A novel ABCC8 (SUR1)-dependent mechanism of metabolism-excitation uncoupling

ATP/ADP-sensing (sulfonylurea receptor (SUR)/K(IR)6)(4) K(ATP) channels regulate the excitability of our insulin secreting and other vital cells via the differential MgATP/ADP-dependent stimulatory actions of their tissue-specific ATP-binding cassette regulatory subunits (sulfonylurea receptors), which counterbalance the nearly constant inhibitory action of ATP on the K(+) inwardly rectifying pore. Mutations in SUR1 that abolish its stimulation have been found in infants persistently releasing insulin. Activating mutations in SUR1 have been shown to cause neonatal diabetes. Here, analyses of K(IR)6.2-based channels with diabetogenic receptors reveal that MgATP-dependent hyper-stimulation of mutant SUR can compromise the ability of K(ATP) channels to function as metabolic sensors. I demonstrate that the channel hyperactivity rises exponentially with the number of hyperstimulating subunits, so small subpopulations of channels with more than two mutant SUR can dominate hyperpolarizing currents in heterozygous patients. I uncovered an attenuated tolbutamide inhibition of the hyperstimulated mutant, which is normally sensitive to the drug under non-stimulatory conditions. These findings show the key role of SUR in sensing the metabolic index in humans and urge others to (re)test mutant SUR/K(IR)6 channels from probands in physiologic MgATP.     

Genomic landscape of CpG rich elements in human

Background

The studies on CpG islands (CGI) and Alu elements functions, evolution, and distribution in the genome started since the discovery in nineteen eighties (1981, 1986, correspondingly). Their highly skewed genome wide distribution implies the non-random retrotransposition pattern. Besides CGIs in gene promoters, CGIs clusters were observed in the homeobox gene regions and in the macrosatellites, but the whole picture of their distribution specifics was not grasped. Attempts to identify any causative features upon their (genome wide) distribution, such as the DNA context mediated preferred insertion sites of Alu repeats, have been made to ascribe their clusters location.

Methods

Recent emergence of high resolution 3D map of human genome allowed segregating the genome into the large scale chromatin domains of naturally observable nuclear subcompartments, or Topologically Associated Domains (TADs), designated by spatial chromatin distribution. We utilized the chromatin map to elucidate relations between large scale chromatin state and CpG rich elements landscape.In the course of analysis it was confirmed that genes, Alu and CGI clusters maintain obvious, albeit different in strength, preference for open chromatin. For the first time it was clearly shown that the clusters density of the Alu and CGIs monotonically depend on the chromatin accessibility rate. In particular, the highest density of these elements is found in A1 euchromatin regions characterized by a high density of small length genes replicating in the early S-phase. It implies that these elements mediate (CGIs) or are a side element (Alus) of chromatin accessibility.

Results

We elucidated that both methylated and non-methylated CGIs display the affinity to chromatin accessibility. As a part of comparative genomics section, we elucidated that the dog’s genome non-canonical structure, outstanding in mammals for its high CGIs abundance compared to gene number, is explained by the presence of dense tandem CGI extended hotspots (500 kb on average) in subtelomeric and pericentromeric regions with highly skewed CG content, and not by CGIs global distribution pattern shift.

Conclusions

The study underlines the close association of CG-rich elements distribution with the newly introduced large scale chromatin state map, proposing a refined standpoint on interrelation of aforementioned genome elements and the chromatin state. To our expertise, the TAD-associated partition model employed in the study is likely the most substantial one regarding CpG rich clusters distribution among the whole genome chromatin/isochores maps available.

     

Reconstruction of ancestral protosplice sites

Most of the eukaryotic protein-coding genes are interrupted by multiple introns. A substantial fraction of introns occupy the same position in orthologous genes from distant eukaryotes, such as plants and animals, and consequently are inferred to have been inherited from the common ancestor of these organisms. In contrast to these conserved introns, many other introns appear to have been gained during evolution of each major eukaryotic lineage. The mechanism(s) of insertion of new introns into genes remains unknown. Because the nucleotides that flank splice junctions are nonrandom, it has been proposed that introns are preferentially inserted into specific target sequences termed protosplice sites. However, it remains unclear whether the consensus nucleotides flanking the splice junctions are remnants of the original protosplice sites or if they evolved convergently after intron insertion. Here, we directly address the existence of protosplice sites by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and accordingly are not subject to selection for splicing efficiency. We show that introns are either predominantly inserted into specific protosplice sites, which have the consensus sequence (A/C)AG/Gt, or that they are inserted randomly but are preferentially fixed at such sites.     

Evidence of splice signal migration from exon to intron during intron evolution

A comparison of the nucleotide sequences around the splice junctions that flank old (shared by two or more major lineages of eukaryotes) and new (lineage-specific) introns in eukaryotic genes reveals substantial differences in the distribution of information between introns and exons. Old introns have a lower information content in the exon regions adjacent to the splice sites than new introns but have a corresponding higher information content in the intron itself. This suggests that introns insert into nonrandom (proto-splice) sites but, during the evolution of an intron after insertion, the splice signal shifts from the flanking exon regions to the ends of the intron itself. Accumulation of information inside the intron during evolution suggests that new introns largely emerge de novo rather than through propagation and migration of old introns.     

RB1 and CDKN2A functional defects resulting in retinoblastoma

Multiplex methylation-sensitive PCR was employed in studying the methylation of the RB1 and CDKN2A/p16 promoter regions in 52 retinoblastomas. Aberrant methylation inactivating RB1 was detected in 14 (27%) tumors. Methylation of p16 was for the first time observed in retinoblastoma (9 tumors, 17%). Both promoters proved to be methylated in two tumors. In four tumors, aberrant methylation was combined with structural defects of both RB1 alleles. Aberrant methylation of the p16 promoter was the second mutation event in two tumors and was not accompanied by RB1 defects in one tumor. Complex testing for RB1 mutations, loss of heterozygosity, and functional inactivation of the two genes revealed a molecular defect in at least one allele in 51 (98%) tumors.     

Subdivision of the tribe Oligaphorurini in the light of new and lesser known species from North-East Russia (Collembola,Onychiuridae, Onychiurinae)

The paper is devoted to a taxonomic review of Oligaphorurini from the north-eastern part of Palaearctic. Two new species, i.e. Oligaphorura ambigua sp. n. and Oligaphorura duocellata sp. n., are described. Four species, Oligaphorura nataliae (Fjellberg, 1987), Oligaphorura interrupta (Fjellberg, 1987), Oligaphorura pingicola (Fjellberg, 1987), and Micraphorura alnus (Fjellberg, 1987), are redescribed on base of the types and new material, and remarks on other species known for the region, Oligaphorura groenlandica (Tullberg, 1876), Oligaphorura ursi (Fjellberg, 1984), Oligaphorura aborigensis (Fjellberg, 1987), and Micraphorura absoloni (Börner, 1901), are given to clarify their generic affiliation. Finally, merits and disadvantages of the current subdivision of the tribe are discussed and a key to the northern species of the tribe is provided.     

The tolbutamide site of SUR1 and a mechanism for its functional coupling to K(ATP) channel closure.

Micromolar concentrations of tolbutamide will inhibit (SUR1/K(IR)6. 2)(4) channels in pancreatic beta-cells, but not (SUR2A/K(IR)6.2)(4) channels in cardiomyocytes. Inhibition does not require Mg(2+) or nucleotides and is enhanced by intracellular nucleotides. Using chimeras between SUR1 and SUR2A, we show that transmembrane domains 12-17 (TMD12-17) are required for high-affinity tolbutamide inhibition of K(ATP) channels. Deletions demonstrate involvement of the cytoplasmic N-terminus of K(IR)6.2 in coupling sulfonylurea-binding with SUR1 to the stabilization of an interburst closed configuration of the channel. The increased efficacy of tolbutamide by nucleotides results from an impairment of their stimulatory action on SUR1 which unmasks their inhibitory effects. The mechanism of inhibition of beta-cell K(ATP) channels by sulfonylureas during treatment of non-insulin-dependent diabetes mellitus thus involves two components, drug-binding and conformational changes within SUR1 which are coupled to the pore subunit through its N-terminus and the disruption of nucleotide-dependent stimulatory effects of the regulatory subunit on the pore. These findings uncover a molecular basis for an inhibitory influence of SUR1, an ATP-binding cassette (ABC) protein, on K(IR)6.2, a ion channel subunit.     

Characteristics of the mitochondrial genome of russian germans]

Nucleotide sequences of the mitochondrial DNA (mtDNA) control region were studied in Germans living in the Altai, Russia. Although this ethnic group has been living in Russia for a long time, the obtained data indicate that its mitochondrial gene pool retains the main characteristics of the Western and Central European gene pools. Regarding the mitochondrial gene pool, Russian Germans were more similar to Germans living in Germany than to Russians with regard to the frequency of the Cambridge nucleotide sequence, frequencies and composition of five European haplotypic groups (classification of Richards et al.), and average intra- and interpopulation pairwise nucleotide differences. However, the mitochondrial gene pool of Altaian Germans also differed from that of Western European populations. The gene pool of Altaian Germans contained the ancestral variants of the main haplotypic groups. To date, these variants have not been found in modern Western and Central European populations, which is apparently due to their lower frequencies. In addition, some previously unknown mtDNA variants with specific nucleotide substitutions were found in Altaian Germans. The obtained results suggest that the modern mitochondrial gene pool of Europeans, including Germans from Germany, was largely affected by the demographic processes that occurred in the past two centuries. The Germans that lived in Russia were relatively isolated and, hence, retained more characteristics of the ancestral gene pool.