , Humans , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Glycolysis/genetics , Tumor Microenvironment , Cell Line, Tumor , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Neutrophils/metabolism , Neutrophils/immunology , Mitochondria/metabolism , Mitochondria/genetics , Oxidative Phosphorylation/drug effects
13.
Cell Commun Signal ; 11: 58, 2013 Aug 14.
Article in English | MEDLINE | ID: mdl-23945128

ABSTRACT

BACKGROUND: Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. FINDINGS: Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation. CONCLUSIONS: The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution.


Subject(s)
Cofilin 1/metabolism , Cucurbitacins/pharmacology , Actin Cytoskeleton/metabolism , Cysteine/metabolism , Humans , MCF-7 Cells , Protein Binding
14.
Rapid Commun Mass Spectrom ; 27(3): 443-50, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23280976

ABSTRACT

RATIONALE: Some large-scale proteomics studies in which strong cation exchange chromatography has been applied are used to determine proteomes and post-translational modification dynamics. Although such datasets favour the characterisation of thousands of modified peptides, e.g., phosphorylated and N-α-acetylated, a large fraction of the acquired spectra remain unexplained by standard proteomics approaches. Thus, advanced data processing allows characterisation of a significant part of these unassigned spectra. METHODS: Our recent investigation of the N-α-acetylation status of plant proteins gave a dataset of choice to investigate further the in-depth characterisation of peptide modifications using Mascot tools associated with relevant validation processes. Such an approach allows to target frequently occurring modifications such as methionine oxidation, phosphorylation or N-α-acetylation, but also the less usual peptide cationisation. Finally, this dataset offers the unique opportunity to determine the overall influence of some of these modifications on the identification score. RESULTS: Although methionine oxidation has no influence and tends to favour the characterisation of protein N-terminal peptides, peptide alkalinisation shows an adverse effect on peptide average score. Nevertheless, peptide cationisation appears to favour the characterisation of protein C-terminal peptides with a limited to no direct influence on the identification score. Unexpectedly, our investigation reveals the unfortunate combination of the molecular weight of N-α-acetylation and potassium cation that mimics the mass increment of a phosphorylation group. CONCLUSIONS: Since these characterisations rely upon computational treatment associated with statistical validation approaches such as 'False discovery rates' calculation or post-translational modification position validation, our investigation highlights the limitation of such treatment which is biased by the initial searched hypotheses.


Subject(s)
Peptide Fragments/analysis , Proteome/analysis , Proteomics/methods , Amino Acid Sequence , Arabidopsis Proteins/analysis , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Databases, Protein , Methionine/analysis , Methionine/chemistry , Methionine/metabolism , Molecular Sequence Data , Oxidation-Reduction , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphorylation , Protein Processing, Post-Translational , Proteome/metabolism , Reproducibility of Results
15.
bioRxiv ; 2023 Nov 04.
Article in English | MEDLINE | ID: mdl-37961535

ABSTRACT

Extracellular vesicles (EVs) are generated by all cells and systemic administration of allogenic EVs derived from epithelial and mesenchymal cells have been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cells derived EVs can be modified to acquire the capacity to induce immune response, we engineered 293T EVs to harbor the immunomodulatory CD80, OX40L and PD-L1 molecules. We demonstrated abundant levels of these proteins on the engineered cells and EVs. Functionally, the engineered EVs efficiently elicit positive and negative co-stimulation in human and murine T cells. In the setting of cancer and auto-immune hepatitis, the engineered EVs modulate T cell functions and alter disease progression. Moreover, OX40L EVs provide additional benefit to anti-CTLA-4 treatment in melanoma-bearing mice. Our work provides evidence that epithelial cell derived EVs can be engineered to induce immune responses with translational potential to modulate T cell functions in distinct pathological settings.

16.
bioRxiv ; 2023 Nov 15.
Article in English | MEDLINE | ID: mdl-38014296

ABSTRACT

The murine helminth parasite Heligmosomoides polygyrus expresses a family of modular proteins which, replicating the functional activity of the immunomodulatory cytokine TGF-ß, have been named TGM (TGF-ß Μimic). Multiple domains bind to different receptors, including TGF-ß receptors TßRI (ALK5) and TßRII through domains 1-3, and prototypic family member TGM1 binds the cell surface co-receptor CD44 through domains 4-5. This allows TGM1 to induce T lymphocyte Foxp3 expression, characteristic of regulatory (Treg) cells, and to activate a range of TGF-ß-responsive cell types. In contrast, a related protein, TGM4, targets a much more restricted cell repertoire, primarily acting on myeloid cells, with less potent effects on T cells and lacking activity on other TGF-ß-responsive cell types. TGM4 binds avidly to myeloid cells by flow cytometry, and can outcompete TGM1 for cell binding. Analysis of receptor binding in comparison to TGM1 reveals a 10-fold higher affinity than TGM1 for TGFßR-I (TßRI), but a 100-fold lower affinity for TßRII through Domain 3. Consequently, TGM4 is more dependent on co-receptor binding; in addition to CD44, TGM4 also engages CD49d (Itga4) through Domains 1-3, as well as CD206 and Neuropilin-1 through Domains 4 and 5. TGM4 was found to effectively modulate macrophage populations, inhibiting lipopolysaccharide-driven inflammatory cytokine production and boosting interleukin (IL)-4-stimulated responses such as Arginase-1 in vitro and in vivo. These results reveal that the modular nature of TGMs has allowed the fine tuning of the binding affinities of the TßR- and co-receptor binding domains to establish cell specificity for TGF-ß signalling in a manner that cannot be attained by the mammalian cytokine.

17.
Sci Adv ; 9(5): eabq1858, 2023 02 03.
Article in English | MEDLINE | ID: mdl-36735782

ABSTRACT

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.


Subject(s)
Glycocalyx , Sialoglycoproteins , Male , Humans , Glycocalyx/metabolism , Sialoglycoproteins/metabolism , Heterografts , Transplantation, Heterologous
18.
bioRxiv ; 2023 Mar 23.
Article in English | MEDLINE | ID: mdl-36993533

ABSTRACT

The mitochondrial genome encodes essential machinery for respiration and metabolic homeostasis but is paradoxically among the most common targets of somatic mutation in the cancer genome, with truncating mutations in respiratory complex I genes being most over-represented1. While mitochondrial DNA (mtDNA) mutations have been associated with both improved and worsened prognoses in several tumour lineages1-3, whether these mutations are drivers or exert any functional effect on tumour biology remains controversial. Here we discovered that complex I-encoding mtDNA mutations are sufficient to remodel the tumour immune landscape and therapeutic resistance to immune checkpoint blockade. Using mtDNA base editing technology4 we engineered recurrent truncating mutations in the mtDNA-encoded complex I gene, Mt-Nd5, into murine models of melanoma. Mechanistically, these mutations promoted utilisation of pyruvate as a terminal electron acceptor and increased glycolytic flux without major effects on oxygen consumption, driven by an over-reduced NAD pool and NADH shuttling between GAPDH and MDH1, mediating a Warburg-like metabolic shift. In turn, without modifying tumour growth, this altered cancer cell-intrinsic metabolism reshaped the tumour microenvironment in both mice and humans, promoting an anti-tumour immune response characterised by loss of resident neutrophils. This subsequently sensitised tumours bearing high mtDNA mutant heteroplasmy to immune checkpoint blockade, with phenocopy of key metabolic changes being sufficient to mediate this effect. Strikingly, patient lesions bearing >50% mtDNA mutation heteroplasmy also demonstrated a >2.5-fold improved response rate to checkpoint inhibitor blockade. Taken together these data nominate mtDNA mutations as functional regulators of cancer metabolism and tumour biology, with potential for therapeutic exploitation and treatment stratification.

19.
Nat Cancer ; 4(3): 344-364, 2023 03.
Article in English | MEDLINE | ID: mdl-36732635

ABSTRACT

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.


Subject(s)
Lysine Acetyltransferases , NF-kappa B , Mice , Animals , NF-kappa B/metabolism , Carnitine O-Palmitoyltransferase/metabolism , Acetylation , Acetyl Coenzyme A/metabolism , Palmitates , Lysine Acetyltransferases/metabolism
20.
Cell Rep ; 41(1): 111442, 2022 10 04.
Article in English | MEDLINE | ID: mdl-36198272

ABSTRACT

The MICAL1 monooxygenase is an important regulator of filamentous actin (F-actin) structures. Although MICAL1 has been shown to be regulated via protein-protein interactions at the autoinhibitory carboxyl terminus, a link between actin-regulatory RHO GTPase signaling pathways and MICAL1 has not been established. We show that the CDC42 GTPase effector PAK1 associates with and phosphorylates MICAL1 on two serine residues, leading to accelerated F-actin disassembly. PAK1 binds to the amino-terminal catalytic monooxygenase and calponin homology domains, distinct from the autoinhibitory carboxyl terminus. Extracellular ligand stimulation leads to PAK-dependent phosphorylation, linking external signals to MICAL1 phosphorylation. Mass spectrometry indicates that MICAL1 co-expression with CDC42 and PAK1 increases MICAL1 association with hundreds of proteins, including the previously described MICAL1-interacting proteins RAB10 and RAB7A. These results provide insights into a redox-mediated pathway linking extracellular signals to cytoskeleton regulation via a RHO GTPase and indicate a means of communication between RHO and RAB GTPases.


Subject(s)
Actins , p21-Activated Kinases , Actin Cytoskeleton/metabolism , Actins/metabolism , Ligands , Mixed Function Oxygenases/metabolism , Serine/metabolism , p21-Activated Kinases/metabolism , rab GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/metabolism
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