Kalina Hristova

Trk-A is a receptor tyrosine kinase (RTK) which plays an essential role in the development and functioning of the nervous system. Trk-A is expressed in neurons and signals in response to two ligands, NGF and NT-3, with very different functional consequences. Thus, NGF and NT-3 are "biased" ligands for Trk-A. Since it has been hypothesized that biased RTK ligands induce differential stabilization of RTK dimers, here we seek to test this hypothesis for NGF and NT-3. In particular, we use Förster Resonance Energy Transfer (FRET) and Fluorescence Intensity Fluctuation (FIF) spectroscopy to assess the strength of Trk-A interactions and Trk-A oligomer size in the presence of the two ligands...

Ligand bias is the ability of ligands to differentially activate certain receptor signaling responses compared to others. It reflects differences in the responses of a receptor to specific ligands and has implications for the development of highly specific therapeutics. While ligand bias has been studied primarily for G protein-coupled receptors (GPCRs), there are also reports of ligand bias for receptor tyrosine kinases (RTKs). However, the understanding of RTK ligand bias is lagging behind the knowledge of GPCR ligand bias...

The synthetically evolved pH-dependent delivery (pHD) peptides are a unique family of peptides that bind to membranes, fold into α-helices, and form macromolecule-sized pores at low concentration in response to acidic pH < 6. They have potential applications in drug delivery and tumor targeting but their activity and membrane selectivity must be optimized. Here, we develop a better understanding of how pHD peptide activity in membranes can be modulated by increasing hydrophobicity and membrane binding without changing amino acid sequence...

The human Epidermal Growth Factor Receptor (EGFR/ERBB1) is a Receptor Tyrosine Kinase (RTK) that forms activated oligomers in response to ligand.  Much evidence indicates that EGFR/ERBB1 also forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent oligomers remain unclear. To examine these features, we use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent protein-labeled forms of EGFR and its paralog, Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) in vesicles derived from mammalian cell membranes...

The rate of transport of small molecule drugs across biological barriers, such as the blood-brain barrier, is often a limiting factor in achieving a therapeutic dose. One proposed strategy to enhance delivery across endothelial or epithelial monolayers is conjugation to cell-penetrating peptides (CPPs); however, very little is known about the design of CPPs for efficient transcellular transport. Here, we report on transcellular transport of a CPP, designated the CL peptide, that increases the delivery of small-molecule cargoes across model epithelium approximately 10-fold...

Receptor tyrosine kinases (RTKs) are single-pass membrane proteins that control vital cell processes such as cell growth, survival, and differentiation. There is a growing body of evidence that RTKs from different subfamilies can interact and that these diverse interactions can have important biological consequences. However, these heterointeractions are often ignored, and their strengths are unknown. In this work, we studied the heterointeractions of nine RTK pairs, epidermal growth factor receptor (EGFR)-EPH receptor A2 (EPHA2), EGFR-vascular endothelial growth factor receptor 2 (VEGFR2), EPHA2-VEGFR2, EPHA2-fibroblast growth factor receptor 1 (FGFR1), EPHA2-FGFR2, EPHA2-FGFR3, VEGFR2-FGFR1, VEGFR2-FGFR2, and VEGFR2-FGFR3, using a FRET-based method...

Piscidins are host-defense peptides (HDPs) from fish that exhibit antimicrobial, antiviral, anti-cancer, anti-inflammatory, and wound-healing properties. They are distinctively rich in histidine and contain an amino terminal copper and nickel (ATCUN) binding motif due to the presence of a conserved histidine at position 3. Metallation lowers their total charge and provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs)...

OBJECTIVE: To study two major molecular alterations in spontaneous abortions (SA) with unexplained etiology - fetal genomic anomalies and the endometrial expression of main angiogenic factors VEGFA/VEGFR2 and chemokines SDF-1/CXCR4. MATERIALS AND METHODS: Whole genome copy number analysis by arrayCGH or Next Generation Sequencing (NGS) was applied for detection of fetal genomic imbalances. The abortive decidua of SA without fetal aneuploidies was further investigated for expression levels of the abovementioned factors using real time PCR analysis...

Here, we discuss the transition model of receptor tyrosine kinase (RTK) activation, which is derived from biophysical investigations of RTK interactions and signaling. The model postulates that (1) RTKs can interact laterally to form dimers even in the absence of ligand, (2) different unliganded RTK dimers have different stabilities, (3) ligand binding stabilizes the RTK dimers, and (4) ligand binding causes structural changes in the RTK dimer. The model is grounded in the principles of physical chemistry and provides a framework to understand RTK activity and to make predictions in quantitative terms...

EGFR is a receptor tyrosine kinase that plays a critical role in cell proliferation, differentiation, survival and migration. Its activating ligand, EGF, has long been believed to stabilize the EGFR dimer. Two research studies aimed at quantitative measurements of EGFR dimerization, however, have led to contradicting conclusions and have questioned this view. Given the controversy, here we sought to measure the dimerization of EGFR in the absence and in the presence of saturating EGF concentrations, and to tease out the effect of ligand on dimer stability, using a FRET-based quantitative method...

Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) controls angiogenesis, and is critically important for normal human development and cancer progression. A recent finding that VEGFR2 can dimerize in the absence of ligand raises the question whether VEGF binds to either VEGFR2 monomers or dimers, or to both. While VEGF-VEGFR2 effective binding constants have been measured, these prior measurements have not discriminated between the association state of the receptor. Since ligand binding is coupled to receptor dimerization, this coupling lends complexity to a seemingly straight-forward problem...

The EPH receptor A2 (EphA2) receptor tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements...

Using synthetic molecular evolution, we previously discovered a family of peptides that cause macromolecular poration in synthetic membranes at low peptide concentration in a way that is triggered by acidic pH. To understand the mechanism of action of these "pHD peptides", here we systematically explored structure-function relationships through measurements of the effect of pH and peptide concentration on membrane binding, peptide structure, and the formation of macromolecular-sized pores in membranes. Both AFM and functional assays demonstrate the peptide-induced appearance of large pores in bilayers...

Receptor tyrosine kinases (RTKs) play important roles in cell growth, motility, differentiation, and survival. These single-pass membrane proteins are grouped into subfamilies based on the similarity of their extracellular domains. They are generally thought to be activated by ligand binding, which promotes homodimerization and then autophosphorylation in trans. However, RTK interactions are more complicated, as RTKs can interact in the absence of ligand and heterodimerize within and across subfamilies. Here, we review the known cross-subfamily RTK heterointeractions and their possible biological implications, as well as the methodologies which have been used to study them...

Receptor tyrosine kinases (RTKs) are cell surface receptors which control cell growth and differentiation, and play important roles in tumorigenesis. Despite decades of RTK research, the mechanism of RTK activation in response to their ligands is still under debate. Here we investigate the interactions that control the activation of the tropomyosin receptor kinase (Trk) family of RTKs in the plasma membrane, using a FRET-based methodology. The Trk receptors are expressed in neuronal tissues, and they guide the development of the central and peripheral nervous systems during development...

[This corrects the article DOI: 10.1038/s42003-018-0017-7.].

The EphA2 receptor tyrosine kinase is capable of activating multiple diverse signaling pathways with roles in processes such as tissue homeostasis and cancer. EphA2 is known to form activated oligomers in the presence of ephrin-A ligands. Here, we characterize the lateral interactions between full-length EphA2 molecules in the plasma membrane in the presence of three types of ligands (dimeric ephrinA1-Fc, monomeric ephrinA1, and an engineered peptide ligand) as well as in the absence of ligand, using a quantitative FRET technique...

G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes...

Pore-forming peptides with novel functions have potential utility in many biotechnological applications. However, the sequence-structure-function relationships of pore forming peptides are not understood well enough to empower rational design. Therefore, in this work, we used synthetic molecular evolution to identify a novel family of peptides that are highly potent and cause macromolecular poration in synthetic lipid vesicles at low peptide concentration and at neutral pH. These unique 26-residue peptides, which we call macrolittins, release macromolecules from lipid bilayer vesicles made from zwitterionic PC lipids at peptide to lipid ratios as low as 1:1000, a property that is almost unprecedented among known membrane permeabilizing peptides...

Phenotypic differences between closely related taxa or populations can arise through genetic variation or be environmentally induced, leading to altered transcription of genes during development. Comparative developmental studies of closely related species or variable populations within species can help to elucidate the molecular mechanisms related to evolutionary divergence and speciation. Studies of Arctic charr ( Salvelinus alpinus ) and related salmonids have revealed considerable phenotypic variation among populations and in Arctic charr many cases of extensive variation within lakes (resource polymorphism) have been recorded...