Engineering of haloalkane dehalogenase enantioselectivity towards βbromoalkanes: Open-solvated versus occluded-desolvated active sites

Enzymatic catalysis is widely used for preparing optically pure chemicals. Natural catalysts have to be often optimized to exhibit sufficient enantioselectivity towards industrially attractive non-natural substrates. Understanding the molecular basis of enzyme–substrate interactions involved in enantiodiscrimination is essential for rational design of selective catalysts. Haloalkane dehalogenases (EC 3.8.1.5) can convert a broad range of halogenated aliphatic compounds to their corresponding alcohols via SN2 mechanism [1]. The very first haloalkane dehalogenase exhibiting high enantioselectivity towards β-brominated alkanes (E-values of up to 174) was DbjA from Bradyrhizobium japonicum USDA110 [2]. This enzyme has a wide open solvent-accessible active site and its enantioselectivity towards β-brominated alkanes is modulated by a surface loop unique to DbjA [2]. Assuming that the active site geometry is crucial for substrate recognition, it was proposed that DbjA’s enantioselectivity could be transferred to closely related, but non-selective DhaA from Rhodococcus rhodochrous NCIMB13064 [1] by active site transplantation [3]. The unique loop fragment from DbjA together with additional 8-point substitutions was inserted to DhaA. Although the crystal structure of resulting variant DhaA12 exhibited identical geometry of the active site and the access tunnel as DbjA, it did not reach identical level of hydration and flexibility and lacked enantioselectivity towards β-bromoalkanes (E-value = 18) [3]. Interestingly, the variant DhaA31 constructed independently with a goal to enhance enzyme activity towards anthropogenic compound 1,2,3-trichlopropane [4], exhibited high enantioselectivity towards 2-bromopentane (E-value = 179) [5] as DbjA (E-value = 174) [2, 3]. DhaA31 contains five mutations, I135F, C176Y, V245F, L246I and Y273F, located in a main and a slot tunnel. Four of five mutations are large and aromatic residues narrowing two access tunnels and occluding the enzyme active site [4]. The level of DhaA31 active site hydration, so important for DbjA’s enantioselectivity [2, 3] is low, suggesting a different structural basis of enantioselectivity towards 2-bromopentane. A systematic study on the molecular basis of enantioselectivity in DbjA, DhaA, and DhaA31 using thermodynamic and kinetic analyses, site-directed mutagenesis, and molecular modeling was carried out. DhaA31 enantioselectivity arises from the hydrophobic substrate’s interactions with the occluded and desolvated active site [5], while DbjA enantioselectivity results from water-mediated interactions of 2-bromopentane with the active site’s hydrophobic wall [2]. Our data imply that enantioselectivity of haloalkane dehalogenases can be achieved by both occluded-desolvated active site and open-solvated active site. The engineering of “DbjA-like” enantioselectivity by modification of the active site hydration remains challenging. References: 1. Koudelakova, T., et al. 2013. Biotechnol. J. 8: 32–45. Prokop, Z., et al. 2010. Angew. Chem. Int. Ed., 49: 6111-6115. Sykora, J., et al. 2014. Nat. Chem. Biol., 10: 428-430. Pavlova, M., et al. 2009. Nat. Chem. Biol., 5: 727-733. Liskova, V., et al. 2017. Angew. Chem. Int. Ed., DOI: 10.1002/anie.201611193

Engineering the protein dynamics of an ancestral luciferase.

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins

Targeting of Deregulated Wnt/β-Catenin Signaling by PRI-724 and LGK974 Inhibitors in Germ Cell Tumor Cell Lines

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/β-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of β-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of β-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/β-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/β-catenin pathway inhibition in GCTs is therefore warranted

Engineering the protein dynamics of an ancestral luciferase

Abstract: Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins

Role of Sodium/Calcium Exchangers in Tumors

The sodium/calcium exchanger (NCX) is a unique calcium transport system, generally transporting calcium ions out of the cell in exchange for sodium ions. Nevertheless, under special conditions this transporter can also work in a reverse mode, in which direction of the ion transport is inverted—calcium ions are transported inside the cell and sodium ions are transported out of the cell. To date, three isoforms of the NCX have been identified and characterized in humans. Majority of information about the NCX function comes from isoform 1 (NCX1). Although knowledge about NCX function has evolved rapidly in recent years, little is known about these transport systems in cancer cells. This review aims to summarize current knowledge about NCX functions in individual types of cancer cells

Oral presentation of paraneoplastic pemphigus as the first sign of tonsillar HPV associated squamous cell carcinoma. A case report.

Background. Paraneoplastic pemphigus (PNP) in the oral cavity is a rare variant of blistering pemphigus disease closely associated with mostly malignant tumors. The diagnosis may even precede an underlying malignancy enabling early detection. Here, we describe a previously unreported case of PNP associated with HPV-related tonsillar squamous cell carcinoma. Methods and Results. A 50-year-old woman was referred to a dentist because of painful oral lesions resembling aphthae major and minor. Later, blisters appeared and an incisional biopsy was performed. Histological examination revealed an unusual coexistence of subepithelial and intraepithelial blisters raising suspicion of paraneoplastic pemphigus. The patient underwent 18F-FDG PET/MRI, showing a metabolically active process in the left palatal tonsil. Diagnostic biopsy revealed HPV type 16 associated tonsillar squamous cell carcinoma. A left tonsillectomy with elective left-sided neck dissection was performed. The postoperative period was complicated by bilateral fluidothorax. Two weeks after radical tumor removal, the mucosal and skin lesions of PNP disappeared. The patient currently shows no evidence of recurrence either of malignancy or PNP eight months after the surgery. Conclusion. PNP is a rare autoimmune blistering disease characterized by polymorphous cutaneous and mucosal lesions associated with internal neoplasms including HPV associated squamous cell carcinoma of a tonsil. In order to identify an occult malignancy, a whole-body PET/CT or PET/MRI scan is recommended. Rarely, accurate patient management may depend on the dentist being familiar with this entity and on interdisciplinary cooperation involving dermatologist, radiologist, pathologist, and pneumologist. A strict patient follow-up is indicated

Type 1 Sodium Calcium Exchanger Forms a Complex with Carbonic Anhydrase IX and Via Reverse Mode Activity Contributes to pH Control in Hypoxic Tumors

Hypoxia and acidosis are among the key microenvironmental factors that contribute to cancer progression. We have explored a possibility that the type 1Na+/Ca2+ exchanger (NCX1) is involved in pH control in hypoxic tumors. We focused on changes in intracellular pH, co-localization of NCX1, carbonic anhydrase IX (CA IX), and sodium proton exchanger type 1 (NHE1) by proximity ligation assay, immunoprecipitation, spheroid formation assay and migration of cells due to treatment with KB-R7943, a selective inhibitor of the reverse-mode NCX1. In cancer cells exposed to hypoxia, reverse-mode NCX1 forms a membrane complex primarily with CA IX and also with NHE1. NCX1/CA IX/NHE1 assembly operates as a metabolon with a potent ability to extrude protons to the extracellular space and thereby facilitate acidosis. KB-R7943 prevents formation of this metabolon and reduces cell migration. Thus, we have shown that in hypoxic cancer cells, NCX1 operates in a reverse mode and participates in pH regulation in hypoxic tumors via cooperation with CAIX and NHE1

Prognostic Value of Apoptosis-Inducing Factor (AIF) in Germ Cell Tumors

Apoptosis is a strictly regulated process essential for preservation of tissue homeostasis. This study aimed to evaluate expression of apoptosis inducing factor (AIF) in testicular germ cell tumors (GCTs) and to correlate expression patterns with clinicopathological variables. Formalin-fixed and paraffin-embedded specimens of non-neoplastic testicular tissue and GCTs obtained from 216 patients were included in the study. AIF expression was detected by immunohistochemistry, scored by the multiplicative quickscore method (QS). Normal testicular tissue exhibits higher cytoplasmic granular expression of AIF compared to GCTs (mean QS = 12.77 vs. 4.80, p p = 0.048). We observed significantly lower AIF expression in GCTs compared to normal testicular tissue, which is an uncommon finding in malignant tumors. AIF downregulation might represent one of the mechanisms of inhibition of apoptosis and promotion of cell survival in GCTs

Engineering Protein Dynamics of Ancestral Luciferase

Insertion-deletion mutations are sources of major functional innovations in naturally evolved proteins, but directed evolution methods rely primarily on substitutions. Here, we report a powerful strategy for engineering backbone dynamics based on InDel mutagenesis of a stable and evolvable template, and its validation in application to a thermostable ancestor of haloalkane dehalogenase and Renilla luciferase. First, extensive multidisciplinary analysis linked the conformational flexibility of a loop-helix fragment to binding of the bulky substrate coelenterazine. The fragment’s key role in extant Renilla luciferase was confirmed by transplanting it into the ancestor. This increased its catalytic efficiency 7,000-fold, and fragment-containing mutants showed highly stable glow-type bioluminescence with 100-fold longer half-lives than the flash-type Renilla luciferase RLuc8, thereby addressing a limitation of a popular molecular probe. Thus, our three-step approach: (i) constructing a robust template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of a dynamic feature, provides a potent strategy for discovering protein modifications with globally disruptive but functionally innovative effects.</p