Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR-ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2

The Iddm14 gene is Tcrbv-13S1A1: Prevention of Autoimmune Diabetes in the Rat with an Allele-Specific Depleting Antibody That Recognizes the Vβ13a T Cell Receptor Beta Chain

To identify new intervention strategies for autoimmune type 1 diabetes (T1D), we investigated several rat models of the disorder. We dissected the powerful Iddm14 diabetes susceptibility locus in eight T1D susceptible vs. resistant rat strains by single nucleotide polymorphism (SNP) haplotyping. We identified an allele of a T cell receptor (TCR) beta chain gene, Tcrb-V13S1A1 (encoding V13βa) as a candidate gene. In three separate trials, treating LEW.1WR1 rats, which are susceptible to T1D, with a depleting anti-Vβ13 monoclonal antibody reduced diabetes frequency from 75% (N=50) to 17% (N=30, p\u3c0.001. Anti-Vβ13 monoclonal antibody also prevented T1D in spontaneously diabetic BBDP rats. We then analyzed the phenotype of infiltrating T cells recovered from the cultured islets of LEW.1WR1 rats exposed to a diabetogenic trigger. Within 5 days, up to 22% of CD4+ T cells recovered from islets were V13β+, most of these CD25+FoxP3-. We also recovered Vβ13 transcripts from pre-diabetic islets and observed a limited number of Jβ variant transcripts, indicating an oligoclonal TCR response to pancreatic beta cells. These data indicate that, in susceptible rats, V13βa on diabetogenic T cells is required to recognize a critical T1D autoantigen. Interestingly, the diabetogenic and non-diabetogenic alleles of Vβ13 have non-conservative sequence differences in both CRR1 and CDR2. The data suggest that it is possible to prevent T1D in the rat with a very narrowly targeted deletional therapy. Preliminary data suggest that a specific alpha chain may preferentially pair with Vβ13a. We are currently generating rat T cell hybridoma clones with which to analyze the interaction of putative autoantigens with a diabetogenic TCR

BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands

TOPBP1 and its fission yeast homologue Rad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 with diverse phospho-ligands, including human RAD9 and Treslin, and Schizosaccharomyces pombe Crb2 and Sld3, to define the determinants of BRCT domain specificity. We use this to identify and characterise previously unknown phosphorylationdependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins

The Missing Heritability in T1D and Potential New Targets for Prevention

Type 1 diabetes (T1D) is a T cell-mediated disease. It is strongly associated with susceptibility haplotypes within the major histocompatibility complex, but this association accounts for an estimated 50% of susceptibility. Other studies have identified as many as 50 additional susceptibility loci, but the effect of most is very modest (odds ratio (OR) 5) and that deletion of V beta 13+ T cells prevents diabetes. A role for the TCR is also suspected in NOD mice, but TCR regions have not been associated with human T1D. To investigate this disparity, we tested the hypothesis in silico that previous studies of human T1D genetics were underpowered to detect MHC-contingent TCR susceptibility. We show that stratifying by MHC markedly increases statistical power to detect potential TCR susceptibility alleles. We suggest that the TCR regions are viable candidates for T1D susceptibility genes, could account for missing heritability, and could be targets for prevention

Glioblastoma Mimicking an Arteriovenous Malformation

Abnormal cerebral vasculature can be a manifestation of a vascular malformation or a neoplastic process. We report the case of a patient with angiography-negative subarachnoid hemorrhage (SAH) who re-presented 3 years later with a large intraparenchymal hemorrhage. Although imaging following the intraparenchymal hemorrhage was suggestive of arteriovenous malformation, the patient was ultimately found to have an extensive glioblastoma associated with abnormal tumor vasculature. The case emphasizes the need for magnetic resonance imaging to investigate angiography-negative SAH in suspicious cases to rule out occult etiologies, such as neoplasm. We also discuss diagnostic pitfalls when brain tumors are associated with hemorrhage and abnormal vasculature

An essential function for the ATR-Activation-Domain (AAD) of TopBP1 in mouse development and cellular senescence

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development

Behavioural and biochemical effects of an ICV injection of streptozotocin in old Lewis rats

Intacerebroventricularly (ICV) injected streptozotocin (STREP) decreases central glucose metabolism and energy metabolism, which has also been observed in patients with dementia. In the present study we examined the behavioral (open-field behavior and two-way active avoidance learning) and biochemical (hippocampal ChAT activity) effects of STREP treatment in old Lewis rats. The results suggest that hippocampal function was affected by STREP. STREP-treated rats acquired the two-way active avoidance task faster than the control rats, which indicates that STREP treatment does not lead to a general learning deficit. Hippocampal ChAT activity was decreased in STREP-treated rats. The present results suggests also that susceptibility to STREP amy not be related to age in Lewis rats

Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA

TopBP1, acting in concert with DNA containing bulky base lesions, stimulates ATR kinase activity under physiologically relevant reaction conditions. Here, we analyze the roles of the three components in ATR activation: DNA, base damage and TopBP1. We show that base adducts caused by a potent carcinogen, benzo[a]pyrene diol epoxide (BPDE), constitute a strong signal for TopBP1-dependent ATR kinase activity on Chk1 and p53. We find that the C-terminus of TopBP1 binds preferentially to damaged DNA and is sufficient to mediate damaged DNA-dependent ATR activation in a manner similar to full-length TopBP1. Significantly, we find that stimulation of ATR by BPDE-damaged DNA exhibits strong dependence on the length of DNA, with essentially no stimulation with fragments of 0.2 kb and reaching maximum stimulation with 2 kb fragments. Moreover, TopBP1 shows preferential binding to longer DNA fragments and, in contrast to previous biochemical studies, TopBP1 binding is completely independent of DNA ends. We find that TopBP1 binds to circular and linear DNAs with comparable affinities and that these DNA forms elicit the same level of TopBP1-dependent ATR activation. Taken together, these findings suggest a cooperative activation mechanism for the ATR checkpoint kinase by TopBP1 and damaged DNA

CHOP Mediates Endoplasmic Reticulum Stress-Induced Apoptosis in Gimap5-Deficient T Cells

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5−/− BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5−/− T cells. Knockdown of CHOP by siRNA protected Gimap5−/− T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells