Interaction of hepatitis B viral proteins with host cell machineries

Hepatitis B virus (HBV) infection is a global public health problem, affecting more than 400 million people worldwide and placing them at high risk of developing chronic liver diseases including cirrhosis and hepatocellular carcinoma. The key factor in viral persistence is covalently closed circular (ccc) DNA, an intracellular HBV replication intermediate that resides in the nucleus of infected cells as a non-integrated plasmid-like molecule and serves as a transcriptional template for HBV. Despite extensive research, the contribution of HBV proteins to the development and regulation of chronic infection remains poorly understood, and strategies for cccDNA degradation and elimination are limited. Our main goal is to understand the role of the HBx protein in stimulation of HBV transcription and development of chronic hepatitis B. HBx is a multifunctional protein, and its biological functions are mainly dependent on pleiotropic protein–protein interactions. The role of HBx in redirection of the DNA-damage binding protein 1 (DDB1), the adaptor of Cullin-RING ubiquitin-E3 (CLR4E3) ligase to target cccDNA transcription restriction Smc5/6 complex-specific proteins for degradation was reported recently (Decorsiere 2016). Our group is working on structural characterization of potential complexes formed during degradation of this restriction factor.

Another project focuses on processing of HBV precore protein precursor (HBe) during replication. HBe is processed stepwise in host cells, and its final product, called p17, is released and serves as a clinical marker for disease severity. We analyze the factors influencing HBe processing and localization and study the role of host factors in HBe subcellular localization.

Functional analysis of enzymes regulating Mycobacterium tuberculosis metabolism

An estimated one-third of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb). However, the biology of Mtb that persists in infected people is poorly understood. Mtb reprograms its metabolism during latent infection in response to the host environment. This metabolic adaptation is a very complex and dynamic process. We perform structural and functional characterization of enzymes from selected pathways of central carbon metabolism and biosynthesis of nucleic bases; in particular, we focus on phosphoenolpyruvate kinase, phosphofructokinases A and B, and pyruvate kinase. We investigate the impacts on the function of these enzymes of different conditions mimicking a changing intracellular environment, changes in levels of metabolites, and interactions with cellular proteins. To identify key enzymes in nucleic acid biosynthesis, we use Mycobacterium smegmatis as a model organism. By generating knock-out mutants of enzymes from both the de novo and salvage pathways, we plan to identify the key enzymes influencing biosynthesis of different bases and use them as targets to develop specific inhibitors.

Carbonic anhydrases of Candida species as targets for development of specific antimycotics

Candida albicans and Candida parapsilosis, opportunistic human pathogens naturally present in the microflora of healthy individuals, represent a serious threat to immunocompromised patients. Pathogenic Candida species have evolved the ability to proliferate in niches as diverse as human skin, gut, blood, and mucosal surfaces. Among other factors, these niches differ in CO2 concentration, which is more than 150-fold higher in blood than in atmospheric air. To cope with such differences, Candida produces carbonic anhydrase (CA), which is encoded by the NCE103 gene and catalyzes reversible hydration of CO2 into bicarbonate. CA is dispensable during bloodstream infections, but it is essential for survival of the fungus on skin or abiotic surfaces. Fungal CAs are structurally unrelated to human CAs, which makes them an ideal target for prophylactic interventions. Our research is focused on structure-function analysis of CA encoded by the NCE103 genes of Candida albicans and Candida parapsilosis and on development of novel inhibitors that may be useful for ointments or disinfectants.

Evolution of enzymes involved in fatty acid biosynthesis

Insect species use pheromones composed of different secondary metabolites for mate finding and mating. Pheromones are specific mixtures of saturated and unsaturated fatty acid (FA)-derived alcohols, aldehydes, esters, hydrocarborns, and epoxides and contribute to evolutionary success. Subtle changes in pheromone composition can represent the origin of new species evolution. The overall goal of this work is to uncover molecular mechanisms for the evolution of pheromone components. We focus on classes of enzymes involved in fatty acyl derivative biosynthesis, such as membrane fatty acyl desaturases (mFADs) and fatty acyl reductases (FARs). mFADs introduce double bonds into FA hydrocarbon chains, and FARs reduce fatty-acyl CoA to fatty alcohols. As model species, we use the tobacco hornworm moth, Manduca sexta (Lepidoptera), and the bumblebees Bombus terrestris, Bombus lucorum, and Bombus lapidarius. We have found that a single amino acid substitution in the kink of the mFAD substrate binding tunnel can lead to synthesis of novel unsaturated FA precursors. Currently, our project is focused on confirming this mechanism of FAD specificity determination and understanding the evolution of new pheromone components and new insect species.

Novel results obtained from analysis of FAR gene expression profiles in pheromone glands of three bumblebee species indicate that these insects possess a separate class of FARs. We are working to characterize these enzymes and determine the structural motifs critical for their functions.

These projects are done in collaboration with the groups of Irena Valterová from IOCB and Aleš Svatoš from the Max Planck Institute for Chemical Ecology in Jena, Germany. Our manuscript “Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase,” published in PNAS in 2015, inspired René Levinski’s theatre performance “Touch the space and continue,” which is currently on the program of the New Stage of the National Theatre in Prague and the City Theatre of Brno.