Ісак Мазепа: перші кроки у великій політиці

Досліджується становлення світоглядних позицій та формування політичних поглядів І. Мазепи.Исследуется становление мировоззренческих позиций и формирование политических взглядов И. Мазепы.Formation world outlook positions and formation of political views of I. Mazepa is investigated

The human cytomegalovirus-encoded G protein- coupled receptor UL33 exhibits oncomodulatory properties

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein- coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies

Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D

Stem cells & developmental biolog

Disruption of constitutive CXCR4 oligomers impairs oncogenic properties in lymphoid neoplasms

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remains elusive. Using a nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and prosurvival signaling via changes in the phosphoproteome, indicating the existence of constitutive CXCR4 oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B cells to venetoclax is of clinical relevance. Taken together, we established a noncanonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that their selective targeting has clinical potential

Clustering-Induced, Clathrin-Mediated Endocytosis (CIC-ME) for Cancer Therapy

Cancer is characterized by uncontrolled growth or proliferation of cells. Besides conventional cancer therapy, antibodies can be used to target tumor-related molecules like the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF) receptor (Met). Unfortunately, this attenuates tumor growth instead of eliminating tumors completely. Improvement can be made by using so-called antibody-drug conjugates (ADC) that combine the tumor-specificity of antibodies with the cytotoxicity of therapeutic agents. The variable domain of the heavy chain of heavy chain only antibodies (VHH or nanobody) are small antibody fragments that display high binding affinity in combination with good tumor penetration. This thesis provides a detailed overview of the use of nanobodies in cancer-therapy. Nanobodies for the delivery of therapeutic molecules are referred to as Nanobullets. Because Nanobullets often act on intracellular processes, they should facilitate both tumor targeting and intracellular delivery. Therefore, a proper understanding on cellular uptake by tumor cells is important. Ligand-induced signaling of both EGFR and Met is silenced by a negative-feedback mechanism consisting of rapid internalization and subsequent degradation of the receptor-ligand complex. This process is mediated by ligand-induced post-translational modifications like phosphorylation, ubiquitination and acetylation. Recently, ligand-induced clustering of EGFR on the plasma membrane was also found to be involved in receptor internalization. In this thesis, the mechanism behind clustering-induced endocytosis of EGFR was studied in more detail. EGFR clustering induces kinase-independent, clathrin-mediated endocytosis (CIC-ME), which is followed by an unconventional, ubiquitin-independent trafficking towards lysosomal degradation. CIC-ME of EGFR requires a previously unrecognized role for the transmembrane dimerization motifs in receptor internalization. Subsequently, this knowledge was put to practice by developing different examples of Nanobullets for intracellular drug delivery. Nanobody-decorated, albumin-based nanoparticles (NANAPs) facilitated the intracellular release of the platinum-linked multikinase inhibitor 17864-Lx in the lysosomes of EGFR-expressing tumor cells. Similarly, anti-Met NANAPs constitute a novel type of biomaterial for lysosomal drug delivery in Met-expressing tumor cells. Finally, nanobody-photosensitizer (PS) conjugates against EGFR exhibited specific and strong cytotoxicity, which could be improved by intracellular delivery of the PS via CIC-ME (100% specific cell death, IC50 of ~1nM). The small size, high specificity and high potency of these Nanobullets make them promising candidates for further in vivo testing

Clustering-Induced, Clathrin-Mediated Endocytosis (CIC-ME) for Cancer Therapy

Cancer is characterized by uncontrolled growth or proliferation of cells. Besides conventional cancer therapy, antibodies can be used to target tumor-related molecules like the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF) receptor (Met). Unfortunately, this attenuates tumor growth instead of eliminating tumors completely. Improvement can be made by using so-called antibody-drug conjugates (ADC) that combine the tumor-specificity of antibodies with the cytotoxicity of therapeutic agents. The variable domain of the heavy chain of heavy chain only antibodies (VHH or nanobody) are small antibody fragments that display high binding affinity in combination with good tumor penetration. This thesis provides a detailed overview of the use of nanobodies in cancer-therapy. Nanobodies for the delivery of therapeutic molecules are referred to as Nanobullets. Because Nanobullets often act on intracellular processes, they should facilitate both tumor targeting and intracellular delivery. Therefore, a proper understanding on cellular uptake by tumor cells is important. Ligand-induced signaling of both EGFR and Met is silenced by a negative-feedback mechanism consisting of rapid internalization and subsequent degradation of the receptor-ligand complex. This process is mediated by ligand-induced post-translational modifications like phosphorylation, ubiquitination and acetylation. Recently, ligand-induced clustering of EGFR on the plasma membrane was also found to be involved in receptor internalization. In this thesis, the mechanism behind clustering-induced endocytosis of EGFR was studied in more detail. EGFR clustering induces kinase-independent, clathrin-mediated endocytosis (CIC-ME), which is followed by an unconventional, ubiquitin-independent trafficking towards lysosomal degradation. CIC-ME of EGFR requires a previously unrecognized role for the transmembrane dimerization motifs in receptor internalization. Subsequently, this knowledge was put to practice by developing different examples of Nanobullets for intracellular drug delivery. Nanobody-decorated, albumin-based nanoparticles (NANAPs) facilitated the intracellular release of the platinum-linked multikinase inhibitor 17864-Lx in the lysosomes of EGFR-expressing tumor cells. Similarly, anti-Met NANAPs constitute a novel type of biomaterial for lysosomal drug delivery in Met-expressing tumor cells. Finally, nanobody-photosensitizer (PS) conjugates against EGFR exhibited specific and strong cytotoxicity, which could be improved by intracellular delivery of the PS via CIC-ME (100% specific cell death, IC50 of ~1nM). The small size, high specificity and high potency of these Nanobullets make them promising candidates for further in vivo testing

Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry

Antibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have explored the capabilities of low-flow sheathless capillary electrophoresis-mass spectrometry (CE-MS) for the high-resolution and sensitive profiling of antibody therapeutics. Near-zero electroosmotic flow was achieved by employing a novel neutral capillary coating that also prevents protein adsorption. CE-MS analysis of intact model proteins using an acidic background electrolyte demonstrated satisfactory performance, with overall migration-time RSDs below 2.2% from three different capillaries tested. For system evaluation, three nanobody preparations, including mono- and bivalent forms, and three monoclonal antibodies (mAbs) were analyzed. Intact nanobodies were resolved from their degradation products, which could be assigned to deamidated, cleaved, and truncated forms at the C-terminal tag. Excellent resolution of isomeric deamidated products was obtained. The mAbs were analyzed intact and after digestion by the endoproteinase IdeS (middle-up approach). CE-MS of intact mAbs provided resolution of clipped species (e.g. light chain and light chain-heavy chain fragments) from the native protein. Moreover, glycoforms containing sialic acids were resolved from their non-sialylated counterparts. For IdeS-digested, F (ab)(2) and Fc/2 portions where efficiently resolved for the three mAbs. Whereas the migration time of the Fc/2 fragments was fairly similar, the migration time of the F (ab)(2) part was strongly varied among the mAbs. For all mAbs, separation of Fc/2 charge variants - including sialylated glycoforms and other post-translational modifications, such as loss of C-terminal lysine or asparagine deamidation - was achieved. This allowed a detailed and reliable assessment of the Fc/2 heterogeneity (18-33 proteoforms) of the three analyzed mAbs. (C) 2018 The Authors. Published by Elsevier B.V.Proteomic

Analysis of EGF receptor oligomerization by homo-FRET

Growth factor receptors are present in the plasma membrane of resting cells as monomers or (pre)dimers. Ligand binding results in higher-order oligomerization of ligand-receptor complexes. To study the regulation of receptor clustering, several experimental techniques have been developed in the last decades. However, many involve invasive approaches that are likely to disturb the integrity of the membrane, thereby affecting receptor interactions. In this chapter, we describe the use of a noninvasive approach to study receptor dimerization and oligomerization. This method is based upon the Förster energy transfer between identical adjacent fluorescent proteins (homo-FRET) and is determined by analyzing the change in fluorescence anisotropy. Homo-FRET takes place within a distance of 10nm, making this an excellent approach for studying receptor-receptor interactions in intact cells. After excitation of monomeric GFP (mGFP) with polarized light, limiting anisotropy values (r(inf)) of the emitted light are determined, where proteins with known cluster sizes are used as references. Dimerization and oligomerization of the epidermal growth factor receptor (EGFR) in response to ligand binding is determined by using receptors that have been fused with mGFP at their C-terminus. In this chapter, we describe the involved technology and discuss the feasibility of homo-FRET experiments for the determination of cluster sizes of growth factor receptors like EGFR