Sorting of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase): identification of a necessary and sufficient Golgi/endosomal retention signal in Stv1p

Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p). To identify residues responsible for sorting of the Golgi isoform of the V-ATPase, a random mutagenesis was performed on the N terminus of Stv1p. Subsequent characterization of mutant alleles led to the identification of a short peptide sequence, W(83)KY, that is necessary for proper Stv1p localization. Based on three-dimensional homology modeling to the Meiothermus ruber subunit I, we propose a structural model of the intact Stv1p-containing V-ATPase demonstrating the accessibility of the W(83)KY sequence to retrograde sorting machinery. Finally, we characterized the sorting signal within the context of a reconstructed Stv1p ancestor (Anc.Stv1). This evolutionary intermediate includes an endogenous W(83)KY sorting motif and is sufficient to compete with sorting of the native yeast Stv1p V-ATPase isoform. These data define a novel sorting signal that is both necessary and sufficient for trafficking of the V-ATPase within the Golgi/endosomal network.     

NMR solution structure of subunit E (fragment E1–69) of the Saccharomyces cerevisiae V1VO ATPase

The N-terminus of V-ATPase subunit E has been shown to associate with the subunits C, G and H, respectively. To understand the assembly of E with its neighboring subunits as well as its N-terminal structure, the N-terminal region, E(1-69), of the Saccharomyces cerevisiae V-ATPase subunit E was expressed and purified. The solution structure of E(1-69) was determined by NMR spectroscopy. The protein is 90.3?? in length and forms an á-helix between the residues 12-68. The molecule is amphipathic with hydrophobic residues at the N-terminus, predicted to interact with subunit C. The polar epitopes of E(1-69) are discussed as areas interacting with subunits G and H.     

Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice

The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.     

Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.     

Tensor GSVD of Patient- and Platform-Matched Tumor and Normal DNA Copy-Number Profiles Uncovers Chromosome Arm-Wide Patterns of Tumor-Exclusive Platform-Consistent Alterations Encoding for Cell Transformation and Predicting Ovarian Cancer Survival

The number of large-scale high-dimensional datasets recording different aspects of a single disease is growing, accompanied by a need for frameworks that can create one coherent model from multiple tensors of matched columns, e.g., patients and platforms, but independent rows, e.g., probes. We define and prove the mathematical properties of a novel tensor generalized singular value decomposition (GSVD), which can simultaneously find the similarities and dissimilarities, i.e., patterns of varying relative significance, between any two such tensors. We demonstrate the tensor GSVD in comparative modeling of patient- and platform-matched but probe-independent ovarian serous cystadenocarcinoma (OV) tumor, mostly high-grade, and normal DNA copy-number profiles, across each chromosome arm, and combination of two arms, separately. The modeling uncovers previously unrecognized patterns of tumor-exclusive platform-consistent co-occurring copy-number alterations (CNAs). We find, first, and validate that each of the patterns across only 7p and Xq, and the combination of 6p+12p, is correlated with a patient’s prognosis, is independent of the tumor’s stage, the best predictor of OV survival to date, and together with stage makes a better predictor than stage alone. Second, these patterns include most known OV-associated CNAs that map to these chromosome arms, as well as several previously unreported, yet frequent focal CNAs. Third, differential mRNA, microRNA, and protein expression consistently map to the DNA CNAs. A coherent picture emerges for each pattern, suggesting roles for the CNAs in OV pathogenesis and personalized therapy. In 6p+12p, deletion of the p21-encoding CDKN1A and p38-encoding MAPK14 and amplification of RAD51AP1 and KRAS encode for human cell transformation, and are correlated with a cell’s immortality, and a patient’s shorter survival time. In 7p, RPA3 deletion and POLD2 amplification are correlated with DNA stability, and a longer survival. In Xq, PABPC5 deletion and BCAP31 amplification are correlated with a cellular immune response, and a longer survival.     

Computational model of blood flow in the aorto-coronary bypass graft

Background

Coronary artery bypass grafting surgery is an effective treatment modality for patients with severe coronary artery disease. The conduits used during the surgery include both the arterial and venous conduits. Long- term graft patency rate for the internal mammary arterial graft is superior, but the same is not true for the saphenous vein grafts. At 10 years, more than 50% of the vein grafts would have occluded and many of them are diseased. Why do the saphenous vein grafts fail the test of time? Many causes have been proposed for saphenous graft failure. Some are non-modifiable and the rest are modifiable. Non-modifiable causes include different histological structure of the vein compared to artery, size disparity between coronary artery and saphenous vein. However, researches are more interested in the modifiable causes, such as graft flow dynamics and wall shear stress distribution at the anastomotic sites. Formation of intimal hyperplasia at the anastomotic junction has been implicated as the root cause of long- term graft failure.Many researchers have analyzed the complex flow patterns in the distal sapheno-coronary anastomotic region, using various simulated model in an attempt to explain the site of preferential intimal hyperplasia based on the flow disturbances and differential wall stress distribution. In this paper, the geometrical bypass models (aorto-left coronary bypass graft model and aorto-right coronary bypass graft model) are based on real-life situations. In our models, the dimensions of the aorta, saphenous vein and the coronary artery simulate the actual dimensions at surgery. Both the proximal and distal anastomoses are considered at the same time, and we also take into the consideration the cross-sectional shape change of the venous conduit from circular to elliptical. Contrary to previous works, we have carried out computational fluid dynamics (CFD) study in the entire aorta-graft-perfused artery domain. The results reported here focus on (i) the complex flow patterns both at the proximal and distal anastomotic sites, and (ii) the wall shear stress distribution, which is an important factor that contributes to graft patency.

Methods

The three-dimensional coronary bypass models of the aorto-right coronary bypass and the aorto-left coronary bypass systems are constructed using computational fluid-dynamics software (Fluent 6.0.1). To have a better understanding of the flow dynamics at specific time instants of the cardiac cycle, quasi-steady flow simulations are performed, using a finite-volume approach. The data input to the models are the physiological measurements of flow-rates at (i) the aortic entrance, (ii) the ascending aorta, (iii) the left coronary artery, and (iv) the right coronary artery.

Results

The flow field and the wall shear stress are calculated throughout the cycle, but reported in this paper at two different instants of the cardiac cycle, one at the onset of ejection and the other during mid-diastole for both the right and left aorto-coronary bypass graft models. Plots of velocity-vector and the wall shear stress distributions are displayed in the aorto-graft-coronary arterial flow-field domain. We have shown (i) how the blocked coronary artery is being perfused in systole and diastole, (ii) the flow patterns at the two anastomotic junctions, proximal and distal anastomotic sites, and (iii) the shear stress distributions and their associations with arterial disease.

Conclusion

The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear-stress variation is observed around the distal anastomotic region. These results can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease.

     

Studies on mutagen-sensitive strains of Drosophila melanogaster I. The enhanced X-ray sensitivity of a mutant strain (ebony) which is UV-sensitive and also deficient in photorepair

Yegorova and colleagues (1978) showed that a mutant strain of Drosophila melanogaster (ebony) was more sensitive to UV-induced killing of embryos and also less proficient in photoreactivating (PR) ability than a wild-type (Canton-S) strain and that the genes governing UV sensitivity and PR ability were different and presumably located on the autosomes. The experiments reported in the present paper were designed to compare the patterns of sensitivity of these 2 strains and their hybrids to X-irradiation. The sensitivity of the larvae to the killing effects of X-irradiation, and of male and female germ-cell stages to the X-ray induction of genetic damage was studied.It was found that the larvae of the ebony strain are more sensitive to X-ray-induced killing than those of the Canton-S strain. The frequencies of radiation-induced dominant lethals and sex-linked recessive lethals are higher in spermatozoa sampled from ebony males than in those of Canton-S males. In spermatozoa sampled from hybrid males, the yields of dominant lethals are no higher than in those sampled from Canton-S males and do not seem to depend on the origin of the X-chromosome. There are no statistically significant differences between the ebony and Canton-S strains in the sensitivity of their spermatozoa to the induction of autosomal translocations.Stage-7 oocytes sampled from ebony females are more sensitive to the X-ray induction of dominant lethality than are those from Canton-S females; oocytes sampled from hybrid females manifest a level of sensitivity that is significantly lower than that in either parental strain. The frequencies of X-chromosome losses induced in in this germ-cell stage are significantly lower in ebony than in Canton-S females at least at the exposure level of 3000 R at which 3 experiments were carried out. There are no measurable differences in the amount of dominant lethality induced in stage-14 oocytes of ebony, Canton-S and hybrid females.When X-irradiated Berlin-K males are mated to ebony or Canton-S females, the yields of dominant lethals are higher when ebony females are used, showing that there is a “maternal effect” for this kind of damage. Such a maternal effect is also found for sex-linked recessive lethals (irradiated Muller-5 males mated to ebony or Canton-S females). However, when irradiated ring-X-chromosome-carrying males are mated to ebony or Canton-S females, the frequencies of paternal sex-chromosome losses (scored as XO males) are lower when ebony females are used.These results have been interpreted on the assumption that the ebony strain is homozygous for recessive, autosomal genes that confer increased radiosensitivity and that the Canton-S strain carries the normal, wild-type alleles for these genes. The higher yields of dominant and recessive lethals in mature spermatozoa and of dominant lethals in stage-7 oocytes are a consequence of an enhanced sensitivity to the mutagenic (in particular, to the chromosome-breaking) effects of X-irradiation and/or of defective repair of radiation-induced genetic damage. The lower yield of XO males from irradiated stage-7 oocytes of ebony females is probably a consequence of a defect in the repair of chromosome-breakage effects, resulting in the conversion of potential X losses in females into dominant lethals. The “maternal effects” for dominant lethals, sex-linked recessive lethals and for the loss of ring-X chromosomes are assumed to have a common causal basis, namely, a defective repair of chromosome-breakage events in the females of the ebony strain.     

Interaction of calmodulin with L-selectin at the membrane interface: implication on the regulation of L-selectin shedding

The calmodulin (CaM) hypothesis of ectodomain shedding stipulates that CaM, an intracellular Ca²⁺-dependent regulatory protein, associates with the cytoplasmic domain of l-selectin to regulate ectodomain shedding of l-selectin on the other side of the plasma membrane. To understand the underlying molecular mechanism, we have characterized the interactions of CaM with two peptides derived from human l-selectin. The peptide ARR18 corresponds to the entire cytoplasmic domain of l-selectin (residues Ala317-Tyr334 in the mature protein), and CLS corresponds to residues Lys280-Tyr334, which contains the entire transmembrane and cytoplasmic domains of l-selectin. Monitoring the interaction by fluorescence spectroscopy and other biophysical techniques, we found that CaM can bind to ARR18 in aqueous solutions or the l-selectin cytoplasmic domain of CLS reconstituted in the phosphatidylcholine bilayer, both with an affinity of approximately 2 μM. The association is calcium independent and dynamic and involves both lobes of CaM. In a phospholipid bilayer, the positively charged l-selectin cytoplasmic domain of CLS is associated with anionic phosphatidylserine (PS) lipids at the membrane interface through electrostatic interactions. Under conditions where the PS content mimics that in the inner leaflet of the cell plasma membrane, the interaction between CaM and CLS becomes undetectable. These results suggest that the association of CaM with l-selectin in the cell can be influenced by the membrane bilayer and that anionic lipids may modulate ectodomain shedding of transmembrane receptors.