Additional file 7: of Multiple independent origins of auto-pollination in tropical orchids (Bulbophyllum) in light of the hypothesis of selfing as an evolutionary dead end

Estimating times of divergence: Secondary calibration approach. (DOCX 25 kb

Bulbophyllum occultum, AFLP-matrix

Bulbophyllum occultum, AFLP-matri

The FKBP-Type Domain of the Human Aryl Hydrocarbon Receptor-Interacting Protein Reveals an Unusual Hsp90 Interaction

The aryl hydrocarbon receptor-interacting protein (AIP) has been predicted to consist of an N-terminal FKBP-type peptidyl-prolyl <i>cis</i>/<i>trans</i> isomerase (PPIase) domain and a C-terminal tetratricopeptide repeat (TPR) domain, as typically found in FK506-binding immunophilins. AIP, however, exhibited no inherent FK506 binding or PPIase activity. Alignment with the prototypic FKBP12 showed a high sequence homology but indicated inconsistencies with regard to the secondary structure prediction derived from chemical shift analysis of AIP^2–166. NMR-based structure determination of AIP^2–166 now revealed a typical FKBP fold with five antiparallel β-strands forming a half β-barrel wrapped around a central α-helix, thus permitting AIP to be also named FKBP37.7 according to FKBP nomenclature. This PPIase domain, however, features two structure elements that are unusual for FKBPs: (i) an N-terminal α-helix, which additionally stabilizes the domain, and (ii) a rather long insert, which connects the last two β-strands and covers the putative active site. Diminution of the latter insert did not generate PPIase activity or FK506 binding capability, indicating that the lack of catalytic activity in AIP is the result of structural differences within the PPIase domain. Compared to active FKBPs, a diverging conformation of the loop connecting β-strand C′ and the central α-helix apparently is responsible for this inherent lack of catalytic activity in AIP. Moreover, Hsp90 was identified as potential physiological interaction partner of AIP, which revealed binding contacts not only at the TPR domain but uncommonly also at the PPIase domain

Rapamycin-inspired macrocycles with new target specificity

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.</p

MM284 reduces infiltration of T-cells and macrophages in autoimmune myocarditis in mice.

<p><b>A</b>, <b>B</b>, Infiltration of T-cells and macrophages was assessed using anti-CD3 and anti-Mac-3 staining. Representative images for each treatment are shown. Data in the right panels show individual scoring results (n ≥ 10), horizontal bars indicate medians, * indicates p < 0.05.</p

Extracellular Cyclophilin A—inhibition by MM284 attenuates migration of human monocytes <i>in vitro</i>.

<p><b>A</b>, Effects of MM284 on Cyclophilin A-induced chemotaxis were studied using a modified Boyden chamber. Migration of human monocytes was assessed using media, SDF-1α (50ng/ml) as positive control, CyPA (200nM), or CyPA (200nM) + MM284 (200; 500; and 800nM). Cells were incubated for 4h at 37°C. Migrated cells were counted and a chemotactic index was calculated (n = 5). <b>B</b>, Adhesion of monocytes under flow conditions to activated human umbilical vein endothelial cells. Data are shown as mean ± SEM. <b>C</b>, Cell membrane permeability of MM284 was assessed using a competition assay. MM284 or NIM811 (a cell permeable CsA derivate) was used to displace Fluo-mCsA (a fluorescently labeled (green), cell permeable CsA derivate) from the cytoplasm of THP1 cells. The presence of Fluo-mCsA in the cytoplasm after treatment was assessed using confocal laser scanning microscopy. * indicates p < 0.05 compared to CyPA 200nM.</p

MM284 reduces cardiac inflammation and fibrosis in autoimmune myocarditis in mice.

<p>Troponin I-induced autoimmune myocarditis was studied in A/J mice. <b>A</b>, Hematoxylin and Eosin staining (H&E) was used to determine the area of cardiac damage 28 days after induction of myocarditis. <b>B</b>, Masson’s Trichrome staining was used to determine the extent of fibrotic remodeling in the myocardium 28 days after induction of experimental autoimmune myocarditis. Representative images for each treatment are shown. Data in the right panels show individual scoring results (n ≥ 10), horizontal bars indicate medians, * indicates p < 0.05. <b>C</b>, 28 days after induction of autoimmune myocarditis, small animal echocardiography was used to evaluate left ventricular function (n = 7). Left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular mass (LV Mass), left ventricular end-systolic volume (LV ESV), left ventricular end-diastolic volume (LV EDV), left ventricular posterior wall in diastole (LVPWd), heart rate, and body weight are shown. n.s. indicates not significant.</p

Extracellular Cyclophilin A is associated with myocardial fibrosis.

<p><b>A</b>, Representative stainings of heart sections from mice 28 days after induction of troponin I-induced autoimmune myocarditis. Sections were stained with Masson’s Trichrome, anti-CyPA, and IgG-control as indicated. Marked area is magnified in the middle panel. <b>B</b>, Representative image of healthy control mice stained with anti-CyPA or IgG control. <b>C</b>, Myocardial stainings from mice 28 days after induction of troponin I-induced autoimmune myocarditis. Myocardial sections were stained with anti-CyPA (green), rhodamin phalloidin (red) for actin cytoskeleton, and ToPro-3 for nuclei (blue), as described in materials and methods. Arrows indicate localization of CyPA in the extracellular space.</p

Influence of MM284 on myocardial TNFα, IL-6 and MMP-9 expression.

<p>Realtime PCR assay was performed to evaluate expression of TNFα (<b>A</b>), IL-6 (<b>B</b>) and MMP-9 (<b>C</b>) in the myocardium. Data are shown as mean of relative expression ± SD, n ≥ 8. * indicates p < 0.05, n.s. indicates not significant.</p