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The subunit composition of the mitochondrial ATP synthase from Saccharomyces cerevisiae was analyzed using blue native gel electrophoresis and high resolution SDS-polyacrylamide gel electrophoresis. We report here the identification of a novel subunit of molecular mass of 6,687 Da, termed subunit j (Su j). An open reading frame of 127 base pairs (ATP18), which encodes for Su j, was identified on chromosome XIII. Su j does not display sequence similarity to ATP synthase subunits from other organisms. Data base searches, however, identified a potential homolog from Schizosaccharomyces pombe with 51% identity to Su j of S. cerevisiae. Su j, a small protein of 59 amino acid residues, has the characteristics of an integral inner membrane protein with a single transmembrane segment. Deletion of the ATP18 gene encoding Su j led to a strain (Deltasu j) completely deficient in oligomycin-sensitive ATPase activity and unable to grow on nonfermentable carbon sources. The presence of Su j is required for the stable expression of subunits 6 and f of the F0 membrane sector. In the absence of Su j, spontaneously arising rho- cells were observed that lacked also ubiquinol-cytochrome c reductase and cytochrome c oxidase activities. We conclude that Su j is a novel and essential subunit of yeast ATP synthase.
SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1- and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a beta-strand that could bind in parallel or antiparallel orientation to the beta2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions.
The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.
Abdominal aortic aneurysm (AAA) is the most common type of aortic aneurysm, which is defined as a dilation of the abdominal aorta over 3.0 cm or more. Surgical repair is the golden standard for the treatment of AAA, in which open surgical repair (OSR) and endovascular aneurysm repair (EVAR) are the main approaches. Technically speaking, the lesion segment of aueurysm is completely replaced by a graft during OSR, while in EVAR, the lesion is insulated by a stentgraft. EVAR is a less invasive treatment than OSR and shows a lower early mortality rate, although the long-term advantages of EVAR over OSR remain inconclusive.
Endoleak, especially the type II endoleak (T2EL), is a common complication after EVAR. According to research, 16-28% of the patients develop a T2EL after EVAR, and it accounts for nearly three in four of all types of endoleaks. Around 30-50% of the T2EL resolved spontaneously during the follow-up, however, it still causes a secondary intervention in many patients. Therefore, it is critical to monitor endoleaks after repair.
Patent aortic branches in the stent-overlapped area and vasa vasorum have been identified as potential sources of blood flow in T2EL. However, the mechanisms of biological changes or remodeling of the aneurysm sac after the repair are still not clear, but they have been considered to play an important role in the development of endoleaks. Unfortunately, it is impossible to obtain a tissue sample of the aortic wall in patients who underwent EVAR.
MicroRNAs (miRNAs) are a class of small single-stranded non-coding RNAs that inhibit the expression of target message RNA (mRNA). miR-29b/29c, miR-155, and miR-15a are miRNAs associated with regulating extracellular matrix (ECM) components, inflammation, and proliferation, respectively. All four miRNAs have been identified as biomarkers of AAA, not only in aneurysm tissue but also extracellular as circulating miRNAs. However, it is still unknown whether they can reflect the biological changes after AAA repair. Thus, we conducted a prospective study to investigate the changes in expression of circulating miR-29b, miR-29c, miR-155, and miR-15a before (T0), 3 days (T1), and 3 months (T2) after AAA repair.
A total of 39 patients were recruited for this study, 17 of whom were repaired by OSR and 22 of whom were repaired by EVAR. Four patients failed the T2 follow-up due to the Covid-19 pandemic. No significant changes were found in the expression of miR-29b, miR-29c, miR-155, and miR-15a. There were also no obvious differences between OSR and EVAR. However, the T1 expression of miR-15a was significantly lower in patients without endoleak after EVAR than in those who developed endoleak after EVAR and those who were repaired by OSR. Unfortunately, these differences did not persist to the T2 follow-up, and no other differences were found among these patients.
In summary, miR-15a is a miRNA that significantly changes in AAA patients. This study demonstrates that the expression of circulating miR-15a is lower in patients without endoleak three days after EVAR, compared to those who had endoleak after EVAR and those who underwent OSR. The results suggest that miR-15a might be involved in the early aortic remodeling after EVAR as an indicator of endoleak.
Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.
The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of salt and water homeostasis. Here, we demonstrate the expression and functional role of cGMP-dependent protein kinases (PKGs) in rat adrenal cortex. Expression of PKG II is restricted to adrenal zona glomerulosa (ZG) cells, whereas PKG I is localized to the adrenal capsule and blood vessels. Activation of the aldosterone system by a low sodium diet up-regulated the expression of PKG II, however, it did not change PKG I expression in adrenal cortex. Both, activation of PKG II in isolated ZG cell and adenoviral gene transfer of wild type PKG II into ZG cells enhanced aldosterone production. In contrast, inhibition of PKG II as well as infection with a PKG II catalytically inactive mutant had an inhibitory effect on aldosterone production. Steroidogenic acute regulatory (StAR) protein that regulates the rate-limiting step in steroidogenesis is a new substrate for PKG II and can be phosphorylated by PKG II in vitro at serine 55/56 and serine 99. Stimulation of aldosterone production by PKG II in contrast to stimulation by PKA did not activate StAR gene expression in ZG cells. The results presented indicate that PKG II activity in ZG cells is important for maintaining basal aldosterone production.
Atrial natriuretic peptide (ANP) plays a key regulatory role in arterial blood pressure homeostasis. We recently generated mice with selective deletion of the ANP receptor, guanylyl cyclase-A (GC-A), in vascular smooth muscle (SMC GC-A knockout (KO) mice) and reported that resting arterial blood pressure was completely normal in spite of clear abolition of the direct vasodilating effects of ANP (Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R. E., Herz, J., and Kuhn M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7142–7147). The purpose of this study was to clarify mechanisms compensating for the missing vasodilator responses to ANP. In particular, we analyzed the effect of the endothelial, cGMP-mediated vasodilators C-type natriuretic peptide and nitric oxide (NO). In isolated arteries from SMC GC-A KO mice, the vasorelaxing sensitivity to sodium nitroprusside and the endothelium-dependent vasodilator, acetylcholine, was significantly greater than in control mice. There was no difference in responses to C-type natriuretic peptide or to the activator of cGMP-dependent protein kinase I, 8-para-chlorophenylthio-cGMP. The aortic expression of soluble GC (sGC), but not of endothelial NO synthase or cGMP-dependent protein kinase I, was significantly increased in SMC GC-A KO mice. Chronic oral treatment with the NO synthase inhibitor Nw-nitro-l-arginine methyl ester increased arterial blood pressure, the effect being significantly enhanced in SMC GC-A KO mice. We conclude that SMC GC-A KO mice exhibit a higher vasodilating sensitivity to NO. This can be attributed to an enhanced expression of sGC, whereas the expression and/or activity levels of downstream cGMP-effector pathways are not involved. Increased vasodilating responsiveness to endothelial NO contributes to compensate for the missing vasodilating effect of ANP in SMC GC-A KO mice.
Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.
Cyclic GMP-dependent protein kinases protein kinase G (PKG) Iα and PKGIβ are major mediators of cGMP signaling in the cardiovascular system. PKGIα is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGIα against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIα or catalytically inactive mutant hPKGIαK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of KATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 μm), HMR1098 (30 μm), or glibenclamide (50 μm), the respective blockers of mitochondrial, sarcolemmal, or both types of KATP channels prior to SI. The necrosis observed in 33.7 ± 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 ± 0.8% by PKGIα (mean ± S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 ± 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 ± 0.6% by PKGIα (mean ± S.E., n = 7, p < 0.001). In addition, PKGIα inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIαK390A mutant showed no protection. PKGIα enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2, inducible nitric-oxide synthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGIα-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGIα-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGIα.
The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1X2CX3X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus alpha-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of approximately 60 mV for the nearby cluster. (ii) Very negative potentials, below -600 mV, correlate with the presence of a bulky side chain in position X4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins.