Synonymous variants in HTRA1 implicated in AMD susceptibility impair its capacity to regulate TGF-ß signaling.

High-temperature requirement A1 (HTRA1) is a secreted serine protease reported to play a role in the development of several cancers and neurodegenerative diseases. Still, the mechanism underlying the disease processes largely remains undetermined. In age-related macular degeneration (AMD), a common cause of vision impairment and blindness in industrialized societies, two synonymous polymorphisms (rs1049331:C>T, and rs2293870:G>T) in exon 1 of the HTRA1 gene were associated with a high risk to develop disease. Here, we show that the two polymorphisms result in a protein with altered thermophoretic properties upon heat-induced unfolding, trypsin accessibility and secretion behavior, suggesting unique structural features of the AMD-risk-associated HTRA1 protein. Applying MicroScale Thermophoresis and protease digestion analysis, we demonstrate direct binding and proteolysis of transforming growth factor ß1 (TGF-ß1) by normal HTRA1 but not the AMD-risk-associated isoform. As a consequence, both HTRA1 isoforms strongly differed in their ability to control TGF-ß mediated signaling, as revealed by reporter assays targeting the TGF-ß1-induced serpin peptidase inhibitor (SERPINE1, alias PAI-1) promoter. In addition, structurally altered HTRA1 led to an impaired autocrine TGF-ß signaling in microglia, as measured by a strong down-regulation of downstream effectors of the TGF-ß cascade such as phosphorylated SMAD2 and PAI-1 expression. Taken together, our findings demonstrate the effects of two synonymous HTRA1 variants on protein structure and protein interaction with TGF-ß1. As a consequence, this leads to an impairment of TGF-ß signaling and microglial regulation. Functional implications of the altered properties on AMD pathogenesis remain to be clarified.

The angiotensin receptor-associated protein Atrap is a stimulator of the cardiac Ca2+-ATPase SERCA2a.

AIMS: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor. METHODS AND RESULTS: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS of the isolated complexes showed that Atrap interacts with the cardiac Ca(2+)-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca(2+) uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca(2+)]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap-/- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap-/- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap-/- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap-/- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap-/- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap-/- (725 ± 48 µL/s) compared with WT mice (1065 ± 122 µL/s; P = 0.01; n = 15). CONCLUSION: We identified Atrap as a novel regulatory protein of the cardiac Ca(2+)-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation.

Hyperthermostable acetyl xylan esterase.

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90 °C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl(-1) in the absence of substrate was about 13 h at 98 °C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100-104 °C and 107.5 °C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.