D1 Repair Research Articles

Comparative analysis of cell adhesion molecules, cell cycle regulatory proteins, mismatch repair genes, cyclooxygenase-2, and DPC4 in carcinomas arising in inflammatory bowel disease and sporadic colon cancer

Colon carcinoma arising in inflammatory bowel disease often exhibits aggressive behavior compared to sporadic carcinomas. The rationale for the different biological behaviors of these two groups of tumors is not fully understood. In this study, we have examined carcinomas arising in inflammatory bowel disease (IBD) and sporadic carcinomas (SCA) for molecular differences that may provide clues for the behavioral disparity of these tumors. Thirty-eight colon carcinomas (12 from ulcerative colitis, 5 from Crohn's disease, and 21 SCA) were analyzed by immunohistochemistry for cell adhesion molecules (E-cadherin, beta-catenin, CD44), cell cycle regulatory proteins (cyclin D1, p27, p21), mismatch repair proteins (hMLH1, hMSH2), cyclooxygenase-2 and DPC4. Carcinomas arising in IBD showed significant decrease in expression of cell adhesion molecules, the cell cycle inhibitor protein, p21, and increased expression of cyclooxygenase-2 compared to sporadic carcinomas. No differences were observed in the expression of cell cycle regulatory proteins p27, cyclin D1, DPC4 and mismatch repair proteins between these two groups of tumors. Decreased expression of p21 as well as adhesion molecules may provide increased impetus for the aggressive behavior of tumors arising in inflammatory bowel disease.

Var2 mutation in Arabidopsis affects D1 degradation

Var2 is one of several FtsH homologues in Arabidopsis thaliana. Var2 mutations cause variegation, and alter photosynthetic function in a number of ways. FtsH proteins are AAA proteases (ATPases associated with a variety of cellular activities), which are widely distributed in prokaryotes and eukaryotes and have numerous roles. We have found that Var2 mutant are affected in the D1 repair cycle following photoinhibition. Composition of the photosynthetic apparatus is comparable in wild-type (WT). The chlorophyll a/b ratios suggest similar-size photosystem (PS) I1 antennae, and fluorescence spectra indicate only slightly different PS1:PSII ratios in low-light-grown plants. Measurement of Fv/Fm, however, suggests that Var2 undergoes greater photoinhibition than WT at low (300 kE/mZ/s) and, markedly, high light (1800 pE/mZ/s). When protein synthesis is prevented (with lincomycin), both Var2 and WT leaves are photoinhibited at high light since neither can synthesise new D1 polypeptide following photooxidative damage. Even without lincomycin, however, Var2 is photoinhibited to twice the extent of WT this key PSII protein is not replaced. Western blots confirm that lincomycin-treated WT leaves exhibit rapid D1 turnover whereas Var2 leaves do not, suggesting that D1 is not degraded.

The role of FtsH proteins in photosystem biosynthesis and turnover in Arabidopsis and Synechocystis

Both Arabidopsis thaliana and Synechocystis PCC 6803 genomes encode several homologues of Escherichia coli FtsH. These proteins are AAA-family proteases (ATPases associated with a variety of cellular activities), which are widely distributed in prokaryotes and eukaryotes. Among their diverse roles, FtsH proteins are important in photosynthesis. Of four found in Synechocystis, one (encoded by open-reading frame slr0228) appears to be involved in photosystem I (PSI) biosynthesis, and it has been suggested that it may also participate in photosystem II (PSII) D1-protein turnover (see S8 poster by P. Silva et al.). In Arabidopsis, mutation of the `Var2?-ftsH leads to severe leaf-variegation [1]. Whereas fluorescence-emission spectra of Synechocystis show a higher PSI:PSII ratio in wild-type (WT) than in slr0228? cells, in Arabidopsis, WT and Var2 have similar PSI:PSII peaks under normal growth conditions. High-light treatment, however, results in greater photoinhibition of Var2-FtsH? plants, measured by Fv/Fm. Fluorescence emission spectra also show reduced recovery from photoinhibitory high light in Var2 compared with WT. These experiments and Western blots indicate that the D1 repair cycle is impeded in the Arabidopsis FtsH mutant as in the cyanobacterium. Sequence similarities in the slr0228 and Var2 ftsH genes indeed suggest they may be closely-related proteins. To study the role of slr0228 in reaction centre biosynthesis, site-directed mutants have been made in PSII? and chlorophyll-synthesis-deficient Synechocystis strains. Further work characterising the role of plant and cyanobacterial FtsH proteins is presented. 1. Chen M, Choi Y, Voytas DF, Rodermel S: Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease. Plant J 2000, 22:303.

The lumenal loop connecting transmembrane helices I and II of the D1 polypeptide is important for assembly of the photosystem two complex

Current structural models indicate that the D1 and D2 polypeptides of the Photosystem two reaction center complex (PS II RC) each span the thylakoid membrane five times. In order to assess the importance of the lumenal extrinsic loop that connects transmembrane helices I and II of D1 we have constructed five deletion mutants and two double mutants in the cyanobaterium Synechocystic sp. PCC 6803. Four of the deletion mutants (Δ59-65, Δ69-74, Δ79-86 and Δ109-110) are obligate photoheterotrophs unable to accumulate D1 in the membrane as assayed by immunoblotting experiments or pulse-labelling experiments using [(35)S]-methionine. In contrast deletion mutant Δ100 which lacks A100 behaved very similarly to the WT control strain in terms of photoautotrophic growth rate, saturated rates of oxygen evolution, flash-induced oxygen evolution, fluorescence induction and decay, and thermoluminescence. Δ100 is the first example of an internal deletion on the lumenal side of the D1 polypeptide that is benign to photosystem two function. Double mutant D103G/E104A also behaves similarly to the WT control strain leading to the conclusion that residues D103 and E104 are unlikely to be involved in ligating the metal ions Mn or Ca(2+), which are needed for photosynthetic oxygen evolution. Double mutant, G109A/G110A, was constructed to assess the significance of this GlyGly motif which is also conserved in the L subunit of purple bacterial reaction centres. The G109A/G110A mutant is able to evolve oxygen at approximately 50-70% of WT rates but is unable to grow phatoautotrophically apparently because of an enhanced sensitivity to photoinactivation than the WT control strain. A photoautotropic revertant was isolated from this strain and shown to result from a mutation that restored the WT codon at position 109. Pulse-chase experiments in cells using [(35)S]-methionine showed that resistance to photoinhibition in the revertant correlated with an enhanced rate of incorporation of D1 into the membrane compared to mutant G109A/G110A. The sensitivity to photoinhibition shown by the G109A/G110A mutant is therefore consistent with a perturbation to the D1 repair cycle possibly at the level of D1 synthesis or incorporation of D1 into the PS II complex.