ERAAP Shapes the Peptidome Associated with Classical and Nonclassical MHC Class I Molecules.

The peptide repertoire presented by classical as well as nonclassical MHC class I (MHC I) molecules is altered in the absence of the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP). To characterize the extent of these changes, peptides from cells lacking ERAAP were eluted from the cell surface and analyzed by high-throughput mass spectrometry. We found that most peptides found in wild-type (WT) cells were retained in the absence of ERAAP. In contrast, a subset of "ERAAP-edited" peptides was lost in WT cells, and ERAAP-deficient cells presented a unique "unedited" repertoire. A substantial fraction of MHC-associated peptides from ERAAP-deficient cells contained N-terminal extensions and had a different molecular composition than did those from WT cells. We found that the number and immunogenicity of peptides associated with nonclassical MHC I was increased in the absence of ERAAP. Conversely, only peptides presented by classical MHC I were immunogenic in ERAAP-sufficient cells. Finally, MHC I peptides were also derived from different intracellular sources in ERAAP-deficient cells.

MHC I-associated peptides preferentially derive from transcripts bearing miRNA response elements.

MHC I-associated peptides (MIPs) play an essential role in normal homeostasis and diverse pathologic conditions. MIPs derive mainly from defective ribosomal products (DRiPs), a subset of nascent proteins that fail to achieve a proper conformation and the physical nature of which remains elusive. In the present study, we used high-throughput proteomic and transcriptomic methods to unravel the structure and biogenesis of MIPs presented by HLA-A and HLA-B molecules on human EBV-infected B lymphocytes from 4 patients. We found that although HLA-different subjects present distinctive MIPs derived from different proteins, these MIPs originate from proteins that are functionally interconnected and implicated in similar biologic pathways. Secondly, the MIP repertoire of human B cells showed no bias toward conserved versus polymorphic genomic sequences, were derived preferentially from abundant transcripts, and conveyed to the cell surface a cell-type-specific signature. Finally, we discovered that MIPs derive preferentially from transcripts bearing miRNA response elements. Furthermore, whereas MIPs of HLA-disparate subjects are coded by different sets of transcripts, these transcripts are regulated by mostly similar miRNAs. Our data support an emerging model in which the generation of MIPs by a transcript depends on its abundance and DRiP rate, which is regulated to a large extent by miRNAs.

14-3-3 proteins function in the initiation and elongation steps of DNA replication in Saccharomyces cerevisiae.

14-3-3s are highly conserved abundant eukaryotic proteins essential for viability, at least in lower eukaryotes. We previously showed that they associate with mammalian and yeast replication origins in a cell-cycle-dependent manner, and are involved in the initiation of DNA replication. Here, we present evidence that 14-3-3 proteins are novel regulators of the initiation and elongation steps of DNA replication in Saccharomyces cerevisiae. The results show that the Bmh2 protein, one of the two 14-3-3 homologues in S. cerevisiae, interacts with Mcm2 and Orc2 proteins, binds to ARS1 maximally at the G1 phase, is essential for plasmid stability, and is required for normal S-phase entry and progression. Furthermore, during G1 phase, the Bmh2 protein is required for the association of MCM proteins with chromatin and their maintenance at replication origins. The results reveal that 14-3-3 proteins function as essential factors for the assembly and maintenance of the pre-replication complex during G1 phase.

14-3-3 cruciform-binding proteins as regulators of eukaryotic DNA replication.

Cruciforms are secondary DNA structures, serving as recognition signals at or near eukaryotic (yeast and mammalian) origins of DNA replication. The cruciform-binding protein is a member of the 14-3-3 protein family and binds to origins of DNA replication in a cell cycle-dependent manner. Five 14-3-3 protein isoforms (beta, gamma, epsilon, zeta and sigma) have been identified as having cruciform binding activity.

Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast.

BACKGROUND: Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner. RESULTS: By expressing the human 14-3-3epsilon, as the sole member of 14-3-3 proteins family in Saccharomyces cerevisiae, we show that 14-3-3epsilon complements the S. cerevisiae Bmh1/Bmh2 double knockout, conserves its cruciform binding activity, and associates in vivo with the yeast replication origins ARS307. Deletion of the alpha5-helix, the potential cruciform binding domain of 14-3-3, decreased the cruciform binding activity of the protein as well as its association with the yeast replication origins ARS307 and ARS1. Furthermore, the mutant cells had a reduced ability to stably maintain plasmids bearing one or multiple origins. CONCLUSION: 14-3-3, a cruciform DNA binding protein, associates with yeast origins of replication and functions as an initiator of DNA replication, presumably through binding to cruciform DNA forming at yeast replicators.