ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins

Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes. Posttranslational modifications are involved in the sorting of specific proteins into exosomes. Here we identify ISGylation as a ubiquitin-like modification that controls exosome release. ISGylation induction decreases MVB numbers and impairs exosome secretion. Using ISG15-knockout mice and mice expressing the enzymatically inactive form of the de-ISGylase USP18, we demonstrate in vitro and in vivo that ISG15 conjugation regulates exosome secretion. ISG15 conjugation triggers MVB co-localization with lysosomes and promotes the aggregation and degradation of MVB proteins. Accordingly, inhibition of lysosomal function or autophagy restores exosome secretion. Specifically, ISGylation of the MVB protein TSG101 induces its aggregation and degradation, being sufficient to impair exosome secretion. These results identify ISGylation as a novel ubiquitin-like modifier in the control of exosome production.We thank Dr K. Knobeloch, Dr A. Garcia-Sastre and Dr M.A. Alonso for providing reagents, and Dr S. Bartlett for assistance with English editing. C.E. is thankful to electron microscopy facility (campus Casanova), CCiT-University of Barcelona. This study was supported by grants SAF2014-55579-R from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, Cardiovascular Network RD12-0042-0056 and PIE13/00041 from Instituto de Salud Carlos III (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER), ERC-2011-AdG 294340-GENTRIS and COST-Action BM1202 to F.S.-M.; grant SAF2014-54623-R, FIS grant PI11/00127 (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and Ministry of Health of Spain, State secretary of R+D and FEDER/FSE) and Bayer Group Grants4Grants (ID 2013-08-0982) to S.G.; and grant BFU2015-66785-P from the Spanish Ministry of Economy and Competitiveness to C.E.; Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). C.V.-B. was supported by FPU programme (Spanish Ministry of Education). M. M. is supported by MS14/00219 from Instituto de Salud Carlos III.S

Mechanisms controlling the secretion and composition of exosomes

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 2-12-201

Post-translational modifications of exosomal proteins

Exosomes mediate intercellular communication and participate in many cell processes such as cancer progression, immune activation or evasion, and the spread of infection. Exosomes are small vesicles secreted to the extracellular environment through the release of intraluminal vesicles contained in multivesicular bodies (MVBs) upon the fusion of these MVBs with the plasma membrane. The composition of exosomes is not random, suggesting that the incorporation of cargo into them is a regulated process. However, the mechanisms that control the sorting of protein cargo into exosomes are currently elusive. Here, we review the post-translational modifications detected in exosomal proteins, and discuss their possible role in their specific sorting into exosomes.This work was supported by SAF2011-25834 fromthe Spanish Ministry of Science and Innovation. INDISNET-S2011/BMD2332 from the Comunidad de Madrid, Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII), ERC2011-AdG 294340-GENTRIS, and COST-Action BN1202. Olga Moreno-Gonzalo and Carolina Villarroya-Beltri were supported by FPU program (Spanish Ministry of Education).S

ISGylation – a key to lock the cell gates for preventing the spread of threats

Interferon stimulated gene 15 (ISG15) is an ubiquitin-like protein whose expression and conjugation to targets (ISGylation) is induced by infection, interferon (IFN)-α and -β, ischemia, DNA damage and aging. Attention has historically focused on the antiviral effects of ISGylation, which blocks the entry, replication or release of different intracellular pathogens. However, recently, new functions of ISGylation have emerged that implicate it in multiple cellular processes, such as DNA repair, autophagy, protein translation and exosome secretion. In this Review, we discuss the induction and conjugation of ISG15, as well as the functions of ISGylation in the prevention of infections and in cancer progression. We also offer a novel perspective with regard to the latest findings on this pathway, with special attention to the role of ISGylation in the inhibition of exosome secretion, which is mediated by fusion of multivesicular bodies with lysosomes. Finally, we propose that under conditions of stress or infection, ISGylation acts as a defense mechanism to inhibit normal protein translation by modifying protein kinase R (PKR, also known as EIF2AK2), while any newly synthesized proteins are being tagged and thus marked as potentially dangerous. Then, the endosomal system is re-directed towards protein degradation at the lysosome, to effectively ‘lock’ the cell gates and thus prevent the spread of pathogens, prions and deleterious aggregates through exosomeThe work of our laboratories is supported by the Spanish Ministerio de Economıa y ́ Competitividad (MINECO) (SAF 2014-55579-R to F.S.-M., SAF 2014-54623 to S.G.), CIBERCV is funded by Instituto de Salud Carlos III and Fondos Fonds Européen de Développement Régional (FEDER); the European Research Council (ERC-2011-AdG294340-GENTRIS to F.S.-M); BIOIMID PIE13/041 and Fundacióla Maratóde TV3 (20152330 31). The CNIC is supported by MINECO and the Pro Centro Nacional de Investigaciones Cardiovasculares Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505

PP2A-B55 phosphatase counteracts Ki-67-dependent chromosome individualization during mitosis

Chromosome clustering; Mitosis; PhosphataseAgrupamiento cromosómico; Mitosis; FosfatasaAgrupament cromosòmic; Mitosi; FosfatasaCell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis.We thank Dr. Imamoto (RIKEN, Japan) and Ana Losada (CNIO, Spain) for providing antibodies. M.S.-F. was supported by the FPU Program from the Spanish Agencia Estatal de Investigación (AEI) of the Ministry of Science and Innovation. M.A.-F. was supported by the Asociación Española contra el Cáncer ( AECC; 2019/INVES19001ALVA ). M.R.-T. was supported by AECC ( POSTD211362RUIZ ). Research in the laboratory of D.W.G. is supported by the Austrian Academy of Sciences, the Vienna Science and Technology Fund (WWTF; projects LS17-003 and LS19-001 ), and the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement 101019039 ). Work in the M.M. laboratory was supported by grants from the AEI-MICIIN ( RTI2018-095582-B-I00 and PID2021-1287260-B-100 ), the iLUNG2 Program ( S2022/BMD-7437 ) from the Comunidad de Madrid, and the iDIFFER Network of Excellence ( RED2022-134792-T ). VHIO acknowledges the Cellex Foundation for providing research facilities and equipment and the CERCA Program from the Generalitat de Catalunya for support. CNIO and VHIO are Severo Ochoa Centers of Excellence ( AEI-MICIU CEX2019-000891-S and CEX2020-001024-S/AEI/10.13039/501100011033 )

Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs

Exosomes are released by most cells to the extracellular environment and are involved in cell-to-cell communication. Exosomes contain specific repertoires of mRNAs, microRNAs (miRNAs) and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe sequence motifs present in miRNAs that control their localization into exosomes. The protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.This work was supported by grants SAF2011-25834, ERC-2011-AdG, BIO2009_07990, BIO2010-17527, COST-Action BM1202, P210/BMD-2305 and Cardiovascular Network RD12-0042-0056 from Instituto Salud Carlos III. C.V.B. was supported by FPU program (Spanish Ministry of Education). M.M. was supported by the Instituto de Salud Carlos III, C.G.-V. by the Comunidad de Madrid and D.J.M.H. by Programa La Caixa.S

Biallelic germline mutations in MAD1L1 induce a syndrome of aneuploidy with high tumor susceptibility.

Germline mutations leading to aneuploidy are rare, and their tumor-promoting properties are mostly unknown at the molecular level. We report here novel germline biallelic mutations in MAD1L1, encoding the spindle assembly checkpoint (SAC) protein MAD1, in a 36-year-old female with a dozen of neoplasias. Functional studies demonstrated lack of full-length protein and deficient SAC response, resulting in ~30 to 40% of aneuploid blood cells. Single-cell RNA analysis identified mitochondrial stress accompanied by systemic inflammation with enhanced interferon and NFκB signaling both in aneuploid and euploid cells, suggesting a non-cell autonomous response. MAD1L1 mutations resulted in specific clonal expansions of γδ T cells with chromosome 18 gains and enhanced cytotoxic profile as well as intermediate B cells with chromosome 12 gains and transcriptomic signatures characteristic of leukemia cells. These data point to MAD1L1 mutations as the cause of a new variant of mosaic variegated aneuploidy with systemic inflammation and unprecedented tumor susceptibility.This work is supported by Spanish Ministry of Science, Juan de la Cierva programme (C.V.-B.); Spanish Ministry of Science and Innovation, Agencia Estatal de Investigacion (MCI-AEI), BIO2017-91272-EXP (S.R.-P.); Spanish National Research and Development Plan, ISCIII, and FEDER, PI17/02303, PI20/01837, and DTS19/00111 (S.R.-P.); Spanish National Research and Development Plan, ISCIII, and FEDER, PI21/01641 (R.T.-R.); Fundacion Cientifica de la Asociacion Espanola contra el Cancer, LABAE20049RODR (S.R.-P.); MCI-AEI/FEDER, RTI2018-095582-B-I00, and RED2018-102723-T (M.M.); Comunidad de Madrid iLUNG and scCANCER programmes, B2017/BMD-3884 and Y2020/BIO-6519 (M.M.); and MCI-AEI, Severo Ochoa Center of Excellence, CEX2019-000891-S (S.R.-P., M.M., and M.U.).S

PP2A-B55 phosphatase counteracts Ki-67-dependent chromosome individualization during mitosis

Summary: Cell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis

Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts

Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate antipathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.We thank Dr. S. Bartlett for assistance with English editing and Dr A. Garcia-Sastre for providing reagents. This study was supported by grants SAF2017/82886-R from the Spanish Ministry of Economy and Competitiveness, CAM S2017/BMD-3671 from the Comunidad de Madrid, CIBER Cardiovascular (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER), ERC-2011-AdG 294340-GENTRIS and COST-Action BM1202 to F.S.-M.; grant SAF2015-65633-R from the Spanish Ministry of Economy and Competitiveness to J.A.E. M.M. is supported by MS14/00219 from Instituto de Salud Carlos III. Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).S