The solution structure of the disulphide-linked homodimer of the human trefoil protein TFF1

AbstractThe trefoil factor family protein, TFF1, forms a homodimer, via a disulphide linkage, that has greater activity in wound healing assays than the monomer. Having previously determined a high-resolution solution structure of a monomeric analogue of TFF1, we now investigate the structure of the homodimer formed by the native sequence. The two putative receptor/ligand recognition domains are found to be well separated, at opposite ends of a flexible linker. This contrasts sharply with the known fixed and compact arrangement of the two trefoil domains of the closely related TFF2, and has significant implications for the mechanism of action and functional specificity of the TFF of proteins

Common cancer-associated imbalances in the DNA damage response confer sensitivity to single agent ATR inhibition

ATR is an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest, DNA damage accumulation and repair were determined following VE-821 exposure. Defects in homologous recombination repair (HRR: ATM, BRCA2 and XRCC3) and base excision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly, the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component, Ku80, caused hypersensitivity to VE-821, but loss of its partner catalytic subunit, DNA-PKcs, did not. Unexpectedly, VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR, determined by RAD51 focus formation, to a greater extent in cells with high DNA-PKcs. Defects in HRR and BER and high DNA-PKcs expression, that are common in cancer, confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine

Does radiation-induced c-MYC amplification initiate breast oncogenesis?

© 2016 Taylor and Francis Group, LLC. The MYC (v-myc avian myelocytomatosis viral oncogene homolog; c-MYC) locus on chromosome 8q is susceptible to high-level amplification following exposure of human breast cells to ionizing radiation, and c-MYC amplification is a common feature of both radiogenic adenocarcinoma and radiogenic angiosarcoma of the breast. Taken together, these observations suggest common breast-specific susceptibility factors that predispose cells to amplification of this critical proto-oncogene and the development of radiogenic cancer in multiple tissue types of this radiosensitive organ

Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor

Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and pathophysiology of nephrolithiasis. We have combined conventional biochemical methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization–time-of-flight MS and electrospray ionization–quadrupole–time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti–C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis

The trefoil factor interacting protein TFIZ1 binds the trefoil protein TFF1 preferentially in normal gastric mucosal cells but the co-expression of these proteins is deregulated in gastric cancer

The gastric tumour suppressor trefoil protein TFF1 is present as a covalently bound heterodimer with a previously uncharacterised protein, TFIZ1, in normal human gastric mucosa. The purpose of this research was firstly to examine the molecular forms of TFIZ1 present, secondly to determine if TFIZ1 binds other proteins apart form TFF1 in vivo, thirdly to investigate if TFIZ1 and TFF1 are co-regulated in normal gastric mucosa and fourthly to determine if their co-regulation is maintained or disrupted in gastric cancer. We demonstrate that almost all human TFIZ1 is present as a heterodimer with TFF1 and that TFIZ1 is not bound to either of the other two trefoil proteins, TFF2 and TFF3. TFIZ1 and TFF1 are co-expressed by the surface mucus secretory cells throughout the stomach and the molecular forms of each protein are affected by the relative abundance of the other. TFIZ1 expression is lost consistently, early and permanently in gastric tumour cells. In contrast, TFF1 is sometimes expressed in the absence of TFIZ1 in gastric cancer cells and this expression is associated with metastasis (lymph node involvement: p = 0.007). In conclusion, formation of the heterodimer between TFIZ1 and TFF1 is a specific interaction that occurs uniquely in the mucus secretory cells of the stomach, co-expression of the two proteins is disrupted in gastric cancer and expression of TFF1 in the absence of TFIZ1 is associated with a more invasive and metastatic phenotype. This indicates that TFF1 expression in the absence of TFIZ1 expression has potentially deleterious consequences in gastric cancer