RESUMEN
The intimate relationship between the epithelium and immune system is crucial for maintaining tissue homeostasis, with perturbations therein linked to autoimmune disease and cancer1-3. Whereas stem cell-derived organoids are powerful models of epithelial function4, they lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident memory T (TRM) cells, a portion of which integrate within the epithelium and continuously survey the barrier. TRM cell migration and interaction with epithelial cells was orchestrated by TRM cell-enriched transcriptomic programs governing cell motility and adhesion. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients. Inflammation was associated with the emergence of an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and potentiated by a T helper-1-like CD4+ population, which initially produced cytokines and subsequently became cytotoxic itself. As a system amenable to direct perturbation, IIOs allowed us to identify the Rho pathway as a new target for mitigation of immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and underlying interlineage immune interactions, IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis and infectious and autoimmune diseases.
Asunto(s)
Linfocitos T CD8-positivos , Células T de Memoria , Organoides , Organoides/inmunología , Humanos , Células T de Memoria/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/citología , Análisis de la Célula Individual , Movimiento Celular/inmunología , Inflamación/inmunología , Inflamación/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/citología , Intestinos/inmunología , Intestinos/citología , Transcriptoma , Células Epiteliales/inmunología , Células Epiteliales/citología , Femenino , Inmunoterapia , Masculino , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/citologíaRESUMEN
Small ubiquitin-like modifiers from the ATG8 family regulate autophagy initiation and progression in mammalian cells. Their interaction with LC3-interacting region (LIR) containing proteins promotes cargo sequestration, phagophore assembly, or even fusion between autophagosomes and lysosomes. Previously, we have shown that RabGAP proteins from the TBC family directly bind to LC3/GABARAP proteins. In the present study, we focus on the function of TBC1D2B. We show that TBC1D2B contains a functional canonical LIR motif and acts at an early stage of autophagy by binding to both LC3/GABARAP and ATG12 conjugation complexes. Subsequently, TBC1D2B is degraded by autophagy. TBC1D2B condensates into liquid droplets upon autophagy induction. Our study suggests that phase separation is an underlying mechanism of TBC1D2B-dependent autophagy induction.
Asunto(s)
Autofagia , Proteínas Activadoras de GTPasa , Proteínas Asociadas a Microtúbulos , Humanos , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Células HeLa , Proteínas Reguladoras de la Apoptosis/metabolismo , Células HEK293 , Unión Proteica , Separación de FasesRESUMEN
The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Lisosomas/metabolismo , Fusión de Membrana/genética , Glicoproteínas de Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Fagosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Autofagia , Proteínas Relacionadas con la Autofagia , Endosomas/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Transducción de Señal , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The RabGAP protein TBC1D5 controls cellular endomembrane trafficking processes and binds the retromer subunit VPS29 and the ubiquitin-like protein ATG8 (LC3). Here, we describe that TBC1D5 also associates with ATG9 and the active ULK1 complex during autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 to late endosomes upon activation of autophagy, whereas inhibition of clathrin-mediated endocytosis or AP2 depletion alters ATG9 trafficking and its association with TBC1D5. Taken together, our data show that TBC1D5 and the AP2 complex are important novel regulators of the rerouting of ATG9-containing vesicular carriers toward sites of autophagosome formation.
Asunto(s)
Complejo 2 de Proteína Adaptadora/fisiología , Proteínas Activadoras de GTPasa/fisiología , Proteínas de la Membrana/metabolismo , Autofagia , Homólogo de la Proteína 1 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Línea Celular Tumoral , Endocitosis , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fagosomas/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de ProteínasRESUMEN
Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.
Asunto(s)
Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Familia de las Proteínas 8 Relacionadas con la Autofagia , Sitios de Unión , Western Blotting , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Receptores de GABA-A/metabolismo , Reticulocitos/citología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 coprecipitates with, and is directly phosphorylated by, mTOR. Using a phosphospecific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Relacionadas con la Autofagia/genética , Lisosomas/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Serina-Treonina Quinasas TOR/genética , Autofagia/genética , Endosomas/genética , Células HeLa , Humanos , Fosforilación/genética , Unión Proteica/genéticaRESUMEN
BACKGROUND: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021). AIM: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland. METHODS: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale. RESULTS: We estimate B.1.1.7 had a transmission fitness advantage of 43-52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021. CONCLUSION: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Suiza/epidemiología , Reino UnidoRESUMEN
A long-standing question in quantitative biology is the relationship between mRNA and protein levels of the same gene. Here, we measured mRNA and protein abundance, the phenotypic state, and the population context in thousands of single human cells for 23 genes by combining a unique collection of cell lines with fluorescently tagged endogenous genomic loci and quantitative immunofluorescence with branched DNA single-molecule fluorescence in situ hybridization and computer vision. mRNA and protein abundance displayed a mean single-cell correlation of 0.732 at steady state. Single-cell outliers of linear correlations are in a specific phenotypic state or population context. This is particularly relevant for interpreting mRNA-protein relationships during acute gene induction and turnover, revealing a specific adaptation of gene expression at multiple steps in single cells. Together, we show that single-cell protein abundance can be predicted by multivariate information that integrates mRNA level with the phenotypic state and microenvironment of a particular cell.
Asunto(s)
Variación Biológica Poblacional , MAP Quinasa Quinasa 4/genética , ARN Mensajero/genética , Línea Celular , Células Cultivadas , Células HeLa , Humanos , MAP Quinasa Quinasa 4/metabolismo , Modelos Teóricos , ARN Mensajero/metabolismo , Análisis de la Célula IndividualRESUMEN
Ubiquitination is crucial for a plethora of physiological processes, including cell survival and differentiation and innate and adaptive immunity. In recent years, considerable progress has been made in the understanding of the molecular action of ubiquitin in signaling pathways and how alterations in the ubiquitin system lead to the development of distinct human diseases. Here we describe the role of ubiquitination in the onset and progression of cancer, metabolic syndromes, neurodegenerative diseases, autoimmunity, inflammatory disorders, infection and muscle dystrophies. Moreover, we indicate how current knowledge could be exploited for the development of new clinical therapies.
Asunto(s)
Enfermedad , Ubiquitinación/efectos de los fármacos , Inestabilidad Genómica , Humanos , Estabilidad Proteica , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
Autophagy is an evolutionarily conserved degradation pathway characterized by dynamic rearrangement of membranes that sequester cytoplasm, protein aggregates, organelles, and pathogens for delivery to the vacuole and lysosome, respectively. The ability of autophagosomal membranes to act selectively toward specific cargo is dependent on the small ubiquitin-like modifier ATG8/LC3 and the LC3-interacting region (LIR) present in autophagy receptors. Here, we describe a comprehensive protein-protein interaction analysis of TBC (Tre2, Bub2, and Cdc16) domain-containing Rab GTPase-activating proteins (GAPs) as potential autophagy adaptors. We identified 14 TBC domain-containing Rab GAPs that bind directly to ATG8 modifiers and that colocalize with LC3-positive autophagy membranes in cells. Intriguingly, one of our screening hits, TBC1D5, contains two LIR motifs. The N-terminal LIR was critical for interaction with the retromer complex and transport of cargo. Direct binding of the retromer component VPS29 to TBC1D5 could be titrated out by LC3, indicating a molecular switch between endosomes and autophagy. Moreover, TBC1D5 could bridge the endosome and autophagosome via its C-terminal LIR motif. During starvation-induced autophagy, TBC1D5 was relocalized from endosomal localization to the LC3-positive autophagosomes. We propose that LC3-interacting Rab GAPs are implicated in the reprogramming of the endocytic trafficking events under starvation-induced autophagy.