Searching for new molecules involved in Anopheles mosquitoes' response to Plasmodium infection.

Plasmodium parasites within mosquitoes are exposed to various physiological processes, such as blood meal digestion activity, the gonotrophic cycle, and host responses preventing the entry of parasites into the midgut wall. However, when in vitro-cultured ookinetes are injected into the hemocoel of mosquitoes, Plasmodium parasites are not affected by the vertebrate host's blood contents and do not pass through the midgut epithelial cells. This infection method might aid in identifying mosquito-derived factors affecting Plasmodium development within mosquitoes. This study investigated novel mosquito-derived molecules related to parasite development in Anopheles mosquitoes. We injected in vitro-cultured Plasmodium berghei (ANKA strain) ookinetes into female and male Anopheles stephensi (STE2 strain) mosquitoes and found that the oocyst number was significantly higher in males than in females, suggesting that male mosquitoes better support the development of parasites. Next, RNA-seq analysis was performed on the injected female and male mosquitoes to identify genes exhibiting changes in expression. Five genes with different expression patterns between sexes and greatest expression changes were identified as being potentially associated with Plasmodium infection. Two of the five genes also showed expression changes with infection by blood-feeding, indicating that these genes could affect the development of Plasmodium parasites in mosquitoes.

Formation of free oocysts in Anopheles mosquitoes injected with Plasmodium ookinetes.

Malaria needs new strategies for its control. Plasmodium spp., the causative agent of malaria, is transmitted by mosquitoes. These parasites develop into oocysts and sporozoites in the body of the mosquitoes. A deeper understanding of oocysts that produce the infectious form of the parasite, sporozoites, can facilitate the development of novel countermeasures. However, the isolation of Plasmodium oocysts is challenging as these are formed between midgut epithelial cells and basal lamina after gametocytes enter the mosquito's body through blood feeding. Further research on oocysts has been impeded by issues related to oocyst isolation. Therefore, in this study, we injected Plasmodium into mosquitoes-an artificial and unique method-and aimed to clarify how oocysts were formed in mosquitoes after Plasmodium injection and whether free oocysts were formed from the mosquito tissue. Plasmodium berghei (ANKA strain) ookinetes cultured in vitro were injected into the thoracic body cavity (hemocoel) of female and male Anopheles stephensi mosquitoes. Oocysts were formed in the body of female and male mosquitoes at 14 days post injection. In addition, oocysts formed as a result of injection developed into sporozoites, which were infectious to mice. These findings suggest that P. berghei can complete its developmental stage in mosquitoes by injection. Some of the oocysts formed were free from mosquito tissue, and it was possible to collect oocysts with minimal contamination of mosquito tissue. These free oocysts can be used for investigating oocyst proteins and metabolism.

Phosphorylation by ATR triggers FANCD2 chromatin loading and activates the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination. We uncover how the phosphorylation events are tightly regulated in cells and that mimicking their constant phosphorylation leads to an uncontrolled active state of FANCD2, which is loaded onto chromosomes in an unrestrained fashion. Taken together, we describe a mechanism where ATR triggers FANCD2/FANCI loading onto chromosomes.

A Highly Conserved Region in BRCA2 Suppresses the RAD51-Interaction Activity of BRC Repeats.

Mammary tumors are the most prevalent type of tumors in female dogs. Breast cancer 2, early onset (BRCA2) malignant mutations are associated with tumorigenesis in humans and dogs. BRCA2 plays a pivotal role in homologous recombination repair by recruiting RAD51 recombinase to DNA damage sites to maintain genome stability. To recruit RAD51, BRCA2 must interact with RAD51 via BRC repeats, but the regulation of this interaction has been unclear. In this study, we focused on a highly conserved region (HCR) near BRC repeats. Using co-immunoprecipitation and mammalian two-hybrid assay, we found that HCR suppressed the RAD51-interaction activity of BRC repeats and that substitutions of HCR phosphorylation sites affected it. In canine tumor samples, we found ten mutations, including a novel HCR mutation (I1110M) from canine tumor samples. The effect of four HCR mutations, including I1110M, on the RAD51-interaction activity of BRC repeats was tested. One of the HCR mutations found in canine mammary tumors increased the interaction, but the two mutations found in human breast cancers decreased it. This study suggested that the HCR regulated the RAD51-interacting activity of BRC repeats through HCR phosphorylation and that mutations in HCR may be related to tumorigenesis in both dogs and humans.

Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition.

Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage.

Plasmodium berghei Brca2 is required for normal development and differentiation in mice and mosquitoes.

BACKGROUND: Malaria is a major global parasitic disease caused by species of the genus Plasmodium. Zygotes of Plasmodium spp. undergo meiosis and develop into tetraploid ookinetes, which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1) are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs. Nevertheless, these have not been reported for Plasmodium. METHODS: A Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 (PbBrca2) and Rad51 (PbRad51) was investigated using a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, PbBrca2 was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony. RESULTS: The candidate protein was conserved among Plasmodium species, and it was indicated that it harbors critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, the candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia with increased ring stage and decreased trophozoite stage counts, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, the morphology of the blood stages in mice and the ookinete stage was comparable to those of the wild type parasites. Transmission electron microscopy results showed that sporogony never progressed in Brca2-knockout parasites. CONCLUSIONS: Brca2 is implicated in nearly all Plasmodium life cycle stages, and especially in sporogony. PbBrca2 contributes to HR during meiosis.

BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents.

Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand crosslinking agents or X-ray irradiation, which induces DNA double-strand breaks. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51 oligomer-containing nucleofilaments. In this study, we investigated CTRBD expression in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, BRCA2 CTRBD expression in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. Under endogenous BRCA2 depletion using shRNA, the sensitivities of the BRCA2-depleted cells with and without the CTRBD did not significantly differ. Thus, the resistance to X-ray irradiation conferred by an exogenous CTRBD required endogenous BRCA2 expression. BRCA2 CTRBD-expressing cells demonstrated effective RAD51 foci formation and increased homologous recombination efficiency, but not nonhomologous end-joining efficiency. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment by increased homologous recombination efficiency. Thus, this peptide may be useful for protecting cells against X-ray irradiation or chemotherapeutic agents.

Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2.

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.

Reduced translation efficiency due to novel splicing variants in 5' untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2.

BACKGROUND: Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination. RESULTS: In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5' untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5' UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1. CONCLUSIONS: This study demonstrates that BRCA2 expression level is regulated via 5' UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

The canine RAD51 mutation leads to the attenuation of interaction with PALB2.

RAD51 forms a complex with BRCA2 and plays a central role in the DNA damage response pathway that is associated with homologous recombination. The structures of RAD51 and its homologues are highly conserved from prokaryotes to higher eukaryotes. Although a large number of BRCA2 mutations have been reported, there are only a few reports on the mutations of RAD51, which have been shown in humans and dogs. However, several mutations of canine RAD51 were identified from mammary gland tumour tissues in a recent study. Some of these mutations seem to have an influence on the homo-oligomerization or interaction with "Partner and localizer of BRCA2" (PALB2). In this study, we cloned the canine PALB2 homologue and investigated the effect on its interaction with the RAD51 mutants to evaluate the alteration in the function of RAD51 mutants. The A209S and T225S mutants of RAD51 show an attenuation of the interaction between RAD51 and PALB2. These results indicate that the canine RAD51 mutations can potentially alter the homologous recombination pathways in response to DNA damage in dogs.

Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage.

Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.

Identification of UHRF2 as a novel DNA interstrand crosslink sensor protein.

The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.

Heme-binding of bovine lactoferrin: the potential presence of a heme-binding capacity in an ancestral transferrin gene.

Lactoferrin (Lf) and transferrin (Tf) are iron-binding proteins that can bind various metal ions. This study demonstrates the heme-binding activity of bovine Lf and Tf using biotinylated hemin. When both proteins were coated on separate plate wells, each directly bound biotinylated hemin. On the other hand, when biotinylated hemin was immobilized on an avidin-coated plate, soluble native Lf bound to the immobilized biotinylated hemin whereas native Tf did not, suggesting that a conformational change triggered by coating on the plate allows the binding of denatured Tf with hemin. Incubation of Lf with hemin-agarose resulted in negligible binding of Lf with biotinylated hemin. Lf in bovine milk also bound to immobilized biotinylated hemin. These results demonstrate that bovine Lf has specific heme-binding activity, which is different from Tf, suggesting that either Tf lost heme-binding activity during its evolution or that Lf evolved heme-binding activity from its Tf ancestral gene. Additionally, Lf in bovine milk may bind heme directly, but may also bind heme indirectly by interaction with other milk iron- and/or heme-binding proteins.

Binding Analysis of Human Immunoglobulin G as a Zinc-Binding Protein.

Human immunoglobulin G (IgG) binding with zinc ions was examined using zinc ions immobilized on chelating Sepharose beads (Zn-beads). Human IgG bound to Zn-beads but not to Sepharose beads (control beads). Mouse, rat, bovine and equine IgGs also bound to Zn-beads, similar to human IgG. The human IgG F(c) fragment showed zinc ion-binding activity whereas the Fab fragment did not. Ethylenediaminetetraacetic acid (EDTA)-treated Zn-beads no longer bound human IgG; however, washing the beads, followed by the addition of zinc ions, restored the binding activity towards human IgG. Zn-beads saturated with human fibrinogen could bind human IgG, and Zn-beads saturated with human IgG could bind fibrinogen. These results suggest that animal IgGs, including human, specifically bind zinc ions, probably through a zinc-binding site in the F(c) fragment and not in the Fab fragment. In addition, IgG and fibrinogen interact with each other and/or bind zinc ions through different mechanisms.

Iron and Ferritin Levels in the Serum and Milk of Bovine Leukemia Virus-Infected Dairy Cows.

Iron metabolism was examined in 15 bovine leukemia virus (BLV)-infected dairy cows (2.6-7.8 years old). BLV infection was detected by measuring serum antibody titer against BLV virus antigen (gp51). The anti-BLV antibody titers of the BLV-infected cows were significantly higher in serum than in milk; a single serum-positive animal lacked detectable anti-BLV antibodies in its milk. Iron and ferritin concentrations also were significantly higher in serum than in milk. Although most of the BLV-infected dairy cows had past or present anamneses (such as inflammatory diseases, including intramammary infection), the milk ferritin concentrations of the infected cows were significantly lower than those of normal cows; serum ferritin concentrations did not differ significantly between these two groups. The anti-BLV antibody titers in milk samples showed significant correlation with serum iron concentrations. These results suggest that BLV infection affects iron homeostasis through iron metabolism in the dairy cow mammary gland.

Molecular cloning of canine co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and investigation of its ability to suppress androgen receptor signalling in androgen-independent prostate cancer.

Although the morbidity of canine prostate cancer is low, the majority of cases present with resistance to androgen therapy and poor clinical outcomes. These pathological conditions are similar to the signs of the terminal stage of human androgen-independent prostate cancer. The co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) is known to be overexpressed in human androgen-independent prostate cancer. However, there is little information about the structure and function of canine SGTA. In this study, canine SGTA was cloned and analysed for its ability to suppress androgen receptor signalling. The full-length open reading frame (ORF) of the canine SGTA gene was amplified by RT-PCR using primers designed from canine-expressed sequence tags that were homologous to human SGTA. The canine SGTA ORF has high homology with the corresponding human (89%) and mouse (81%) sequences. SGTA dimerisation region and tetratricopeptide repeat (TPR) domains are conserved across the three species. The ability of canine SGTA to undergo homodimerisation was demonstrated by a mammalian two-hybrid system and a pull-down assay. The negative impact of canine SGTA on androgen receptor (AR) signalling was demonstrated using a reporter assay in androgen-independent human prostate cancer cell lines. Pathological analysis showed overexpression of SGTA in canine prostate cancer, but not in hyperplasia. A reporter assay in prostate cells demonstrated suppression of AR signalling by canine SGTA. Altogether, these results suggest that canine SGTA may play an important role in the acquisition of androgen independence by canine prostate cancer cells.

Reduced canine BRCA2 expression levels in mammary gland tumors.

BACKGROUND: Mammary tumors are the most common tumor type in intact female dogs. Recently, the breast cancer 2 early onset (BRCA2) gene was proposed to be associated with tumorigenesis in dogs. The expression level of BRCA2 is important for its DNA repair function in mammalian cells, and its expression level is linked to tumorigenesis in mammary tissue. However, the expression of canine BRCA2 in mammary tumors is unclear. RESULTS: BRCA2 mRNA levels were compared between seven mammary gland samples and seventeen mammary tumor samples isolated from dogs. The expression level of canine BRCA2 in mammary tumor samples was lower than levels in mammary gland samples. We attempted to identify why the BRCA2 expression level was decreased in mammary tumor samples by promoter sequencing analysis; however, we did not find any mutations in the canine BRCA2 promoter that altered BRCA2 transcription levels. We did detect two types of BRCA2 splice variants in 8 mammary tumor samples. One of the variants induced a frame-shift mutation that could lead to nonsense-mediated mRNA decay, a ubiquitous cellular mechanism that eliminates mRNA containing a premature termination codon. CONCLUSIONS: Reduced expression of canine BRCA2 mRNA in mammary tumor samples is a possible mechanism to explain mammary tumor development in dogs. One possible reason for reduced BRCA2 mRNA levels in these tumor samples was nonsense-mediated mRNA decay, not mutations in the BRCA2 promoter region. While it remains unclear why canine BRCA2 expression levels are reduced in mammary tumor samples, this study found that the expression level of BRCA2 was associated with canine mammary tumorigenesis.

Analysis of the binding of bovine and human fibrinogen to ferritin: evidence that fibrinogen is a common ferritin-binding protein in mammals.

Both human and horse fibrinogen are heme-binding proteins, and horse fibrinogen also exhibits heme-mediated ferritin binding. This study found that bovine and human fibrinogen are heme-mediated ferritin-binding proteins and demonstrated direct binding of bovine ferritin to protoporphyrin (PPIX) and its derivatives or to Zn ions. Binding of bovine and human fibrinogen to bovine spleen ferritin coated on microtiter plate wells was detected using an anti-human fibrinogen antibody, and this binding was inhibited in a dose-dependent manner by hemin (iron-PPIX) and also inhibited by Zn-PPIX. PPIX showed less of an inhibitory effect on the binding of bovine and human fibrinogen to bovine ferritin. The inhibitory effect of Sn-PPIX was similar to that of PPIX, but with respect to human fibrinogen, PPIX did not inhibit the binding of human fibrinogen to ferritin. Bovine fibrinogen immobilized on CNBr-activated Sepharose 4B beads showed affinity for hemin, Sn-PPIX, Zn-PPIX, and iron-free PPIX in the order Sn-PPIX < iron-free PPIX < hemin < Zn-PPIX. The fibrinogen beads also directly bound to zinc ions. These results suggest that bovine fibrinogen is a heme- and zinc-binding protein and that binding of circulating mammalian fibrinogen to ferritin is heme mediated.

Polymorphisms of canine BRCA2 BRC repeats affecting interaction with RAD51.

Mutations in the breast cancer susceptibility gene BRCA2 leading to the failure of interactions with the recombinase RAD51 are associated with an increased risk of cancer in humans. This interaction depends on the eight BRC repeat (BRC1-8) sequences in BRCA2. We previously reported that canine BRC3 has two polymorphisms (T1425P and K1435R) influencing the interaction with RAD51, and 1435R was identified in mammary tumor dog samples. In this study, we investigated the sequence variations of BRC3 and 4 in 236 dogs of five breeds. Allele frequencies of 1425P and 1435R were 0.063 and 0.314, respectively, and there was no other polymorphism in the sequenced region. A mammalian two-hybrid assay using BRC3-4 sequences demonstrated that 1425P allele reduced the binding strength with RAD51 but 1435R had no effect. These results may provide an insight into the functions of not only individual but also multiple BRC repeats of BRCA2 in dogs.

UHRF1 is a sensor for DNA interstrand crosslinks and recruits FANCD2 to initiate the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway is critical for the cellular response to toxic DNA interstrand crosslinks (ICLs). Using a biochemical purification strategy, we identified UHRF1 as a protein that specifically interacts with ICLs in vitro and in vivo. Reduction of cellular levels of UHRF1 by RNAi attenuates the FA pathway and sensitizes cells to mitomycin C. Knockdown cells display a drastic reduction in FANCD2 foci formation. Using live-cell imaging, we observe that UHRF1 is rapidly recruited to chromatin in response to DNA crosslinking agents and that this recruitment both precedes and is required for the recruitment of FANCD2 to ICLs. Based on these results, we describe a mechanism of ICL sensing and propose that UHRF1 is a critical factor that binds to ICLs. In turn, this binding is necessary for the subsequent recruitment of FANCD2, which allows the DNA repair process to initiate.