Numerous proteins, many essential for the DNA replication machinery, interact with proliferating cell nuclear antigen (PCNA) through the PCNA-interacting peptide (PIP) sequence called the PIP box. We have previously shown that the oxidative demethylase human AlkB homologue 2 (hABH2) colocalizes with PCNA in replication foci. In this study, we show that hABH2 interacts with a posttranslationally modified PCNA via a novel PCNA-interacting motif, which we term AlkB homologue 2 PCNA-interacting motif (APIM). We identify APIM in >200 other proteins involved in DNA maintenance, transcription, and cell cycle regulation, and verify a functional APIM in five of these. Expression of an APIM peptide increases the cellular sensitivity to several cytostatic agents not accounted for by perturbing only the hABH2–PCNA interaction. Thus, APIM is likely to mediate PCNA binding in many proteins involved in DNA repair and cell cycle control during genotoxic stress.
CD8+ T cells are critical in human type 1 diabetes and in the NOD mouse. In this study, we elucidated the natural history of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD8+ T cells in NOD diabetes using MHC-tetramer technology. IGRP206–214-specific T cells in the peripheral lymphoid tissue increased with age, and their numbers correlated with insulitis progression. IGRP206–214-specific T cells in the peripheral lymphoid tissue expressed markers of chronic Ag stimulation, and their numbers were stable after diagnosis of diabetes, consistent with their memory phenotype. IGRP206–214-specific T cells in NOD mice expand, acquire the phenotype of effector-memory T cells in the islets, and emigrate to the peripheral lymphoid tissue. Our observations suggest that enumeration of effector-memory T cells of multiple autoantigen specificities in the periphery of type 1 diabetic subjects could be a reliable reporter for progression of islet pathology.
Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung ؉/؉ and Ung ؊/؊ backcrossed mice. Interestingly, human cells displayed ϳ15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ϳ8-fold higher in mouse cells, constituting ϳ50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung ؊/؊ mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity.
In type 1 diabetes, many effector mechanisms damage the β cell, a key one being perforin/granzyme B production by CD8+ T cells. The death receptor pathway has also been implicated in β cell death, and we have therefore generated NOD mice that express a dominant-negative form of the Fas-associated death domain protein (FADD) adaptor to block death receptor signaling in β cells. Islets developed normally in these animals, indicating that FADD is not necessary for β cell development as it is for vasculogenesis. β cells from the transgenic mice were resistant to killing via the Fas pathway in vitro. In vivo, a reduced incidence of diabetes was found in mice with higher levels of dominant-negative FADD expression. This molecule also blocked signals from the IL-1R in culture, protecting isolated islets from the toxic effects of cytokines and also marginally reducing the levels of Fas up-regulation. These data support a role for death receptors in β cell destruction in NOD mice, but blocking the perforin/granzyme pathway would also be necessary for dominant-negative FADD to have a beneficial clinical effect.
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