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- Hein, Z, et al.
(författare)
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Peptide-independent stabilization of MHC class I molecules breaches cellular quality control
- 2014
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Ingår i: Journal of cell science. - : The Company of Biologists. - 1477-9137 .- 0021-9533. ; 127:13Pt 13, s. 2885-2897
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Tidskriftsartikel (refereegranskat)abstract
- The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I are characterized by their conformational mobility in the F pocket region of the peptide binding site. We have created a novel variant of an MHC-I protein, Kb-Y84C, in which two alpha helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound high-affinity peptide. Kb-Y84C shows a remarkable increase in the binding affinity to its light chain, β2m, and bypasses all three cellular quality control steps. Our data demonstrate that coupling between peptide and β2m binding to the MHC-I heavy chain is mediated by conformational dynamics, that support of the folded conformation of MHC-I by β2m plays a decisive role in passing the ER to cell surface transport quality controls, and that β2m association is also tested by the cell surface quality control that leads to MHC-I endocytosis.
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- Schmidt, M, et al.
(författare)
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Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing
- 2003
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Ingår i: Annals of the New York Academy of Sciences (HEMATOPOIETIC STEM CELLS 2002: GENETICS AND FUNCTION: Fourth International Symposium). - 0077-8923. ; 996, s. 112-121
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Konferensbidrag (refereegranskat)abstract
- The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro-, lenti- and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification-mediated PCR (LAM-PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid-phase. Using LAM-PCR, we show the previously unfeasible in vivo retro-, lenti- and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID-X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.
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