Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.     

Designing Molecular Dynamics Simulations to Shift Populations of the Conformational States of Calmodulin

We elucidate the mechanisms that lead to population shifts in the conformational states of calcium-loaded calmodulin (Ca²⁺-CaM). We design extensive molecular dynamics simulations to classify the effects that are responsible for adopting occupied conformations available in the ensemble of NMR structures. Electrostatic interactions amongst the different regions of the protein and with its vicinal water are herein mediated by lowering the ionic strength or the pH. Amino acid E31, which is one of the few charged residues whose ionization state is highly sensitive to pH differences in the physiological range, proves to be distinctive in its control of population shifts. E31A mutation at low ionic strength results in a distinct change from an extended to a compact Ca²⁺-CaM conformation within tens of nanoseconds, that otherwise occur on the time scales of microseconds. The kinked linker found in this particular compact form is observed in many of the target-bound forms of Ca²⁺-CaM, increasing the binding affinity. This mutation is unique in controlling C-lobe dynamics by affecting the fluctuations between the EF-hand motif helices. We also monitor the effect of the ionic strength on the conformational multiplicity of Ca²⁺-CaM. By lowering the ionic strength, the tendency of nonspecific anions in water to accumulate near the protein surface increases, especially in the vicinity of the linker. The change in the distribution of ions in the vicinal layer of water allows N- and C- lobes to span a wide variety of relative orientations that are otherwise not observed at physiological ionic strength. E31 protonation restores the conformations associated with physiological environmental conditions even at low ionic strength.     

O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1

Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C(1)XSXPC(2). We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C(1)XSX(A/P)C(2). We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.     

Major genes for Na+ exclusion, Nax1 and Nax2 (wheat HKT1;4 and HKT1;5), decrease Na+ accumulation in bread wheat leaves under saline and waterlogged conditions

Two major genes for Na(+) exclusion in durum wheat, Nax1 and Nax2, that were previously identified as the Na(+) transporters TmHKT1;4-A2 and TmHKT1;5-A, were transferred into bread wheat in order to increase its capacity to restrict the accumulation of Na(+) in leaves. The genes were crossed from tetraploid durum wheat (Triticum turgidum ssp. durum) into hexaploid bread wheat (Triticum aestivum) by interspecific crossing and marker-assisted selection for hexaploid plants containing one or both genes. Nax1 decreased the leaf blade Na(+) concentration by 50%, Nax2 decreased it by 30%, and both genes together decreased it by 60%. The signature phenotype of Nax1, the retention of Na(+) in leaf sheaths resulting in a high Na(+) sheath:blade ratio, was found in the Nax1 lines. This conferred an extra advantage under a combination of waterlogged and saline conditions. The effect of Nax2 on lowering the Na(+) concentration in bread wheat was surprising as this gene is very similar to the TaHKT1;5-D Na(+) transporter already present in bread wheat, putatively at the Kna1 locus. The results indicate that both Nax genes have the potential to improve the salt tolerance of bread wheat.     

Movement mechanics as a determinate of muscle structure, recruitment and coordination

During muscle contractions, the muscle fascicles may shorten at a rate different from the muscle-tendon unit, and the ratio of these velocities is its gearing. Appropriate gearing allows fascicles to reduce their shortening velocities and allows them to operate at effective shortening velocities across a range of movements. Gearing of the muscle fascicles within the muscle belly is the result of rotations of the fascicles and bulging of the belly. Variable gearing can also occur as a result of tendon length changes that can be caused by changes in the relative timing of muscle activity for different mechanical tasks. Recruitment patterns of slow and fast fibres are crucial for achieving optimal muscle performance, and coordination between muscles is related to whole limb performance. Poor coordination leads to inefficiencies and loss of power, and optimal coordination is required for high power outputs and high mechanical efficiencies from the limb. This paper summarizes key studies in these areas of neuromuscular mechanics and results from studies where we have tested these phenomena on a cycle ergometer are presented to highlight novel insights. The studies show how muscle structure and neural activation interact to generate smooth and effective motion of the body.     

Mutations in TBC1D24, a Gene Associated With Epilepsy,Also Cause Nonsyndromic Deafness DFNB86

Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness.