Biomedical subjects

Dale Mierke

Publications and source records attributed to Dale Mierke.

5 recordsLinked to original sources

PubMed2007

Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells.

Bradykinin (BK) and angiotensin II (AngII) often have opposite roles in cardiovascular diseases. Our aim here was to construct hybrid receptors which bind AngII but signal as BK. Various sequences of the intracellular face of the AngII type I receptor, AT1R, were replaced with corresponding sequences from the bradykinin B2 receptor (BKB2R). The hybrids demonstrated a number of signaling characteristics of the BKB2R. For example, the hybrids demonstrated BK as opposed to AngII like phosphorylation of Akt and JNK. The hybrids containing the BKB2R intracellular loop 2 (IC2) displayed minimal G-protein, Galphai/Galphaq, linked signaling. Computer based molecular models suggested that Ser-Met-Gly from the IC2 of the BKB2R is detrimental for the Galphai/Galphaq coupled functions of this hybrid. The return of Lys-Ser-Arg of the AT1R to this hybrid led to almost full recovery of Galphai and Galphaq activation. The design and production of AT1/BKB2 hybrid receptors is a potential approach in the treatment of hypertension related diseases where the presence of AngII, its AT1 receptor and the consequent signal transduction has proven detrimental.

Amino Acid Sequence↗

PubMed2006

N-methylated cyclic pentaalanine peptides as template structures.

The N-methylation of cyclic peptides can be used to modify the activity and/or selectivity of biologically active peptides. As N-methylation introduces different flexibility and lipophilicity, it can also improve the bioavailability (the ADMET profile). To search for conformationally constrained cyclic peptides, a library of 30 different N-methylated peptides with the basic sequence cyclo(-D-Ala-L-Ala4-) was synthesized. Based on the NMR analysis, seven of these peptides exhibited single conformations (>98%). The structural features of these peptides were determined by a combination of NMR and distance geometry and then further refined by molecular dynamics simulations in an explicit DMSO solvent box. The structures provided from these efforts can now serve as templates for the rational design of cyclic pentapeptides with a distinct backbone conformation or for "spatial screening" to explore the bioactive conformation of medically important peptide systems.

Alanine↗

PubMed2005

Coulombic and hydrophobic interactions in the first intracellular loop are vital for bradykinin B2 receptor ligand binding and consequent signal transduction.

The role of the first intracellular loop (IC1) in the function of the rat bradykinin B2 receptor (BKB2R) was probed. On the basis of the bovine rhodopsin X-ray structure, the BKB2R IC1 consists of six residues: (60)HKTNCT. Exchange of this sequence with the bradykinin B1 receptor IC1 (PRRQLN) resulted in a chimera which bound bradykinin and signaled as wild-type (WT) BKB2R. In contrast, a chimera containing the IC1 of rat angiotensin II type Ia receptor (AT1aR) (YMKLKT) did not bind BK nor signal in response to BK at a concentration as high as 5 microM. ELISA illustrated that this receptor was still processed and inserted into the plasma membrane. Employing portions of the IC1, we observed that (60)HKT of BKB2R could be exchanged as a group with either the BKB1R (PRR) or AT1aR (YMK) with no change in receptor binding or signaling activities. When only the YM of AT1aR replaced the HK of BKB2R, leaving the N-terminal portion of IC1 without a positively charged residue, binding and signaling were reduced by more than 70%. When only N63 was replaced with the corresponding leucine of AT1aR, binding and signaling were ablated. In fact, replacement of the entire IC1 with the AT1aR except for N63 resulted in binding and signaling as WT BKB2R. However, N63 could be replaced by glutamine (in BKB1R) or aspartate and continued to function as WT BKB2R. NMR data indicated that the BKB2R IC1 extends beyond the bovine rhodopsin prototype to include HKTNCTVAEI. When E68 was exchanged with a serine (in AT1aR), ligand binding decreased by 60% and PI turnover decreased by 69%. Molecular modeling points to a strict requirement for a hydrophilic residue at position 63 (N) at the middle of the IC1 and a Coulombic charge interaction between the positive charges (H60 and K61) at the N-terminus and a negative charge (E68) at the C-terminus of the IC1.

Amino Acid Sequence↗

PubMed2005

Architecture of recombination intermediates visualized by in-gel FRET of lambda integrase-Holliday junction-arm DNA complexes.

Lambda integrase (Int) mediates recombination between attachment sites on phage and Escherichia coli DNA. Int is assisted by accessory protein-induced DNA loops in bridging pairs of distinct "arm-type" and "core-type" DNA sites to form synapsed recombination complexes that subsequently recombine by means of a Holliday junction (HJ) intermediate. An in-gel FRET assay was developed and used to measure 15 distances between six points in two Int-HJ complexes containing arm-DNA oligonucleotides, and 3D maps of these complexes were derived by distance-geometry calculations. The maps reveal unexpected positions for the arm-type DNAs relative to core sites on the HJ and a new Int conformation in the HJ tetramer. The results show how the position of arm DNAs determines the bias of catalytic activities responsible for directional resolution, provide insights into the organization of Int higher-order complexes, and lead to models of the structure of the full HJ recombination intermediates.

Bacteriophage lambda↗

PubMed2004

K317, R319, and E320 within the proximal C-terminus of the bradykinin B2 receptor form a motif important for phospholipase C and phospholipase A2 but not connective tissue growth factor related signaling.

We showed previously that large domain exchanges between the bradykinin B2 (BKB2) and angiotensin II type 1a (AT1a) receptors can result in functional hybrids. However, when we proceeded to exchange the entire bradykinin B2 receptor (BKB2R) C-terminal tail with the AT1aR C-terminus, the hybrid, while continuing to bind BK and be endocytosed as wild type (WT) BKB2R, lost much of its ability to activate phosphatidylinositol (PI) turnover or the release of arachidonic acid (ARA). In this study, we constructed chimeric receptors within the proximal C-terminus between the BKB2R and AT1aR or bradykinin B1 receptor (BKB1R). The mutant and WT receptor cDNAs were stably transfected into Rat-1 cells. Also, point mutations were generated to evaluate the role of the individual residues within this region. These chimeric studies revealed that the proximal portion of the BKB2R C-tail is crucial for G protein-linked BKB2R functions. This region could not be swapped with the AT1aR to obtain a BK activated PI turnover or ARA release. Further studies demonstrated that the distal portion (325-330) of this region is exchangeable; however, the middle portion (317-324) is not. Small motif exchanges within this section identified the KSR and EVY motifs as crucial for G(alphaq), G(alphai) related signaling of the BKB2R. Point mutations then showed that the charged amino acids K317, R319, and E320 are the residues critical for linking to PI turnover and ARA release. However, these proximal chimeras showed normal receptor uptake. Interestingly, while apparently not activating G protein-linked signaling, the proximal tail AT1aR exchange mutant and the entire C-terminus exchange hybrid continued to cause a substantial bradykinin effected increase in connective tissue growth factor (CTGF) mRNA level, as WT BKB2R.

Amino Acid Motifs↗