Allosteric inhibition of hypoxia inducible factor-2 with small molecules.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of protumorigenic pathways. Though transcription factors are typically considered 'undruggable', the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high sequence identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2-driven tumors, such as renal cell carcinomas.

An E3 ligase possessing an iron-responsive hemerythrin domain is a regulator of iron homeostasis.

Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of genes responsible for iron uptake, release, use, and storage through the actions of the iron regulatory proteins IRP1 and IRP2. However, the manner in which iron levels are sensed to affect IRP2 activity is poorly understood. We found that an E3 ubiquitin ligase complex containing the FBXL5 protein targets IRP2 for proteasomal degradation. The stability of FBXL5 itself was regulated, accumulating under iron- and oxygen-replete conditions and degraded upon iron depletion. FBXL5 contains an iron- and oxygen-binding hemerythrin domain that acted as a ligand-dependent regulatory switch mediating FBXL5's differential stability. These observations suggest a mechanistic link between iron sensing via the FBXL5 hemerythrin domain, IRP2 regulation, and cellular responses to maintain mammalian iron homeostasis.

On the mechanism of quinol oxidation at the QP site in the cytochrome bc1 complex: studied using mutants lacking cytochrome bL or bH.

To elucidate the mechanism of bifurcated oxidation of quinol in the cytochrome bc1 complex, Rhodobacter sphaeroides mutants, H198N and H111N, lacking heme bL and heme bH, respectively, were constructed and characterized. Purified mutant complexes have the same subunit composition as that of the wild-type complex, but have only 9-11% of the electron transfer activity, which is sensitive to stigmatellin or myxothiazol. The Em values for hemes bL and bH in the H111N and H198N complexes are -95 and -35 mV, respectively. The pseudo first-order reduction rate constants for hemes bL and bH in H111N and H198N, by ubiquiniol, are 16.3 and 12.4 s(-1), respectively. These indicate that the Qp site in the H111N mutant complex is similar to that in the wild-type complex. Pre-steady state reduction rates of heme c1 by these two mutant complexes decrease to a similar extent of their activity, suggesting that the decrease in electron transfer activity is due to impairment of movement of the head domain of reduced iron-sulfur protein, caused by disruption of electron transfer from heme bL to heme bH. Both mutant complexes produce as much superoxide as does antimycin A-treated wild-type complex. Ascorbate eliminates all superoxide generating activity in the intact or antimycin inhibited wild-type or mutant complexes.

Domain conformational switch of the iron-sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH.

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc(1) complex in the past have led to the formulation of the "protonmotive Q-cycle" mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q(P) site with both electrons transferred simultaneously to ISP and cyt b(L) when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc(1) demonstrates that the reduced ISP-ED moves to the c(1)-position to reduce cyt c(1) only after the reduced cyt b(L) is oxidized by cyt b(H). However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q(P) site inhibitors, Pm and Pf, under various redox states of the bc(1) complex, suggest that the electron transfer from heme b(L) to b(H) is the driving force for the releasing of the reduced ISP-ED from the b-position to c(1)-position to reduce cyt c(1).

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex.

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.

[Amplification, cloning and sequence analysis of spider dragline silk cDNA].

Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.

[The advance of tumor development mechanism applying mTR-/- mouse model].

Telomerase is Ribonucleoprotein complex in eukaryocyte, which is composed of telomerase reverse transcriptase(TERT) and telomerase RNA. Telomerase is a special DNA polymerase which can extend the terminal of DNA and maintain the length of telomere. TERT have reverse transcriptase activity. Telomerase activity do not examine in most somatic cell and primary cell, but most tumor cell have strong telomerase activity. It was think that the telomerase has strong relation with tumor occurrences. In this article,the author instruct the correlation of G-strand and P53 in tumor occurrences.