Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance

Wei, Jiajie; Gibbs, James S.; Hickman, Heather D.; Cush, Stephanie S.; Bennink, Jack R.; Yewdell, Jonathan W.

doi:10.1074/jbc.m115.658062

Cited by 17 publications

(17 citation statements)

References 41 publications

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“…2). Although the auto-catalytic cleavage of GFP in NP-S-GFP (28), responsible for ~50% of its DRiP dependent K b -SIINFEKL generation, makes it a highly unusual substrate, we also observe MLN7243-resistant (or even enhanced) presentation of a two IAV peptides generated from unmodified viral proteins in IAV infected-cells, NP 147–155 , previously described as proteasome dependent (48), and PB1 703–711 , whose proteasome-dependence can be inferred by its dependence on immunoproteasome subunit expression (4). …”

Section: Discussionmentioning

confidence: 99%

“…Immunoblotting was performed as described (28) using rabbit anti-histone H3 (Cell signaling), mouse anti-poly Ub antibody (clone FK1) (Enzo), IRDye 800CW anti-rabbit antibody (LI-COR) and IRDye 680LT anti-mouse antibody (LI-COR).…”

Section: Methodsmentioning

confidence: 99%

“…The kinetics of K b -SIINFEKL presentation was determined as described (28). To study the kinetics of endogenous peptide presentation, cells were treated in ice-cold citric acid buffer (0.13 M citric acid, 0.061 M Na 2 HPO 4 , 0.15 M NaCl, ph 3) at 1 X 10 7 cells/ml for 120 s, washed three times with PBS, and resuspended in culture media containing drugs specified in figure legends.…”

Section: Methodsmentioning

confidence: 99%

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Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands

Wei

Zanker

Carluccio

et al. 2017

The Journal of Immunology

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CD8+ T cell immunosurveillance is based on recognizing oligopeptides presented by MHC class I molecules. Despite decades of study, the importance of protein ubiquitylation to peptide generation remains uncertain. Here, we examine the ability of MLN7243, a recently described ubiquitin activating enzyme E1-inhibitor, to block overall cytosolic peptide generation and generation of specific peptides from vaccinia- and influenza A virus-encoded proteins. We show that MLN7243 rapidly inhibits ubiquitylation in a variety of cell lines, and can profoundly reduce generation of cytosolic peptides. Kinetic analysis of specific peptide generation reveals that ubiquitylation of defective ribosomal products (DRiPs) is rate limiting in generating class I peptide complexes. More generally, our findings demonstrate that the requirement for ubiquitylation in MHC class I restricted antigen processing varies with class I allomorph, cell type, source protein, and peptide context. Thus, both ubiquitin-dependent and independent pathways robustly contribute to MHC class I based immunosurveillance.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands

Wei

Zanker

Carluccio

et al. 2017

The Journal of Immunology

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“…A number of immunogenic MHC-I-restricted peptides have been identified within VACV genes that are classified as late genes. Notably, Ag presentation by virus-infected cells (direct presentation) most often uses recently synthesized, rapidly degraded Ag, which may not be correctly folded, as substrate for the manufacture of immunogenic peptides (3,34,42,43). Therefore, the levels of mRNA from which these substrates can be generated are more relevant to Ag presentation than the levels of intact correctly folded protein, which they can also generate.…”

Section: Late Gene Products Are Antigenic and Immunogenic To T Cd8+mentioning

confidence: 99%

“…These peptide-MHC-I complexes (p-MHC-I) can be generated via two spatially distinct pathways. Virus-infected cells present peptides derived primarily from a subset of viral proteins that are rapidly degraded in a process known as direct presentation (2,3). Alternatively, long-lived protein substrates may be transferred from virus-infected cells to professional APC (pAPC), where they are processed and presented by uninfected cells via the cross-presentation pathway (4)(5)(6).…”

Section: Introductionmentioning

confidence: 99%

Analysis of MHC Class I Processing Pathways That Generate a Response to Vaccinia Virus Late Proteins

et al. 2019

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Use of recombinant viral vectors encoding nonnative Ags is an attractive mechanism for the generation of protective Ab, CD4 + T cell (T CD4+ ), and CD8 + T cell (T CD8+ ) responses in vivo following immunization. However, the life cycle and tropism of the viral vector, and its interactions with various components of the immune system, must be fully understood to maximize the efficacy of any vaccination strategies. Ab and T CD4+ responses typically target native Ags driven by late promoters in vaccinia virus (VACV)-based vectors. However, it has been demonstrated that model Ags driven by late promoters in recombinant VACV vectors do not stimulate T CD8+ responses, whereas identical Ags driven by early promoters stimulate strong responses. Conversely, T CD8+ can be generated against some natural late VACV Ags. We explored this dichotomy by investigating the Ag presentation pathways responsible for presentation of natural late VACV Ags in mice. We found that all of the late VACV Ags we examined could be cross-primed (i.e., presented by uninfected professional APC), as well as directly presented by infected dendritic cell populations. However, one Ag was only presented by professional APC populations and was not the target of a protective T CD8+ response. Therefore, there is no generalized blockade in Ag presentation of late VACV Ags, and expression of nonnative Ags driven by a late promoter allows production of large quantities of Ag that may allow simultaneous targeting of both T CD4+ and Ab responses, as well as T CD8+ responses, in the future. ImmunoHorizons, 2019, 3: 559-572.

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PA28γ, the ring that makes tumors invisible to the immune system?

Cascio

2024

Biochimie

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Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance

Cited by 17 publications

References 41 publications

Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands

Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands

Analysis of MHC Class I Processing Pathways That Generate a Response to Vaccinia Virus Late Proteins

PA28γ, the ring that makes tumors invisible to the immune system?

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