Biophysics and bioinformatics reveal structural differences of the two peripheral stalk subunits in chloroplast ATP synthase

ATP synthases convert an electrochemical proton gradient into rotational movement to produce the ubiquitous energy currency adenosine triphosphate. Tension generated by the rotational torque is compensated by the stator. For this task, a peripheral stalk flexibly fixes the hydrophilic catalytic part F1 to the membrane integral proton conducting part F(O) of the ATP synthase. While in eubacteria a homodimer of b subunits forms the peripheral stalk, plant chloroplasts and cyanobacteria possess a heterodimer of subunits I and II. To better understand the functional and structural consequences of this unique feature of photosynthetic ATP synthases, a procedure was developed to purify subunit I from spinach chloroplasts. The secondary structure of subunit I, which is not homologous to bacterial b subunits, was compared to heterologously expressed subunit II using CD and FTIR spectroscopy. The content of alpha-helix was determined by CD spectroscopy to 67% for subunit I and 41% for subunit II. In addition, bioinformatics was applied to predict the secondary structure of the two subunits and the location of the putative coiled-coil dimerization regions. Three helical domains were predicted for subunit I and only two uninterrupted domains for the shorter subunit II. The predicted length of coiled-coil regions varied between different species and between subunits I and II.     

Identification of liver‐derived bone morphogenetic protein (BMP)‐9 as a potential new candidate for treatment of colorectal cancer

Colorectal cancer (CRC) is a high‐incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)‐9 and to find out whether the application of BMP‐9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP‐target gene ID1, especially in combination with low expression of the BMP‐inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T‐Orgs) and corresponding non‐malignant areas (N‐Orgs) of three patients. The N‐Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP‐9 stimulation of organoids promoted an enrichment of tumour‐suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP‐9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1.In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP‐9 enhances this ratio, even in the presence of noggin. Thus, BMP‐9 is identified as a novel target for the development of improved anti‐cancer therapies of patients with CRC.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Murine DNA cytosine C(5)-methyltransferase: in vitro studies of de novo methylation spreading

The preference of murine DNA (cytosine-5)-methyltransferase (Dnmt1) for single stranded DNA substrates is increased up to 50-fold by the presence of a proximal 5-methyl cytosine (5(me)C). This modulation is distance-dependent and is due to an enhanced binding affinity and minor changes in catalytic efficiency. No modulation was observed with double stranded DNA. Modulation requires that the 5(me)C moiety be attached to the DNA strand containing the CpG methylation target. Our results support a model in which 5(me)C binding by the enzyme occurs to at least one site outside the region involved in CpG recognition. No modulation in response to 5(me)C is observed with the bacterial enzyme M.SssI, which lacks the large N-terminal regulatory domain found in Dnmt1. We suggest that this allosteric modulation involves the N-terminal domain of Dnmt1.     

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1^C40A, Panx1^C346A, and Panx1^C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1^C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.