Discovery of a Unique Set of Dog-Seroreactive Coccidioides Proteins Using Nucleic Acid Programmable Protein Array.

Valley Fever (VF), caused by fungi in the genus Coccidioides, is a prevalent disease in southwestern and western parts of the United States that affects both humans and animals, such as dogs. Although the immune responses to infection with Coccidioides spp. are not fully characterized, antibody-detection assays are used in conjunction with clinical presentation and radiologic findings to aid in the diagnosis of VF. These assays often use Complement Fixation (CF) and Tube Precipitin (TP) antigens as the main targets of IgG and IgM reactivity, respectively. Our group previously reported evidence of over 800 genes expressed at the protein level in C. posadasii. However, antibody reactivity to the majority of these proteins has never been explored. Using a new, high-throughput screening technology, the Nucleic Acid Programmable Protein Array (NAPPA), we screened serum specimens from dogs against 708 of these previously identified proteins for IgG reactivity. Serum from three separate groups of dogs was analyzed and revealed a small panel of proteins to be further characterized for immuno-reactivity. In addition to CF/CTS1 antigen, sera from most infected dogs showed antibody reactivity to endo-1,3-betaglucanase, peroxisomal matrix protein, and another novel reactive protein, CPSG_05795. These antigens may provide additional targets to aid in antibody-based diagnostics.

The Fanconi anemia proteins FANCD2 and FANCJ interact and regulate each other's chromatin localization.

Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment.

Functional and physical interaction between the mismatch repair and FA-BRCA pathways.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and an increased risk for leukemia and cancer. Fifteen proteins thought to function in the repair of DNA interstrand crosslinks (ICLs) comprise what is known as the FA-BRCA pathway. Activation of this pathway leads to the monoubiquitylation and chromatin localization of FANCD2 and FANCI. It has previously been shown that FANCJ interacts with the mismatch repair (MMR) complex MutLα. Here we show that FANCD2 interacts with the MMR proteins MSH2 and MLH1. FANCD2 monoubiquitylation, foci formation and chromatin loading are greatly diminished in MSH2-deficient cells. Human or mouse cells lacking MSH2 or MLH1 display increased sensitivity and radial formation in response to treatment with DNA crosslinking agents. Studies in human cell lines and Drosophila mutants suggest an epistatic relationship between FANCD2, MSH2 and MLH1 with regard to ICL repair. Surprisingly, the interaction between MSH2 and MLH1 is compromised in multiple FA cell lines, and FA cell lines exhibit deficient MMR. These results suggest a significant role for MMR proteins in the activation of the FA pathway and repair of ICLs. In addition, we provide the first evidence for a defect in MMR in FA cell lines.

The E3 ubiquitin ligase RAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.