PLANT U-BOX PROTEIN10 Regulates MYC2 Stability in Arabidopsis

MYC2 is an important regulator for jasmonic acid (JA) signaling, but little is known about its posttranslational regulation. Here, we show that the MYC2 C-terminal region interacted with the PLANT U-BOX PROTEIN10 (PUB10) armadillo repeats in vitro. MYC2 was efficiently polyubiquitinated by PUB10 with UBC8 as an E2 enzyme and the conserved C249 in PUB10 was required for activity. The inactive PUB10(C249A) mutant protein retained its ability to heterodimerize with PUB10, thus blocking PUB10 E3 activity as a dominant-negative mutant. Both MYC2 and PUB10 were nucleus localized and coimmunoprecipitation experiments confirmed their interaction in vivo. Although unstable in the wild type, MYC2 stability was enhanced in pub10, suggesting destabilization by PUB10. Moreover, MYC2 half-life was shortened or prolonged by induced expression of PUB10 or the dominant-negative PUB10(C249A) mutant, respectively. Root growth of pub10 seedlings phenocopied 35S:MYC2 seedlings and was hypersensitive to methyl jasmonate, whereas 35S:PUB10 and jin1-9 (myc2) seedlings were hyposensitive. In addition, the root phenotype conferred by MYC2 overexpression in double transgenic plants was reversed or enhanced by induced expression of PUB10 or PUB10(C249A), respectively. Similar results were obtained with three other JA-regulated genes, TAT, JR2, and PDF1.2. Collectively, our results show that MYC2 is targeted by PUB10 for degradation during JA responses.     

Decomposing PPI networks for complex discovery

Background

Protein complexes are important for understanding principles of cellular organization and functions. With the availability of large amounts of high-throughput protein-protein interactions (PPI), many algorithms have been proposed to discover protein complexes from PPI networks. However, existing algorithms generally do not take into consideration the fact that not all the interactions in a PPI network take place at the same time. As a result, predicted complexes often contain many spuriously included proteins, precluding them from matching true complexes.

Results

We propose two methods to tackle this problem: (1) The localization GO term decomposition method: We utilize cellular component Gene Ontology (GO) terms to decompose PPI networks into several smaller networks such that the proteins in each decomposed network are annotated with the same cellular component GO term. (2) The hub removal method: This method is based on the observation that hub proteins are more likely to fuse clusters that correspond to different complexes. To avoid this, we remove hub proteins from PPI networks, and then apply a complex discovery algorithm on the remaining PPI network. The removed hub proteins are added back to the generated clusters afterwards. We tested the two methods on the yeast PPI network downloaded from BioGRID. Our results show that these methods can improve the performance of several complex discovery algorithms significantly. Further improvement in performance is achieved when we apply them in tandem.

Conclusions

The performance of complex discovery algorithms is hindered by the fact that not all the interactions in a PPI network take place at the same time. We tackle this problem by using localization GO terms or hubs to decompose a PPI network before complex discovery, which achieves considerable improvement.

     

Identification of genes with allelic imbalance on 6p associated with nasopharyngeal carcinoma in southern Chinese

Nasopharyngeal carcinoma (NPC) is a malignancy of epithelial origin. The etiology of NPC is complex and includes multiple genetic and environmental factors. We employed case-control analysis to study the association of chromosome 6p regions with NPC. In total, 360 subjects and 360 healthy controls were included, and 233 single nucleotide polymorphisms (SNPs) on 6p were examined. Significant single-marker associations were found for SNPs rs2267633 (p = 4.49 × 10(-5)), rs2076483 (most significant, p = 3.36 × 10(-5)), and rs29230 (p=1.43 × 10(-4)). The highly associated genes were the gamma-amino butyric acid B receptor 1 (GABBR1), human leukocyte antigen (HLA-A), and HLA complex group 9 (HCG9). Haplotypic associations were found for haplotypes AAA (located within GABBR1, p-value = 6.46 × 10(-5)) and TT (located within HLA-A, p = 0.0014). Further investigation of the homozygous genotype frequencies between cases and controls suggested that micro-deletion regions occur in GABBR1 and neural precursor cell expressed developmentally down-regulated 9 (NEDD9). Quantitative real-time polymerase chain reaction (qPCR) using 11 pairs of NPC biopsy samples confirmed the significant decline in GABBR1 and NEDD9 mRNA expression in the cancer tissues compared to the adjacent non-tumor tissue (p<0.05). Our study demonstrates that multiple chromosome 6p susceptibility loci contribute to the risk of NPC, possibly though GABBR1 and NEDD9 loss of function.     

FTO and MC4R gene variants are associated with obesity in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women. It is also associated with metabolic disturbances that place women at increased risk for obesity and type 2 diabetes. There is strong evidence for familial clustering of PCOS and a genetic predisposition. However, the gene(s) responsible for the PCOS phenotypes have not been elucidated. This two-phase family-based and case-control genetic study was designed to address the question of whether SNPs identified as susceptibility loci for obesity in genome-wide association studies (GWAS) are also associated with PCOS and elevated BMI. Members of 439 families having at least one offspring with PCOS were genotyped for 15 SNPs previously shown to be associated with obesity. Linkage and association with PCOS was assessed using the transmission/disequilibrium test (TDT). These SNPs were also analyzed in an independent case-control study involving 395 women with PCOS and 176 healthy women with regular menstrual cycles. Only one of these 15 SNPs (rs2815752 in NEGR1) was found to have a nominally significant association with PCOS (χ² = 6.11, P = 0.013), but this association failed to replicate in the case-control study. While not associated with PCOS itself, five SNPs in FTO and two in MC4R were associated with BMI as assessed with a quantitative-TDT analysis, several of which replicated association with BMI in the case-control cohort. These findings demonstrate that certain SNPs associated with obesity contribute to elevated BMI in PCOS, but do not appear to play a major role in PCOS per se. These findings support the notion that PCOS phenotypes are a consequence of an oligogenic/polygenic mechanism.     

MRI study of minor physical anomaly in childhood autism implicates aberrant neurodevelopment in infancy

Background

MPAs (minor physical anomalies) frequently occur in neurodevelopmental disorders because both face and brain are derived from neuroectoderm in the first trimester. Conventionally, MPAs are measured by evaluation of external appearance. Using MRI can help overcome inherent observer bias, facilitate multi-centre data acquisition, and explore how MPAs relate to brain dysmorphology in the same individual. Optical MPAs exhibit a tightly synchronized trajectory through fetal, postnatal and adult life. As head size enlarges with age, inter-orbital distance increases, and is mostly completed before age 3 years. We hypothesized that optical MPAs might afford a retrospective ‘window’ to early neurodevelopment; specifically, inter-orbital distance increase may represent a biomarker for early brain dysmaturation in autism.

Methods

We recruited 91 children aged 7–16; 36 with an autism spectrum disorder and 55 age- and gender-matched typically developing controls. All children had normal IQ. Inter-orbital distance was measured on T1-weighted MRI scans. This value was entered into a voxel-by-voxel linear regression analysis with grey matter segmented from a bimodal MRI data-set. Age and total brain tissue volume were entered as covariates.

Results

Intra-class coefficient for measurement of the inter-orbital distance was 0.95. Inter-orbital distance was significantly increased in the autism group (p = 0.03, 2-tailed). The autism group showed a significant relationship between inter-orbital distance grey matter volume of bilateral amygdalae extending to the unci and inferior temporal poles.

Conclusions

Greater inter-orbital distance in the autism group compared with healthy controls is consistent with infant head size expansion in autism. Inter-orbital distance positively correlated with volume of medial temporal lobe structures, suggesting a link to “social brain” dysmorphology in the autism group. We suggest these data support the role of optical MPAs as a “fossil record” of early aberrant neurodevelopment, and potential biomarker for brain dysmaturation in autism.     

Regulation of cullin RING E3 ubiquitin ligases by CAND1 in vivo

Cullin RING ligases are multi-subunit complexes consisting of a cullin protein which forms a scaffold onto which the RING protein Rbx1/2 and substrate receptor subunits assemble. CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo. Two models have been proposed to explain this requirement: (i) CAND1 sequesters cullin proteins and thus prevents autoubiquitination of substrate receptors, and (ii) CAND1 is required to promote the exchange of bound substrate receptors. Using mammalian cells, we show that CAND1 is predominantly cytoplasmically localized and that cullins are the major CAND1 interacting proteins. However, only small amounts of CAND1 bind to Cul1 in cells, despite low basal levels of Cul1 neddylation and approximately equal cytoplasmic endogenous protein concentrations of CAND1 and Cul1. Compared to F-box protein substrate receptors, binding of CAND1 to Cul1 in vivo is weak. Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding. In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.     

Comparative profiling of primary colorectal carcinomas and liver metastases identifies LEF1 as a prognostic biomarker

Purpose

We sought to identify genes of clinical significance to predict survival and the risk for colorectal liver metastasis (CLM), the most common site of metastasis from colorectal cancer (CRC).

Patients and Methods

We profiled gene expression in 31 specimens from primary CRC and 32 unmatched specimens of CLM, and performed Significance Analysis of Microarrays (SAM) to identify genes differentially expressed between these two groups. To characterize the clinical relevance of two highly-ranked differentially-expressed genes, we analyzed the expression of secreted phosphoprotein 1 (SPP1 or osteopontin) and lymphoid enhancer factor-1 (LEF1) by immunohistochemistry using a tissue microarray (TMA) representing an independent set of 154 patients with primary CRC.

Results

Supervised analysis using SAM identified 963 genes with significantly higher expression in CLM compared to primary CRC, with a false discovery rate of <0.5%. TMA analysis showed SPP1 and LEF1 protein overexpression in 60% and 44% of CRC cases, respectively. Subsequent occurrence of CLM was significantly correlated with the overexpression of LEF1 (chi-square p = 0.042), but not SPP1 (p = 0.14). Kaplan Meier analysis revealed significantly worse survival in patients with overexpression of LEF1 (p<0.01), but not SPP1 (p = 0.11). Both univariate and multivariate analyses identified stage (p<0.0001) and LEF1 overexpression (p<0.05) as important prognostic markers, but not tumor grade or SPP1.

Conclusion

Among genes differentially expressed between CLM and primary CRC, we demonstrate overexpression of LEF1 in primary CRC to be a prognostic factor for poor survival and increased risk for liver metastasis.     

Exposure of RNA templates and encapsidation of spliced viral RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173)

Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nuclease-resistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.