A cooperative action of the ATP-dependent import motor complex and the inner membrane potential drives mitochondrial preprotein import

The import of mitochondrial preproteins requires an electric potential across the inner membrane and the hydrolysis of ATP in the matrix. We assessed the contributions of the two energy sources to the translocation driving force responsible for movement of the polypeptide chain through the translocation channel and the unfolding of preprotein domains. The import-driving activity was directly analyzed by the determination of the protease resistances of saturating amounts of membrane-spanning translocation intermediates. The ability to generate a strong translocation-driving force was solely dependent on the activity of the ATP-dependent import motor complex in the matrix. For a sustained import-driving activity on the preprotein in transit, an unstructured N-terminal segment of more than 70 to 80 amino acid residues was required. The electric potential of the inner membrane was required to maintain the import-driving activity at a high level. The electrophoretic force of the potential exhibited only a limited capacity to unfold preprotein domains. We conclude that the membrane potential increases the probability of a dynamic interaction of the preprotein with the import motor. Polypeptide translocation and unfolding are mainly driven by the inward-directed translocation activity based on the functional cooperation of the import motor components.     

Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.     

Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta

Irradiation of the heart and vasculature can cause a spectrum of cardiovascular complications, including increased risk of myocardial infarction or coronary heart disease. Although irradiation is implicated in oxidant stress and chronic inflammation, the underlying molecular mechanisms have not been elucidated. We tested the hypothesis that irradiation-initiated upregulation of xanthine oxidase (XO), a primary source of cardiovascular reactive oxygen species, contributes to endothelial dysfunction and increased vascular stiffness. Twenty-two, 3-month-old Sprague–Dawley male rats were gamma-irradiated at the following doses: 0, 50, 160, and 500 cGy. Rats exposed to 500 cGy showed a significant increase in endothelial XO expression and a twofold increase in XO activity, compared to the 0 cGy controls. Endothelial function was investigated ex vivo through vascular tension dose–responses to the endothelial dependent vasodilator, acetylcholine. Endothelial-dependent relaxation in aorta of the 500 cGy exposed rats was significantly attenuated from the control group. Remarkably, specific inhibition of XO with oxypurinol restored the relaxation response to that of the control. Furthermore, these ex vivo results are reflected in vivo through alterations in vascular stiffness, as measured by pulse wave velocity (PWV). As early as 1-day post-exposure, rats exhibited a significant increase in PWV from pre-exposure. The PWV of irradiated rats (50, 160, and 500 cGy) were greater than those of 0 cGy control rats at 1 day, 1 and 2 weeks. The sham and irradiated rats possessed equivalent pre-exposure PWV, with sham showing no change over 2 weeks. Thus, these findings suggest that early upregulation of XO contributes to oxidative stress and endothelial nitro-redox imbalance with resultant endothelial dysfunction and altered vascular mechanics. Furthermore, these data identify XO as a potential molecular target for attenuating irradiation-induced cardiovascular injury.     

Transforming activity of purinergic receptor P2Y, G-protein coupled, 2 revealed by retroviral expression screening

Colorectal cancer (CRC) is one of the leading causes of cancer death in humans. In order to identify novel cancer-promoting genes in CRC, we here constructed a retroviral cDNA expression library from a CRC cell line RKO, and used it for a focus formation assay with mouse 3T3 fibroblasts, leading to the identification of 42 independent cDNAs. One of such cDNAs turned out to encode purinergic receptor P2Y, G-protein coupled, 2 (P2RY2). The oncogenic potential of P2RY2 was confirmed in vitro with the focus formation assay as well as soft agar-growth assay, and also in vivo with a tumorigenicity assay in nude mice. While our P2RY2 cDNA encodes a protein with two amino-acid substitutions compared to the reported one, we have confirmed that the wild-type P2RY2 has a strong transforming potential as well. These results indicate an unexpected role of P2RY2 in the carcinogenesis of human cancers.     

Understanding the changes in the circular dichroism of light harvesting complex II upon varying its pigment composition and organization

In this work we modeled the circular dichroism (CD) spectrum of LHCII, the main light harvesting antenna of photosystem II of higher plants. Excitonic calculations are performed for a monomeric subunit, taken from the crystal structure of trimeric LHCII from spinach [Liu, Z. F., Yan, H. C., Wang, K. B., Kuang, T. Y., Zhang, J. P., Gui, L. L., An, X. M., and Chang, W. R. (2004) Nature 428, 287-292]. All of the major features of the CD spectrum above 450 nm are satisfactorily reproduced, and possible orientations of the Chl and carotenoid transition dipole moments are identified. The obtained modeling parameters are used to simulate the CD spectra of two complexes with altered pigment composition: a mutant lacking Chls a 611-612 and a complex lacking the carotenoid neoxanthin. By removing the relevant pigment(s) from the structure, we are able to reproduce their spectra, which implies that the alteration does not disturb the overall structure. The CD spectrum of trimeric LHCII shows a reversed relative intensity of the two negative bands around 470 and 490 nm as compared to monomeric LHCII. The simulations reproduce this reversal, indicating that it is mainly due to interactions between chromophores in different monomeric subunits, and the trimerization does not induce observable changes in the monomeric structure. Our simulated spectrum resembles one of two different trimeric CD spectra reported in literature. We argue that the differences in the experimental trimeric CD spectra are caused by changes in the strength of the monomer-monomer interactions due to the differences in detergents used for the purification of the complexes.     

Effects of PKA phosphorylation of cardiac troponin I and strong crossbridge on conformational transitions of the N-domain of cardiac troponin C in regulated thin filaments

Regulation of cardiac muscle function is initiated by binding of Ca2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine-tuned by phosphorylation of cTnI. The objective of the present study is to use a new F?rster resonance energy transfer-based structural marker to distinguish structural and kinetic effects of Ca2+ binding, crossbridge interaction, and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin, and actin. Changes in the distance between Cys13 and Cys51 induced by Ca2+ binding/dissociation were determined by FRET-sensed Ca2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes.     

Misexpression screen in Drosophila melanogaster aiming to reveal novel factors involved in formation of body parts

To identify novel factors that lead a fly imaginal disc to adopt its developmental fate, we carried out a modular dominant misexpression screen in imaginal discs. We have identified two factors that appear to change the fate of the respective body structure and appear to lead to the transformation of a body part. In one mutant line, notum tissue, normally derived from wing imaginal tissue, formed close to the site of the sternopleural bristles, which are leg disc derivatives. In the other line, the arista is transformed into a tubular structure, resembling an abnormal leg. We found that ectopic expression of abrupt was responsible for this potential transformation of the arista.     

Experimental manipulation of timing of breeding suggests laying order instead of breeding synchrony affects extra-pair paternity in house sparrows

The timing of breeding may not only affect breeding patterns such as the overlap of chick rearing period with the peak in food availability but also the opportunity for extra-pair mating. A negative relationship has been predicted between extra-pair paternity and breeding synchrony, assuming that male extra-pair activity is traded against mate guarding and parenting duties. In contrast, if female ability to assess male quality is temporally constrained, sperm competition might be a positive function of breeding synchrony. Here we manipulated the progress of nesting by nest material exchange within nesting aggregations to see whether the timing of breeding affects extra-pair paternity in house sparrows. We found that late broods within nesting clusters contained extra-pair young more often than early broods, but breeding synchrony did not turn out to be a significant predictor of extra-pair paternity. Our study indicates that temporal constraints of male extra-pair activity may account for extra-pair paternity levels, but it is also possible that late-breeding females may accept extra-pair copulations to ensure egg fertilization.