Peroxiredoxins wear many hats: Factors that fashion their peroxide sensing personalities.

Peroxiredoxins (Prdxs) sense and assess peroxide levels, and signal through protein interactions. Understanding the role of the multiple structural and post-translational modification (PTM) layers that tunes the peroxiredoxin specificities is still a challenge. In this review, we give a tabulated overview on what is known about human and bacterial peroxiredoxins with a focus on structure, PTMs, and protein-protein interactions. Armed with numerous cellular and atomic level experimental techniques, we look at the future and ask ourselves what is still needed to give us a clearer view on the cellular operating power of Prdxs in both stress and non-stress conditions.

Discovery of a novel lactate dehydrogenase tetramerization domain using epitope mapping and peptides.

Despite being initially regarded as a metabolic waste product, lactate is now considered to serve as a primary fuel for the tricarboxylic acid cycle in cancer cells. At the core of lactate metabolism, lactate dehydrogenases (LDHs) catalyze the interconversion of lactate to pyruvate and as such represent promising targets in cancer therapy. However, direct inhibition of the LDH active site is challenging from physicochemical and selectivity standpoints. However, LDHs are obligate tetramers. Thus, targeting the LDH tetrameric interface has emerged as an appealing strategy. In this work, we examine a dimeric construct of truncated human LDH to search for new druggable sites. We report the identification and characterization of a new cluster of interactions in the LDH tetrameric interface. Using nanoscale differential scanning fluorimetry, chemical denaturation, and mass photometry, we identified several residues (E62, D65, L71, and F72) essential for LDH tetrameric stability. Moreover, we report a family of peptide ligands based on this cluster of interactions. We next demonstrated these ligands to destabilize tetrameric LDHs through binding to this new tetrameric interface using nanoscale differential scanning fluorimetry, NMR water-ligand observed via gradient spectroscopy, and microscale thermophoresis. Altogether, this work provides new insights on the LDH tetrameric interface as well as valuable pharmacological tools for the development of LDH tetramer disruptors.

Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX.

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.

Large crystal growth for neutron protein crystallography.

The use of neutron protein crystallography (NPX) is expanding rapidly, with most structures determined in the last decade. This growth is stimulated by a number of developments, spanning from the building of new NPX beamlines to the availability of improved software for structure refinement. The main bottleneck preventing structural biologists from adding NPX to the suite of methods commonly used is the large volume of the individual crystals required for a successful experiment. A survey of deposited NPX structures in the Protein Data Bank shows that about two-thirds came from crystals prepared using vapor diffusion, while batch and dialysis-based methods all-together contribute to most of the remaining one-third. This chapter explains the underlying principles of these protein crystallization methods and provides practical examples that may help others to successfully prepare large crystals for NPX.

RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata.

The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.

Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative.

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.

Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide.

The small molecule inhibitor acetazolamide (AZM) was conjugated to a set of designed polypeptides and the resulting conjugates were evaluated for their affinity to Human Carbonic Anhydrase II (HCA II) using surface plasmon resonance. The dissociation constant of the AZM-HCA II complex was 38nM and that of the AZM conjugated polypeptide (4-C10L17-AZM) to HCA II was found to be 4nM, an affinity enhancement of a factor of 10 due to polypeptide conjugation. For Human Carbonic Anhydrase IX (HCA IX) the dissociation constant of AZM was 3nM, whereas that of the 4-C10L17-AZM conjugate was 90pM, a 33-fold affinity enhancement. This dramatic affinity increase due to polypeptide conjugation was achieved for a small molecule ligand with an already high affinity to the target protein. This supports the concept that enhancements due to polypeptide conjugation are not limited to small molecule ligands that bind proteins in the mM to µM range but may be used also for nM ligands to provide recognition elements with dissociation constants in the pM range. Evaluations of two HCA IX constructs that do not carry the proteoglycan (PG) domain did not show significant affinity differences between AZM and the polypeptide conjugate, providing evidence that the improved binding of 4-C10L17-AZM to HCA IX emanated from interactions between the polypeptide segment and the PG domain found only in one carbonic anhydrase, HCA IX.

Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests.