Tapasin edits peptides on MHC class I molecules by accelerating peptide exchange.

The endoplasmic reticulum (ER) protein tapasin is essential for the loading of high-affinity peptides onto MHC class I molecules. It mediates peptide editing, i.e. the binding of peptides of successively higher affinity until class I molecules pass ER quality control and exit to the cell surface. The molecular mechanism of action of tapasin is unknown. We describe here the reconstitution of tapasin-mediated peptide editing on class I molecules in the lumen of microsomal membranes. We find that in a competitive situation between high- and low-affinity peptides, tapasin mediates the binding of the high-affinity peptide to class I by accelerating the dissociation of the peptide from an unstable intermediate of the binding reaction.

Mutational analysis of bacteriophage T4 AsiA: involvement of N- and C-terminal regions in binding to sigma(70) of Escherichia coli in vivo.

The T4 AsiA is an anti-sigma factor encoded by an early gene of bacteriophage T4. AsiA has been shown to inhibit T4 early promoters in vitro and expression of this protein from a plasmid causes transcriptional shut off in the host cells leading to cell death. By reasoning that mutant AsiA expression in Escherichia coli will not inhibit the host transcription and hence lead to healthy colony formation, a strategy was developed wherein inactive or partially active mutants of AsiA could be isolated. These mutants were tested for their ability to bind to sigma(70) in vivo in E. coli, monitored as a relative toxicity assay, co-purification of sigma(70), inhibition of [3H-uridine] incorporation and also in the yeast two hybrid system. A good correlation was found between the loss of toxicity of AsiA to E. coli cells and the inability of mutant AsiAs to bind to sigma(70) It was observed that deletion of C-terminal 17 amino acid residues of AsiA did not affect the activity whereas a mutant asiA lacking C-terminal 28 amino acid residues had the toxicity reduced to a large extent, suggesting that amino acid residues between 64 and 73 played a role in binding to AsiA. A mutant with a deletion of 34 amino acids in the C-terminus did not show any toxicity to E. coli cells. In the N-terminal region, deletion of five amino acid residues was tolerated but extending the deletion to ten amino acids abolished the AsiA activity completely. The conversion of glutamic acid (E10) to either leucine, serine, glutamine, tyrosine or alanine did not affect the toxicity to a great extent suggesting that a negative charge at E10 is not critical for interaction with sigma(70). The results of our in vivo studies suggest that the primary sigma(70) binding site of AsiA is in N-terminus, but, it requires the presence of C-terminal 64-73 amino acid residues for effective binding in vivo.

IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1ß (IL-1ß) secretion. Txnip gene deletion reduces pancreatic ß cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1α RNase inhibitors suppress TXNIP production to block IL-1ß secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.

Peptide-receptive major histocompatibility complex class I molecules cycle between endoplasmic reticulum and cis-Golgi in wild-type lymphocytes.

Prior to binding to a high affinity peptide and transporting it to the cell surface, major histocompatibility complex class I molecules are retained inside the cell by retention in the endoplasmic reticulum (ER), recycling through the ER-Golgi intermediate compartment and possibly the cis-Golgi, or both. Using fluorescence microscopy and a novel in vitro COPII (ER-to-ER-Golgi intermediate compartment) vesicle formation assay, we find that in both lymphocytes and fibroblasts that lack the functional transporter associated with antigen presentation, class I molecules exit the ER and reach the cis-Golgi. Intriguingly, in wild-type T1 lymphoma cells, peptide-occupied and peptide-receptive class I molecules are simultaneously exported from ER membranes with similar efficiencies. Our results suggest that binding of high affinity peptide and exit from the ER are not coupled, that the major histocompatibility complex class I quality control compartment extends into the Golgi apparatus under standard conditions, and that peptide loading onto class I molecules may occur in post-ER compartments.