Pulse‐Chase Analysis for Studying Protein Synthesis and Maturation

Fritzsche, Susanne; Springer, Sebastian

doi:10.1002/0471140864.ps3003s78

Cited by 11 publications

(7 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In pulse-chase and thermostability analyses, newly synthesized HLA-B*27:05 molecules were labeled with 35 S-labeled methionine and -cysteine (EasyTag EXPRESS- 35 S protein labeling mix, PerkinElmer) as described previously [ 24 ]. Briefly, cells were pulse labeled for 7 min and chased in complete growth medium containing excess amounts of unlabeled methionine and cysteine for 30, 60, 120, and 240 min, respectively.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface

et al. 2018

Self Cite

View full text Add to dashboard Cite

HLA-B*27:05 is associated with the development of autoimmune spondyloarthropathies, but the precise causal relationship between the MHC haplotype and disease pathogenesis is yet to be elucidated. Studies focusing on the structure and cellular trafficking of HLA-B*27:05 implicate several links between the onset of inflammation and the unusual conformations of the molecule inside and at the surface of antigen presenting cells. Several lines of evidence emphasize the emergence of those unnatural protein conformations under conditions where peptide loading onto B*27:05 is impaired. To understand how cellular factors distinguish between poorly loaded molecules from the optimally loaded ones, we have investigated the intracellular transport, folding, and cell surface expression of this particular B27 subtype. Our findings show that B*27:05 is structurally unstable in the absence of peptide, and that an artificially introduced disulfide bond between residues 84 and 139 conferred enhanced conformational stability to the suboptimally loaded molecules. Empty or suboptimally loaded B*27:05 can escape intracellular retention and arrive at the cell surface leading to the appearance of increased number of β2m-free heavy chains. Our study reveals a general mechanism found in the early secretory pathways of murine and human cells that apply to the quality control of MHC class I molecules, and it highlights the allotype-specific structural features of HLA-B*27:05 that can be associated with aberrant antigen presentation and that might contribute to the etiology of disease.

show abstract

Section: Methodsmentioning

confidence: 99%

“…The supernatant was equilibrated in 1 mL wash buffer and HA-tagged HLA-B*27:05 molecules were re-immunoprecipitated with anti-HA coupled to Protein A-agarose beads. Immune complexes were washed once with wash buffer and treated with Endoglycosidase F1 as described [ 24 ].…”

Section: Methodsmentioning

confidence: 99%

Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…6 D ). The increase in MHC class I signal over the course of the experiment can be explained by ongoing postpulse folding, since the antibody used is specific for the folded conformation ( 96 , 97 ).…”

Section: Resultsmentioning

confidence: 99%

“…Importantly, the abundance and kinetics of folded MHC class I molecules were not affected in ATP13A1 KO cells (Figure 6D). The increase in MHC class I signal over the course of the experiment can be explained by ongoing post-pulse folding, since the antibody used is specific for the folded conformation (96,97).…”

Section: Atp13a1 Deficiency Affects Mr1 Levels At or Immediately Afte...mentioning

confidence: 99%

The P5-type ATPase ATP13A1 modulates major histocompatibility complex I-related protein 1 (MR1)-mediated antigen presentation

Kulicke¹,

Zan²,

Hein³

et al. 2022

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

show abstract

“…Whole cell lysates of Nthyori-CV cells were prepared as described above. To cleave N-linked glycans, 100 µg of protein in a total volume of 27 µL each was treated with Endoglycosidase F1 or Peptide-N-Glycosidase F as described before [77]. Briefly, samples were incubated with denaturing buffer (final concentration 0.5% SDS and 40 mM DTT) at 95 • C for 10 min in order to linearize the proteins and increase their accessibility to the enzymes.…”

Section: Enzymatic De-glycosylation Of N-linked Glycoproteinsmentioning

confidence: 99%

Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe

Al-Hashimi

Venugopalan

Rehders

et al. 2020

IJMS

Self Cite

View full text Add to dashboard Cite

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

show abstract

Pulse‐Chase Analysis for Studying Protein Synthesis and Maturation

Cited by 11 publications

References 27 publications

Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface

Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface

The P5-type ATPase ATP13A1 modulates major histocompatibility complex I-related protein 1 (MR1)-mediated antigen presentation

Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe

Contact Info

Product

Resources

About