Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

N1-methyladenine (1-MeA)3 is formed in DNA by reaction with S n 2 methylating agents such as methyl methanesulfonate and naturally occurring methyl halides (1-3). The S n 2 methyl halides are among the most abundant environmental methylating agents released from biomass burning and from decaying vegetation. Exposure to alkylating agents could also occur from food, occupational hazards, and chemotherapeutic treatments (1).1-MeA is highly cytotoxic because the N1 atom is engaged in Watson-Crick (W-C) base pairing and its modification by a methyl group impairs W-C base pairing and blocks normal DNA replication. In Escherichia coli, AlkB repairs 1-MeA by oxidative demethylation, which liberates formaldehyde from the methylated base and results in complete reversal of the damage (4, 5). In humans, there are nine potential AlkB homologs, two of which, ABH2 and ABH3, can repair the same spectrum of DNA lesions as AlkB (6, 7); ABH2, however, is the primary housekeeping enzyme in humans for repairing 1-MeA (8). Mouse embryonic fibroblast lines derived from ABH2 null mice are highly defective in repairing 1-MeA residues generated in response to methyl methanesulfonate treatment. Because in the absence of any exposure to alkylating agents, 1-MeA residues accumulate over time in the genomic DNA of livers from ABH2 null mice, endogenous DNA methylation contributes to their generation (8).Previously, we reported on the genetic control of translesion synthesis (TLS) opposite UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts and opposite thymine glycol (Tg), which is the most common oxidation product of thymine (9 -12). Of the two UV lesions, cyclobutane pyrimidine dimer does not significantly affect the ability of two pyrimidines to form a correct W-C base pair with the purine bases, and it has only a modest effect on DNA structure (13); by contrast, a (6-4) pyrimidine-pyrimidone photoproduct induces a large structural distortion in DNA. It confers a 44º bend in the DNA helix and the 3ЈT is oriented perpendicular to the 5ЈT in the (6-4) TT photoproduct (14 -16). The Tg lesion also has no significant effect on the ability of oxidized T to form a correct base pair with an A; however, because of the addition of hydroxyl groups at C5 and C6 on Tg, the damaged base becomes non-planar and that prevents the base 5Ј to Tg from stacking above it (17-20). Consequently, Tg presents a strong block to extension of synthesis from the Tg:A base pair. Despite the fact that these DNA lesions differ vastly in their effects on DNA structure and on base pairing, they generate only ϳ2% mutagenic TLS products in human cells (10 -12). This is rather surprising in view of the fact that the various TLS DNA polymerases (Pols) synthesize DNA with a low fidelity (21).Here we identify the TLS Pols that promote replication through the 1-MeA lesion in human cells and show that TLS opposite this lesion is mediated by three independent pathways, involving Pols and in one pathway and Pols and , respectively, in the other tw...

Mammals do not regenerate axons in their central nervous system (CNS) spontaneously. This phenomenon is the cause of numerous medical conditions after damage to nerve fibers in the CNS of humans. The study of the mechanisms of nerve regeneration in other vertebrate animals able to spontaneously regenerate axons in their CNS is essential for understanding nerve regeneration from a scientific point of view, and for developing therapeutic approaches to enhance nerve regeneration in the CNS of humans. RICH proteins are a novel group of proteins implicated in nerve regeneration in the CNS of teleost fish, yet their mechanisms of action are not well understood. A number of mutant versions of the zebrafish RICH protein (zRICH) were generated and characterized at biochemical and cellular levels in our laboratory. With the aim of understanding the effects of RICH proteins in neuronal axon outgrowth, stable transfectants derived from the neuronal model PC12 cell line expressing zRICH Wild-Type or mutant versions of zRICH were studied. Results from differentiation experiments suggest that RICH proteins enhance neuronal plasticity by facilitating neurite branching. Biochemical co-purification results have demonstrated that zRICH binds to the cytoskeletal protein tubulin. The central domain of the protein is sufficient for tubulin binding, but a mutant version of the protein lacking the terminal domains, which cannot bind to the plasma membrane, was not able to enhance neurite branching. RICH proteins may facilitate axon regeneration by regulating the axonal cytoskeleton and facilitating the formation of new neurite branches.

Bcl-2 is an anti-apoptotic protein that inhibits apoptosis elicited by multiple stimuli in a large variety of cell types. BMRP (also known as MRPL41) was identified as a Bcl-2 binding protein and shown to promote apoptosis. Previous studies indicated that the amino-terminal two-thirds of BMRP contain the domain(s) required for its interaction with Bcl-2, and that this region of the protein is responsible for the majority of the apoptosis-inducing activity of BMRP. We have performed site-directed mutagenesis analyses to further characterize the BMRP/Bcl-2 interaction and the pro-apoptotic activity of BMRP. The results obtained indicate that the 13-17 amino acid region of BMRP is necessary for its binding to Bcl-2. Further mutagenesis of this motif shows that amino acid residue aspartic acid (D) 16 of BMRP is essential for the BMRP/Bcl-2 interaction. Functional analyses conducted in mammalian cells with BMRP site-directed mutants BMRP(13Ala17) and BMRP(D16A) indicate that these mutants induce apoptosis through a caspase-mediated pathway, and that they kill cells slightly more potently than wild-type BMRP. Bcl-2 is still able to counteract BMRP(D16A)-induced cell death significantly, but not as completely as when tested against wild-type BMRP. These results suggest that the apoptosis-inducing ability of wild-type BMRP is blocked by Bcl-2 through several mechanisms.