Miha Pavšič

Dual concentration-dependent activity of thyroglobulin type-1 domain of testican: specific inhibitor and substrate of cathepsin L

Abstract The thyroglobulin type-1 (Tg-1) domain is a protein module that occurs in a variety of secreted and membrane proteins and is recognised as a potent inhibitor of cysteine peptidases. We present here some properties of the Tg-1 domain of human testican, a modularly organised proteoglycan secreted mainly by brain cells, the exact in vivo function of which is not yet clear. The domain was prepared as a recombinant protein in a Pichia pastoris expression system and its activity was demonstrated by specific and selective inhibition of cathepsin L (K i=0.14 nM). Interaction at high enzyme and inhibitor concentrations resulted in degradation of the domain by cathepsin L, which was not observed under conditions used for the determination of kinetic parameters. No inhibitory activity could be detected for cathepsin K, but it exhibited a very similar degradation pattern. Homology modelling provided a good explanation for the different behaviour observed with the two enzymes. Firstly, the steric fit between the interfaces of testican domain and cathepsin L is stabilised by numerous favourable forces, while no such interactions are evident in the complex with cathepsin K, and repulsive interactions even prevent access of the domain to the active site of papain. Secondly, the prolonged first loop of the domain occupies a position near the catalytic cysteine residue in a more substrate-like manner, enabling cleavage of the Gly22-Ala23 bond.

Crystal structure and its bearing towards an understanding of key biological functions of EpCAM

EpCAM (epithelial cell adhesion molecule), a stem and carcinoma cell marker, is a cell surface protein involved in homotypic cell-cell adhesion via intercellular oligomerization and proliferative signalling via proteolytic cleavage. Despite its use as a diagnostic marker and being a drug target, structural details of this conserved vertebrate-exclusive protein remain unknown. Here we present the crystal structure of a heart-shaped dimer of the extracellular part of human EpCAM. The structure represents a cis-dimer that would form at cell surfaces and may provide the necessary structural foundation for the proposed EpCAM intercellular trans-tetramerization mediated by a membrane-distal region. By combining biochemical, biological and structural data on EpCAM, we show how proteolytic processing at various sites could influence structural integrity, oligomeric state and associated functionality of the molecule. We also describe the epitopes of this therapeutically important protein and explain the antigenicity of its regions.

Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)

Background: EPCAM was described as a cell adhesion molecule involved in regulation of proliferation through regulated intramembrane proteolysis. Results: Proteolysis was characterized in-depth, but cleavage or knock-out of HEPCAM did not affect adhesion. Conclusion: Direct adhesion through HEPCAM is questionable. Significance: Unraveling cleavage sites of EPCAM is crucial for developing inhibitors; however, its cell adhesion function may reveal context dependence. Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

A simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins in soluble form in Escherichia coli.

Cysteine cathepsins are major players in numerous physiologic and pathologic processes and important drug targets. Several different expression systems have been developed for the production of these enzymes. Here we describe a novel, simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins. Recombinant procathepsin V was expressed in soluble form in the cytoplasm of Escherichia coli and purified in one step by immobilized nickel ion-affinity chromatography, yielding approximately 0.7 mg procathepsin V per liter bacterial culture. The recombinant proenzyme was then autocatalytically activated in vitro by incubation at pH 4.0 and 30 °C. The yield of proenzyme conversion was over 95% and the mature enzyme exhibited potent activity towards several commonly used synthetic substrates. The same protocol also proved successful in the production of several other cysteine procathepsins, such as cathepsin B, demonstrating that this procedure is widely applicable for the production of recombinant papain-like cysteine peptidases.

Biochemical and preliminary X-ray characterization of the tumor-associated calcium signal transducer 2 (Trop2) ectodomain

Trop2 is a stem/progenitor cell marker, which is also upregulated in several human carcinomas. The largest part of the molecule, recognized by several monoclonal antibodies, is represented by the extracellular part (ectodomain) and is composed of three modules. The aim of our work was to prepare the ectodomain of Trop2 in quantities sufficient for structural and functional studies. We used the Spodoptera frugiperda (Sf9) insect cell expression system to prepare the Trop2 ectodomain (Trop2EC) in two forms - wt glycosylated (gTrop2EC) and mutant non-glycosylated form (Trop2EC(Δ/N)). Recombinant protein was purified from cell culture supernatants using two subsequent nickel ion-affinity chromatographies with a final yield of 15-17mg of purified recombinant protein per liter of culture. Size-exclusion chromatography together with MALS and chemical crosslinking were used to demonstrate for the first time that the Trop2 ectodomain forms a dimer. Both gTrop2EC and Trop2EC(Δ/N) exhibit similar biochemical properties, however the solubility of Trop2EC(Δ/N) is much lower (less than 1mg/ml). For the purpose of structural studies, we crystallized the glycosylated form gTrop2EC. The native dataset was collected with a resolution of 2.94Å and will be used in ongoing work for phasing and structure solution to further understand the role of Trop2 and the structure-function relation between Trop2 and the epithelial cell adhesion molecule (EpCAM).

Expression, crystallization and preliminary X-ray characterization of the human epithelial cell-adhesion molecule ectodomain

The epithelial cell-adhesion molecule (EpCAM; CD326) is a transmembrane glycoprotein involved in epithelial cell-cell adhesion, cell proliferation and differentiation. Its elevated expression level in various carcinomas is exploited by several antitumour therapies that are at various stages of clinical development. The 35 kDa polypeptide chain of EpCAM is divided into a large extracellular part, a transmembrane helix and a short cytoplasmic tail. The modular extracellular part of human EpCAM was cloned and mutated to prevent N-linked glycosylation. After expression in insect cells and purification using standard chromatographic techniques, the extracellular part was crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 86.83, b = 50.16, c = 66.56 Å, β = 127.9°. The crystal diffracted to 1.95 Å resolution and contained one molecule in the asymmetric unit.

Structure and calcium-binding studies of calmodulin-like domain of human non-muscle alpha-actinin-1

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

EpCAM homo-oligomerization is not the basis for its role in cell-cell adhesion

Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays. Second, we also show that EpCAM forms stable dimers on the surface of a cell with pre-formed cell-cell contacts using FLIM-FRET; however, no inter-cellular homo-oligomers were detectable. Thus, our study provides clear evidence that EpCAM indeed does not function as a homophilic cell adhesion molecule and therefore calls for a significant revision of its role in both normal and cancerous tissues. In the light of this, we strongly support the previously suggested name Epithelial Cell Activating Molecule instead of the Epithelial Cell Adhesion Molecule.

The cytosolic tail of the tumor marker protein Trop2 - a structural switch triggered by phosphorylation

Trop2 is a transmembrane signaling glycoprotein upregulated in stem and carcinoma cells. Proliferation-enhancing signaling involves regulated intramembrane proteolytic release of a short cytoplasmic fragment, which is later engaged in a cytosolic signaling complex. We propose that Trop2 function is modulated by phosphorylation of a specific serine residue within this cytosolic region (Ser303), and by proximity effects exerted on the cytosolic tail by Trop2 dimerization. Structural characterization of both the transmembrane (Trop2TM) and cytosolic regions (Trop2IC) support this hypothesis, and shows that the central region of Trop2IC forms an α-helix. Comparison of NMR structures of non-phosphorylated and phosphorylated forms suggest that phosphorylation of Trop2IC triggers salt bridge reshuffling, resulting in significant conformational changes including ordering of the C-terminal tail. In addition, we demonstrate that the cytosolic regions of two Trop2 subunits can be brought into close proximity via transmembrane part dimerization. Finally, we show that Ser303-phosphorylation significantly affects the structure and accessibility of functionally important regions of the cytosolic tail. These observed structural features of Trop2 at the membrane-cytosol interface could be important for regulation of Trop2 signaling activity.

EpCAM ectodomain EpEX is a ligand of EGFR that counteracts EGF-mediated epithelial-mesenchymal transition through modulation of phospho-ERK1/2 in head and neck cancers

Head and neck squamous cell carcinomas (HNSCCs) are characterized by outstanding molecular heterogeneity that results in severe therapy resistance and poor clinical outcome. Inter- and intratumoral heterogeneity in epithelial-mesenchymal transition (EMT) was recently revealed as a major parameter of poor clinical outcome. Here, we addressed the expression and function of the therapeutic target epidermal growth factor receptor (EGFR) and of the major determinant of epithelial differentiation epithelial cell adhesion molecule (EpCAM) in clinical samples and in vitro models of HNSCCs. We describe improved survival of EGFRlow/EpCAMhigh HNSCC patients (n = 180) and provide a molecular basis for the observed disparities in clinical outcome. EGF/EGFR have concentration-dependent dual capacities as inducers of proliferation and EMT through differential activation of the central molecular switch phosphorylated extracellular signal–regulated kinase 1/2 (pERK1/2) and EMT transcription factors (EMT-TFs) Snail, zinc finger E-box-binding homeobox 1 (Zeb1), and Slug. Furthermore, soluble ectodomain of EpCAM (EpEX) was identified as a ligand of EGFR that activates pERK1/2 and phosphorylated AKT (pAKT) and induces EGFR-dependent proliferation but represses EGF-mediated EMT, Snail, Zeb1, and Slug activation and cell migration. EMT repression by EpEX is realized through competitive modulation of pERK1/2 activation strength and inhibition of EMT-TFs, which is reflected in levels of pERK1/2 and its target Slug in clinical samples. Accordingly, high expression of pERK1/2 and/or Slug predicted poor outcome of HNSCCs. Hence, EpEX is a ligand of EGFR that induces proliferation but counteracts EMT mediated by the EGF/EGFR/pERK1/2 axis. Therefore, the emerging EGFR/EpCAM molecular cross talk represents a promising target to improve patient-tailored adjuvant treatment of HNSCCs.