Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes

In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and alpha-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1-3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity [4]. The membrane association of early endosomal antigen 1 (EEA1), a specific marker of early endosomes [5,6], has recently been shown to be similarly dependent on PI 3-kinase activity [7], and we therefore postulated that it might be involved in endosome fusion. Here, we present evidence that EEA1 has an important role in determining the efficiency of endosome fusion in vitro. Both the carboxy-terminal domain of EEA1 (residues 1098-1411) and specific antibodies against EEA1 inhibited endosome fusion when included in an in vitro assay. Furthermore, depletion of EEA1, both from the membrane fraction used in the assay by washing with salt and from the cytosol using an EEA1-specific antibody, resulted in inhibition of endosome fusion. The involvement of EEA1 in endosome fusion accounts for the sensitivity of the endosome fusion assay to inhibitors of PI 3-kinase.

Possible association between CTLA4 DNA polymorphisms and early onset type 1 diabetes in a UK population

Linkage and association has been reported between CTLA4 DNA markers and susceptibility to type 1 diabetes in some populations, but not others. We performed case-control and family-based association studies to assess if the CTLA4 A49G and intron 1 C/T polymorphisms were associated with development of early onset type 1 diabetes in the Northern Ireland population. The distribution of A49G and C/T alleles in cases (n = 144) was similar to those observed in controls (n = 307). In contrast, significant distortions in allele transmissions from informative parents to probands were observed for both the A49G (P = 0.02) and C/T (P = 0.01) polymorphisms employing 297 nuclear families. Our results suggest that the CTLA4 gene may play a minor role in the overall genetic predisposition to type 1 diabetes in this UK population.

Double-enhancement strategy: a practical approach to a femto-molar level detection of prostate specific antigen--1-antichymotrypsin (PSA/ACT complex) for SPR immunosensing

Prostate specific antigen-a1-antichymotrypsin was detected by a double-enhancement strategy involving the exploitation of both colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP biocatalyzed oxidation. The AuNPs were synthesized and conjugated with horse-radish peroxidase-PSA polyclonal antibody by physisorption. Using the protein-colloid for SPR-based detection of the PSA/ACT complex showed their enhancement as being consistent with other previous studies with regard to AuNPs enhancement, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the signal. The limit of detection was found at as low as 0.027 ng/ml of the PSA/ACT complex (or 300 fM), which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Nutritonal status of adult ewes during early and mid-pregnancy. 1. Effects of plane of nutrition on ewe reproduction and offspring performance to weaning

The objective of this study was to determine the effects of plane of nutrition during early and mid-pregnancy on the performance of mature ewes and their offspring. From day 0 to day 39 post mating (early pregnancy, EP), 82 multiparous ewes were fed to provide either 60% (low, L), 100% (medium, M) or 200% (high, H) of predicted metabolisable energy (ME) requirements for maintenance, following a synchronised mating. From day 40 to day 90 (mid-pregnancy, MP), ewes were provided with either 80% (M) or 140% (H) of ME requirements. After 90 days of gestation, all ewes were fed to meet requirements for late pregnancy. During EP, mean live weight (LW) and body condition score (BCS) change of ewes were -6.3, -0.8 and +6.0kg and -0.02, +0.10 and +0.22 units in the L-, M- and H-EP treatments, respectively. During MP, mean LW and BCS change were -0.8 and +4.9kg and -0.09 and +0.09 units in the M- and H-MP treatments, respectively (P 0. 05) on conception rate, although there tended to be an inverse relationship (P = 0.085) between plane of nutrition in EP and plasma progesterone concentrations at day 42 of gestation. EP nutrition influenced foetal development with lambs from ewes offered diet L-EP being smaller (P

Relationships between EEA1 binding partners and their role in endosome fusion

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.

Regulation of endosome fusion

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.

ARMS2 Increases the Risk of Early and Late Age-Related Macular Degeneration in the European Eye Study

OBJECTIVE:
To study associations between severity stages of early and late age-related macular degeneration (AMD) and genetic variations in age-related maculopathy susceptibility 2 (ARMS2) and complement factor H (CFH) and to investigate potential interactions between smoking and ARMS2.
DESIGN:
Population-based, cross-sectional European Eye Study in 7 countries in Europe.
PARTICIPANTS:
Four thousand seven hundred fifty participants, 65 years of age and older, recruited through random sampling.
METHODS:
Participants were classified on the basis of the more severely affected eye into 5 mutually exclusive AMD severity stages ranging from no AMD, 3 categories of early AMD, and late AMD. History of cigarette smoking was available and allowed classification into never, former, and current smokers, with the latter 2 groups combined into a single category of ever smokers for analysis. Genotyping was performed for single nucleotide polymorphisms rs10490924 and rs4146894 in ARMS2 and rs1061170 in CFH. Associations were analyzed by logistic regression.
MAIN OUTCOME MEASURES:
Odds ratios (ORs) for stage of AMD associated with genetic variations in ARMS2 and CFH and interactions between ARMS2 and smoking status.
RESULTS:
Early AMD was present in 36.4% and late AMD was present in 3.3% of participants. Data on both genotype and AMD were available for 4276 people. The ORs for associations between AMD stage and ARMS2 increased monotonically with more severe stages of early AMD and were altered little by adjustment for potential confounders. Compared with persons with no AMD, carriers of the TT genotype for rs10490924 in ARMS2 had a 10-fold increase in risk of late AMD (P<3 × 10(-20)). The ORs for associations with CFH were similar for stage 3 early AMD and late AMD. Interactions between rs10490924 in ARMS2 and smoking status were significant in both unadjusted and adjusted models (P = 0.001). The highest risk was observed in those doubly homozygous for rs10490924 and rs1061170 in CFH (OR, 62.3; 95% confidence interval, 16-242), with P values for trend ranging from 0.03 (early AMD, stage 1) to 1 × 10(-26) (late AMD).
CONCLUSIONS:
A strong association was demonstrated between all stages of AMD and genetic variation in ARMS2, and a significant gene-environment interaction with cigarette smoking was confirmed.

Phylogeographical analyses of shellfish viruses: inferring a geographical origin for ostreid herpesviruses OsHV-1 (Malacoherpesviridae)

Mortality episodes have regularly been affecting the shellfish industry throughout its history. Some of these mortalities, especially in the oyster industry, have been attributed to herpesviruses. Purification of viral particles and molecular characterization have led to the development of routine monitoring, as well as improved taxonomic classification. Ostreid herpesviruses (Malacoherpesviridae), mostly affecting Pacific oysters Crassostrea gigas, have been sporadically recorded in the French oyster industry since the early 1990s (OsHV-1 'reference'). From 2008, a new variant of ostreid herpesvirus (OsHV-1 mu Var) has emerged and seriously impacted oyster production in France and other European countries. Consequently, the presence of ostreid herpesviruses has been monitored in different oyster producing areas around the world. The present study compiles molecular data that are available from survey efforts and takes a biogeographical approach, in order to infer an origin for ostreid herpesviruses. The highest genotype diversity was found in East Asia, despite a lower survey effort in that area than in Europe. Genotype network analyses show that both populations of ostreid herpesviruses present in Europe (OsHV-1 'reference' and OsHV-1 mu Var) are closely related to genotypes recorded in Asia. Moreover, ostreid herpesviruses have been detected in wild and symptom-free populations of various Asian native Crassostrea species. In the rest of the world, ostreid herpesvirus genotypes were recorded from cultivated C. gigas, and mostly associated with mortality episodes. Results of this study are therefore highly suggestive of an Asian origin for these viruses, which can be pathogenic under farming conditions. It also highlights the risks of European stock improvements, by means of overseas shellfish imports.

T-box 2 represses NDRG1 through an EGR1-dependent mechanism to drive the proliferation of breast cancer cells

T-box 2 (TBX2) is a transcription factor involved in mammary development and is known to be overexpressed in a subset of aggressive breast cancers. TBX2 has previously been shown to repress growth control genes such as p14(ARF) and p21(WAF1/cip1). In this study we show that TBX2 drives proliferation in breast cancer cells and this is abrogated after TBX2 small interfering RNA (siRNA) knockdown or after the expression of a dominant-negative TBX2 protein. Using microarray analysis we identified a large cohort of novel TBX2-repressed target genes including the breast tumour suppressor NDRG1 (N-myc downregulated gene 1). We show that TBX2 targets NDRG1 through a previously undescribed mechanism involving the recruitment of early growth response 1 (EGR1). We show EGR1 is required for the ability of TBX2 to repress NDRG1 and drive cell proliferation. We show that TBX2 interacts with EGR1 and that TBX2 requires EGR1 to target the NDRG1 proximal promoter. Abrogation of either TBX2 or EGR1 expression is accompanied by the upregulation of cell senescence and apoptotic markers. NDRG1 can recapitulate these effects when transfected into TBX2-expressing cells. Together, these data identify a novel mechanism for TBX2-driven oncogenesis and highlight the importance of NDRG1 as a growth control gene in breast tissue. Oncogene (2010) 29, 3252-3262; doi: 10.1038/onc.2010.84; published online 29 March 2010

TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.

TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.