Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection

Sanyal, Sumana; Ashour, Joseph; Maruyama, Takeshi; Altenburg, Arwen F.; Cragnolini, Juan J.; Bilate, Angelina M.; Avalos, Ana M.; Kundrat, Lenka; Garcı́a-Sastre, Adolfo; Ploegh, Hidde L.

doi:10.1016/j.chom.2013.10.011

Cited by 56 publications

(63 citation statements)

References 44 publications

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“…To measure their cellular localization on TCR stimulation, cells were either mock-treated or stimulated with anti-CD3 for 20 min. Cytosol fractions were separated from the residual subcellular organelles using perfringolysin O (PFO), a pore-forming toxin that selectively permeabilizes the plasma membrane without damaging the internal membranes, as described elsewhere (43)(44)(45)(46)(47). In resting cells, Usp12 and Usp46 are localized primarily to the nucleus; however, when stimulated with anti-CD3, both exhibited significant enrichment in the cytosol fractions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

Jahan

Lestra

Swee

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and timedependent translocation of Usp12 from the nucleus to the cytosol. Usp12 −/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12 −/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.Usp12 | TCR signaling | deubiquitylases

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Section: Resultsmentioning

confidence: 99%

Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

Jahan

Lestra

Swee

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, exosomes are enriched in ISGylation targets, such as TSG101 (ref. 40) and heat-shock proteins41.…”

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confidence: 99%

ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins

et al. 2016

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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.

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“…We generated modified versions of GPA and Kell to render them suitable for modification by sortase, and it is likely that other RBC membrane proteins will lend themselves to similar treatment. Although sortase-mediated cell-surface labeling has been exploited previously [e.g., to explore trafficking of influenza glycoproteins (18,19)], here we apply this method to primary cells as a platform for diagnostic or therapeutic purposes. We used a sortase A variant from S. aureus active at 0°C (20) to reduce the risk of inflicting cellular damage in the course of labeling.…”

Section: Sortagging and Cytosolic Modification Of Terminally Differenmentioning

confidence: 99%

Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes

Shi

Kundrat

Pishesha³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. 2 with a favorable surface-to-volume ratio; and (vi) the absence of a nucleus, mitochondria, and any DNA. Thus, any modification made to the DNA of RBC precursors is eliminated upon their enucleation and cannot lead to abnormal growth or tumorigenicity after their transfusion into a recipient.Engineered RBCs have been generated using encapsulation (2-4), by noncovalent attachment of foreign peptides, or through installation of proteins by fusion to a monoclonal antibody specific for an RBC surface protein (5, 6).However, modified RBCs have limitations if intended for application in vivo. Encapsulation allows the entrapment of sizable quantities of material but does so at the expense of disrupting plasma membrane integrity, with a concomitant reduction in circulatory half life of the modified RBCs. Osmosis-driven entrapment limits the chemical nature of materials that can be encapsulated successfully, the site of release is difficult to control, and encapsulated enzymes are functional only at the final destination, compromising reusability at other sites (5, 6). Targeting of cargo to RBCs by fusion to an RBC-specific antibody, (e.g., anti-glycophorin antibody), has limitations because this mode of attachment to the RBC is noncovalent and dissociates readily, thus reducing circulatory half life and mass of cargo available for delivery (5, 6). Other developments that exploit RBCs for targeted delivery include nanoparticles enveloped by an RBC-mimicking membrane and RBC-shaped polymers (1). The short in vivo survival rate of these RBC-inspired carriers (∼7 d maximum) may limit their therapeutic utility.There is a need to develop new methodology for engineering RBCs so that they can carry a wide variety of useful cargoes to specific locations in the body. We describe an approach that involves minimal modification of the RBCs, with preservation of plasma membrane integrity. The method involves sortase-mediated site-specific covalent attachment of payloads to specific RBC surface proteins.Bacterial sortases are transpeptidases capable of modifying suitably modified proteins in a covalent and site-specific manner (7,8). Sortase A from Staphylococcus aureus recognizes an LPXTG motif positioned close to the substrate's C terminus and cleaves between T and G to form a covalent acyl-enzyme intermediate. This intermediate is resolved by a nucleophilic N-terminal glycine residue on an appropriately designed probe (9) with concomitant formation of a peptide bond between substrate and probe. Conversely, a protein may be labeled at its N terminus by extending it with suitably exposed g...

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Type I Interferon Imposes a TSG101/ISG15 Checkpoint at the Golgi for Glycoprotein Trafficking during Influenza Virus Infection

Cited by 56 publications

References 44 publications

Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins

Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes

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