Optical control of the antigen translocation by synthetic photo-conditional viral inhibitors

Braner, Markus; Koller, Nicole; Knauer, Julia; Herbring, Valentina; Hank, Susanne; Wieneke, Ralph; Tampé, Robert

doi:10.1039/c8sc04863k

Cited by 8 publications

(8 citation statements)

References 25 publications

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“…The supernatant was collected as HLS9 and transferred to a clean tube. To prepare HLM and HLS9 were further centrifuged at 300,000g for 20 minutes at 4°C using an Optima MAX ultracentrifuge with a MLA-80 rotor (Beckman Coulter, Indianapolis, IN) (Braner et al, 2018). The pellet (HLM) formed after the centrifugation was then resuspended in PBS and transferred to a clean tube for storage.…”

Section: Preparation Of Hls9 and Hlmmentioning

confidence: 99%

Comparative Proteomics Analysis of Human Liver Microsomes and S9 Fractions

Wang

Shi

et al. 2019

Drug Metab Dispos

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Human liver microsomes (HLM) and human liver S9 fractions (HLS9) are commonly used to study drug metabolism in vitro. However, a quantitative comparison of HLM and HLS9 proteomes is lacking, resulting in the arbitrary selection of one hepatic preparation over another and in difficulties with data interpretation. In this study, we applied a label-free global absolute quantitative proteomics method to the analysis of HLS9 and the corresponding HLM prepared from 102 individual human livers. A total of 3137 proteins were absolutely quantified, and 3087 of those were determined in both HLM and HLS9. Protein concentrations were highly correlated between the two hepatic preparations (R 5 0.87, P < 0.0001). We reported the concentrations of 98 drug-metabolizing enzymes (DMEs) and 51 transporters, and demonstrated significant differences between their abundances in HLM and HLS9. We also revealed the proteinprotein correlations among these DMEs and transporters and the sex effect on the HLM and HLS9 proteomes. Additionally, HLM and HLS9 displayed distinct expression patterns for protein markers of cytosol and various cellular organelles. Moreover, we evaluated the interindividual variability of three housekeeping proteins, and identified five proteins with low variation across individuals that have the potential to serve as new internal controls for western blot experiments. In summary, these results will lead to better understanding of data obtained from HLM and HLS9 and assist in in vitro-in vivo extrapolations. Knowing the differences between HLM and HLS9 also allows us to make better-informed decisions when choosing between these two hepatic preparations for in vitro drug metabolism studies. SIGNIFICANCE STATEMENTThis investigation revealed significant differences in protein concentrations of drug-metabolizing enzymes and transporters between human liver microsomes and S9 fractions. We also determined the protein-protein correlations among the drugmetabolizing enzymes and transporters and the sex effect on the proteomes of these two hepatic preparations. The results will help interpret data obtained from these two preparations and allow us to make more informed decisions when choosing between human liver microsomes and S9 fractions for in vitro drug metabolism studies.

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Section: Preparation Of Hls9 and Hlmmentioning

confidence: 99%

Comparative Proteomics Analysis of Human Liver Microsomes and S9 Fractions

Wang

Shi

et al. 2019

Drug Metab Dispos

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“…Screening ideal positions for photocleavable backbone modifications in the active region of ICP47 revealed Met15 being the optimal site preserving full TAP inhibition [9] . Since synthesis of full‐length ICP47 with an unnatural amino acid at Met15 is not accessible via SPPS in acceptable yields, we produced pc‐ICP47 SBP in two fragments for a native chemical ligation and subsequent desulfurization (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…[8] A flexible loop region (residues [13][14][15] between the two α-helices was identified as an optimal position for insertion of photocleavable β-amino acids (Figure 1A). [9] Light is an orthogonal and contactless trigger to modulate molecular and cellular processes with minimal side effects. While a 54 aa synthetic photoconditional (pc-)ICP47 2-55 allowed for spatiotemporal control of the PLC in vitro, [9] it cannot be used as a bait to isolate the native PLC from human cells for mechanistic and structural studies.…”

Section: Introductionmentioning

confidence: 99%

“…Light is an orthogonal and contactless trigger to modulate molecular and cellular processes with minimal side effects. While a 54 aa synthetic photoconditional (pc‐)ICP47 2–55 allowed for spatiotemporal control of the PLC in vitro, [9] it cannot be used as a bait to isolate the native PLC from human cells for mechanistic and structural studies [10] . On the contrary, previous isolation strategies based on full‐length viral inhibitors irreversibly inactivated the purified PLC, prohibiting downstream activity assays [3, 10, 11] …”

Section: Introductionmentioning

confidence: 99%

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Semisynthetic Viral Inhibitor for Light Control of the MHC I Peptide Loading Complex

2022

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The immune system detects virally or malignantly transformed cells via peptide-loaded major histocompatibility complex class I (pMHC I) molecules on the cell surface. MHC I molecules are loaded with cargo peptides in the endoplasmic reticulum (ER) by the highly dynamic multiprotein peptide loading complex (PLC). Here, we developed a semisynthetic approach to generate a photocleavable immune modulator ICP47 of Herpes simplex virus. Using this nanotool, we revealed key mechanistic events of the purified PLC, such as peptide binding and translocation coupled to ATP hydrolysis, triggered by light. We established a singleorganelle flow cytometry assay to monitor light-controlled activation of the antigen processing machinery in native ER membranes. This photochemical modulation opens new opportunities for a comprehensive mechanistic analysis of the antigen processing machinery in vitro and native membrane environment.

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“…Nevertheless, both strategies are incapable to restore function of the target protein and, therefore, do not represent an ideal approach. As inhibitors allow the arrest of protein activity while maintaining the native cellular environment, we previously reported a method to arrest and re-activate TAP via a photo-conditional variant of the viral inhibitor ICP47 41 . However, this approach has one major drawback: the photo-conditional immune suppressor is a synthetic cell-impermeable compound, which requires delivery into the cytosol.…”

Section: Discussionmentioning

confidence: 99%

Light control of the peptide-loading complex synchronizes antigen translocation and MHC I trafficking

et al. 2021

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Antigen presentation via major histocompatibility complex class I (MHC I) molecules is essential to mount an adaptive immune response against pathogens and cancerous cells. To this end, the transporter associated with antigen processing (TAP) delivers snippets of the cellular proteome, resulting from proteasomal degradation, into the ER lumen. After peptide loading and editing by the peptide-loading complex (PLC), stable peptide-MHC I complexes are released for cell surface presentation. Since the process of MHC I trafficking is poorly defined, we established an approach to control antigen presentation by introduction of a photo-caged amino acid in the catalytic ATP-binding site of TAP. By optical control, we initiate TAP-dependent antigen translocation, thus providing new insights into TAP function within the PLC and MHC I trafficking in living cells. Moreover, this versatile approach has the potential to be applied in the study of other cellular pathways controlled by P-loop ATP/GTPases.

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Optical control of the antigen translocation by synthetic photo-conditional viral inhibitors

Abstract: By designing and engineering photo-conditional viral inhibitors, spatiotemporal control of the transporter associated with antigen processing TAP was sustained, allowing the on-demand antigen translocation in human immune cell lines and primary cells by light.

Cited by 8 publications

References 25 publications

Comparative Proteomics Analysis of Human Liver Microsomes and S9 Fractions

Comparative Proteomics Analysis of Human Liver Microsomes and S9 Fractions

Semisynthetic Viral Inhibitor for Light Control of the MHC I Peptide Loading Complex

Light control of the peptide-loading complex synchronizes antigen translocation and MHC I trafficking

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