Thomas Schwend

Insights into Egg Coat Assembly and Egg-Sperm Interaction from the X-Ray Structure of Full-Length ZP3.

ZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 Å resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-glycan indicate how sperm binding could trigger downstream events via intramolecular signaling.

Crystal structure of the ZP-N domain of ZP3 reveals the core fold of animal egg coats

Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first, crucial step of mammalian fertilization. Zona pellucida filament components ZP3 and ZP2 act as sperm receptors, and mice lacking either of the corresponding genes produce oocytes without a zona pellucida and are completely infertile. Like their counterparts in the vitelline envelope of non-mammalian eggs and many other secreted eukaryotic proteins, zona pellucida subunits polymerize using a ‘zona pellucida (ZP) domain’ module, whose conserved amino-terminal part (ZP-N) was suggested to constitute a domain of its own. No atomic structure has been reported for ZP domain proteins, and there is no structural information on any conserved vertebrate protein that is essential for fertilization and directly involved in egg–sperm binding. Here we describe the 2.3 ångström (Å) resolution structure of the ZP-N fragment of mouse primary sperm receptor ZP3. The ZP-N fold defines a new immunoglobulin superfamily subtype with a β-sheet extension characterized by an E′ strand and an invariant tyrosine residue implicated in polymerization. The structure strongly supports the presence of ZP-N repeats within the N-terminal region of ZP2 and other vertebrate zona pellucida/vitelline envelope proteins, with implications for overall egg coat architecture, the post-fertilization block to polyspermy and speciation. Moreover, it provides an important framework for understanding human diseases caused by mutations in ZP domain proteins and developing new methods of non-hormonal contraception.

Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage

Uromodulin (or Tamm-Horsfall protein) is the most abundant protein in human urine under physiological conditions. Little is known about the molecular mechanism of uromodulin secretion. By extensive Mass Spectrometry analyses we mapped the C-termini of human and murine urinary proteins demonstrating that urinary uromodulin is generated by a conserved C-terminal proteolytic cleavage and retains its entire ZP domain.

Estrogen receptor β-deficient female mice develop a bladder phenotype resembling human interstitial cystitis

Interstitial cystitis/painful bladder syndrome is a disease seen mostly in women, and symptoms tend to be worse premenopausally or during ovulation. The four cardinal symptoms of interstitial cystitis/painful bladder syndrome are bladder pain, urgency, frequency, and nocturia. Estrogen has been implicated in the etiology of this disease, but the role of the two estrogen receptors (ER), ERα and ERβ, has not been investigated. We found that, in the bladders of WT mice, ERβ is expressed in the basal cell layer of the urothelium. Bladders of male ERβ−/− mice were intact and morphologically indistinguishable from those of their WT littermates. However, in female ERβ−/− mice, there was ulceration and atrophy of bladder urothelium concomitant with infiltration of γδ T cells concentrated in the areas of atrophy and shedding of urothelium. The data support the idea that activated γδ T cells are causing the damage to the urothelium. The hyperactivity of T cells may be because of an imbalance between ERα and ERβ signaling in female ERβ−/− mice. Our data suggest that reduced ERβ signaling might have a role in the pathogenesis of interstitial cystitis, and ERβ could be a candidate for a target of medical therapy.

Different Roles of Estrogen Receptors α and β in the Regulation of E-Cadherin Protein Levels in a Mouse Mammary Epithelial Cell Line

Two estrogen receptors (ERalpha and ERbeta) are found throughout the mammary gland. Evidence indicates that, while ERalpha transduces proliferation signals, ERbeta opposes this effect and is necessary for epithelial differentiation. Using mouse mammary epithelial cells, we have previously shown that activation of ERbeta opposes ERalpha-induced proliferation and increases apoptosis. Furthermore, stable knockdown of ERbeta resulted in loss of growth contact inhibition. In this work, we report that loss of ERbeta is associated with a decrease of E-cadherin protein levels through different posttranscriptional regulatory mechanisms. Ligand activation of ERalpha induced E-cadherin extracellular shedding and internalization only in the absence of ERbeta, followed by lysosomal degradation. Loss of ERbeta also led to an increase of E-cadherin uptake in a ligand-independent manner through mechanisms that required caveolae formation. Proteasome activity was necessary for both mechanisms to operate. Increased E-cadherin internalization correlated with the up-regulation of beta-catenin transcriptional activity and impaired morphogenesis on Engelbreth-Holm-Swarm matrix. Taken together, these results emphasize the role of epithelial ERbeta in maintaining cell adhesion and a differentiated phenotype and highlight the potential importance of ERbeta for the design of specific agonists for use in breast cancer therapy.

Structural basis of SUFU–GLI interaction in human Hedgehog signalling regulation

Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation.

Proteomics Analysis of the Estrogen Receptor α Receptosome

The estrogen receptors (ERs) are ligand-dependent transcription factors that activate transcription by binding to estrogen response elements. Estrogen-mediated effects are tissue- and cell type-specific, determined by the cofactor recruitment to the ERs among other factors. To understand these differences in estrogen action, it is important to identify the various compositions of the ER complexes (ER receptosomes). In this report, we describe a fast and efficient method for the isolation of the ERα receptosome for proteomics analysis. Using immobilized estrogen response element on a Sepharose column in combination with two-dimensional electrophoresis and MALDI-TOF MS, significant amounts of proteins could be isolated and identified. Differences in ERα complex composition with the ER ligands 17β-estradiol, 4-hydroxytamoxifen, and ICI-182,780 could also be observed. Thus, this approach provides an easy and relevant way of identifying ERα cofactor and transcription factor recruitment under different conditions.