Characterization of BARD1 targeting and dynamics at the centrosome: the role of CRM1, BRCA1 and the Q564H mutation

BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time > 500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~ 20 s). The ~ 25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.     

AMP-activated protein kinase β-subunit requires internal motion for optimal carbohydrate binding

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.     

Modelling the potential impact of climate variability and change on species regeneration potential in the temperate forests of South‐Eastern Australia

The sensitivity of early plant regeneration to environmental change makes regeneration a critical stage for understanding species response to climate change. We investigated the spatial and temporal response of eucalypt trees in the Central Highland region of south eastern Australia to high and low climate change scenarios. We developed a novel mechanistic model incorporating germination processes, TACA‐GEM, to evaluate establishment probabilities of five key eucalypt species, Eucalyptus pauciflora, Eucalyptus delegatensis, Eucalyptus regnans, Eucalyptus nitens and Eucalyptus obliqua. Changes to regeneration potential at landscape and site levels were calculated to determine climate thresholds. Model results demonstrated that climate change is likely to impact plant regeneration. We observed increases and decreases in regeneration potential depending on the ecosystem, indicating that some species will increase in abundance in some forest types, whilst other forest types will become inhabitable. In general, the dry forest ecosystems were most impacted, whilst the wet forests were least impacted. We also observed that species with seed dormancy mechanisms, like E. pauciflora and E. delegatensis, are likely to be at higher risk than those without. Landscape‐ and site‐level analysis revealed heterogeneity in species response at different scales. On a landscape scale, a 4.3 °C mean temperature increase and 22% decline in precipitation (predicted for 2080) is predicted to be a threshold for large spatial shifts in species regeneration niches across the study region, while a 2.6 °C increase and 15% decline in precipitation (predicted for 2050) will likely result in local site‐level shifts. Site‐level analysis showed that considerable declines in regeneration potential for E. delegatensis, E. pauciflora and E. nitens were modelled to occur in some ecosystems by 2050. While overall model performance and accuracy was good, better understanding of effects from extreme events and other underlying processes on regeneration will improve modelling and development of species conservation strategies.     

Hwanggeumchal sorghum induces cell cycle arrest, and suppresses tumor growth and metastasis through Jak2/STAT pathways in breast cancer xenografts

Background

Cancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer.

Methodology/Principal Findings

Preliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both in vivo and in vitro to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model.

Conclusions/Significance

Usage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer.     

The naphthylamine palladium(II) template induced E-Z isomerism of exocyclic vinyl group in five-membered P-N chelates: detailed spectroscopic studies

The exocyclic CC bond E-Z isomerism of chelating Ph₂PC(CHPh)-CHNAr in organopalladium complexes containing orthometallated [(S)-1-(dimethylamino)ethyl]naphthalene is reported. In dilute solutions of non-coordinating CH₂Cl₂ or CHCl₃, all the original E-isomers, in which the CHPh phenyl rings are located trans to PPh₂ moieties were partly converted to their Z-isomers. The isomerism was found to be dependent on temperature, concentration and solvent. At higher temperature, the Z-isomers were transformed completely back to their original E-isomers. Removal of the chiral auxiliaries of the E-Z mixtures by concentrated HCl, gave only the dichloro complexes of the E-isomers. The E-Z isomerization processes were well established by detailed spectroscopic studies, including ³¹P NMR, ¹H NMR and 2D ¹H-¹H ROESY NMR studies. It is noteworthy that the dichloro complexes and free P-N ligands did not show such isomerization processes, indicating that the isomerization processes were triggered by the orthopalladated naphthylamine moiety.