Adenosine nucleotide binding sites on Complex V from diverse organisms

Using in silico docking approaches, we scan the various subunits of Complex V (FoF1ATPase) for putative adenosine nucleotide binding sites. We find that multiple generic ADP/ATP binding sites are present on the alpha-beta binding sites and a conserved ATP binding site is present on the epsilon subunit. These findings support the murburn model of Complex V.

COPE is differentially expressed in the brain metastases of patients with metastatic breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes to discover genes associated with brain metastasis in patients with metastatic breast cancer. We found that the epsilon subunit of the coatomer protein complex, COPE, was among the genes whose expression was most different in the brain metastases of patients with metastatic breast cancer as compared to normal breast tissues. COPE mRNA was present at increased quantities in brain metastatic tissues as compared to primary tumors of the breast. Up-regulation of COPE expression may contribute to metastasis of tumor cells from the breast to the brain in humans with metastatic breast cancer.

Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

DNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.

A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP

Adenosine 5’ triphosphate (ATP) is a universal intracellular energy source1 and an evolutionarily ancient2 extracellular signal3–5. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responded to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. iATPSnFRs represent promising new reagents for imaging ATP dynamics.

THE ROLE OF GENETIC TESTING IN PATIENTS WITH CONGENITAL MYASTHENIA

The role of genetic testing in patients with congenital myasthenia Tsimakidi C. MD, Magrepli-Loli N. MD Neurology Department, Aghia Sophia Pediatric Hospital Background: Congenital myasthenic syndromes form a group of inherited disorders with dysfunction of neuromuscular transmission, characterized by fatigable weakness of skeletal muscle with onset shortly after birth or in early childhood. According to the defect of each subtype, they are classified as presynaptic, synaptic or postsynaptic. The diagnosis is based on a combination of the patient’s history, electrophysiological testing, response to acetylcholine-inhibitors, muscle biopsy and genetic testing. Methods/results: We present the cases of three Greek patients (one female, two males) of Gypsy ethnic origin, who have been diagnosed with congenital myasthenia at infancy and are being treated with pyridostimine ever since. All three patients presented with progressive ptosis, external ophthalmoparesis, swallowing and respiratory problems at infancy. They had positive response to the administration of edrophonium and started treatment with pyridostigmine. At present they are seven to twelve years of age and present mild symptoms, mostly frequent respiratory infections.We took blood samples from these patients and their parents and had it analyzed. The mutation ε1267delG was identified in an homozygous state in all of them. The parents were carriers of the mutation. Conclusions: The identified mutation is situated in exon 12 of the AChR epsilon subunit and leads to autosomal recessive congenital myasthenia with variable phenotype. The mutation is quite frequent among patients of Gypsy origin, which complies with findings of similar studies (3). The identification of the mutation confirms the diagnosis as it’s not unusual for those patients to being wrongly diagnosed and treated (e.g. for myasthenia Gravis). Apart from that, the type of the mutation and the identification of the carriers enables us to promote effective genetic counseling. Bibliography: 1. Hantai D., Richard H., Koenig J., Eymard B. Congenital myasthenic syndromes. Curr Opin Neurol 2004; 17:539-551 2. Engel AG, Shen XM, Selcen D, Sine SM. Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. Lancet Neurol. 2015 Apr;14(4):420-34. doi: 10.1016/S1474-4422(14)70201-7 3. Abicht A, Stucka R, Karcagi V, Herczegfalvi A, Horvath R, Mortier W, Schara U, Ramaekers V, Jost W, Brunner J, Janssen G, Seidel U, Schlotter B, Muller-Felber W, Pongratz D, Rudel R, Lochmuller H. A common mutation in congenital myasthenic patients of Gypsy ethnic origin. Neurology 1999 Oct 22; 53(7):1564-9