Genny Degani

Neutrophil Attack Triggers Extracellular Trap-Dependent Candida Cell Wall Remodeling and Altered Immune Recognition.

Pathogens hide immunogenic epitopes from the host to evade immunity, persist and cause infection. The opportunistic human fungal pathogen Candida albicans, which can cause fatal disease in immunocompromised patient populations, offers a good example as it masks the inflammatory epitope β-glucan in its cell wall from host recognition. It has been demonstrated previously that β-glucan becomes exposed during infection in vivo but the mechanism behind this exposure was unknown. Here, we show that this unmasking involves neutrophil extracellular trap (NET) mediated attack, which triggers changes in fungal cell wall architecture that enhance immune recognition by the Dectin-1 β-glucan receptor in vitro. Furthermore, using a mouse model of disseminated candidiasis, we demonstrate the requirement for neutrophils in triggering these fungal cell wall changes in vivo. Importantly, we found that fungal epitope unmasking requires an active fungal response in addition to the stimulus provided by neutrophil attack. NET-mediated damage initiates fungal MAP kinase-driven responses, particularly by Hog1, that dynamically relocalize cell wall remodeling machinery including Chs3, Phr1 and Sur7. Neutrophil-initiated cell wall disruptions augment some macrophage cytokine responses to attacked fungi. This work provides insight into host-pathogen interactions during disseminated candidiasis, including valuable information about how the C. albicans cell wall responds to the biotic stress of immune attack. Our results highlight the important but underappreciated concept that pattern recognition during infection is dynamic and depends on the host-pathogen dialog.

Enzymatic and Non-Enzymatic Detoxification of 4-Hydroxynonenal : Methodological Aspects and Biological Consequences

Abstract 4‐Hydroxynonenal (HNE), an electrophilic end‐product deriving from lipid peroxidation, undergoes a heterogeneous set of biotransformations including enzymatic and non‐enzymatic reactions. The former mostly involve red‐ox reactions on the HNE oxygenated functions (phase I metabolism) and GSH conjugations (phase II) while the latter are due to the HNE capacity to spontaneously condense with nucleophilic sites within endogenous molecules such as proteins, nucleic acids and phospholipids. The overall metabolic fate of HNE has recently attracted great interest not only because it clearly determines the HNE disposal, but especially because the generated metabolites and adducts are not inactive molecules (as initially believed) but show biological activities even more pronounced than those of the parent compound as exemplified by potent pro‐inflammatory stimulus induced by GSH conjugates. Similarly, several studies revealed that the non‐enzymatic reactions, initially considered as damaging processes randomly involving all endogenous nucleophilic reactants, are in fact quite selective in terms of both reactivity of the nucleophilic sites and stability of the generated adducts. Even though many formed adducts retain the expected toxic consequences, some adducts exhibit well‐defined beneficial roles as documented by the protective effects of sublethal concentrations of HNE against toxic concentrations of HNE. Clearly, future investigations are required to gain a more detailed understanding of the metabolic fate of HNE as well as to identify novel targets involved in the biological activity of the HNE metabolites. These studies are and will be permitted by the continuous progress in the analytical methods for the identification and quantitation of novel HNE metabolites as well as for proteomic analyses able to offer a comprehensive picture of the HNE‐induced adducted targets. On these grounds, the present review will focus on the major enzymatic and non‐enzymatic HNE biotransformations discussing both the molecular mechanisms involved and the biological effects elicited. The review will also describe the most important analytical enhancements that have permitted the here discussed advancements in our understanding of the HNE metabolic fate and which will permit in a near future an even better knowledge of this enigmatic molecule. Graphical abstract Figure. No Caption available. HighlightsRecent advances of enzymatic and non‐enzymatic detoxification of HNE are described.Increasing importance in the biological activity of HNE metabolites.Good analytical strategies are available for elucidating HNE detoxification pathways.Critical balance between protective or damaging role of HNE.

Catalytic properties of Phr family members of cell wall glucan remodeling enzymes: implications for the adaptation of Candida albicans to ambient pH.

Fungal wall formation is a dynamic process involving several categories of enzymes. The GH72 family of β(1,3)-glucanosyltransferases is essential for the determination of cell shape, for cell integrity and for virulence in pathogenic fungi. Candida albicans has five GH72 genes: PHR1 and PHR2 are pH dependent, the first being expressed at pH ≥ 6 and repressed at lower pH and the second regulated in the opposite manner, PGA4 is transcribed independently of pH whereas PHR3 and PGA5 have low expression levels. To characterize the catalytic properties of Phr1p-2p and probe the activity of Pga4p, we heterologously expressed these proteins and used a fluorescent assay based on the transfer of oligosaccharyl units from a donor to a sulforhodamine-labeled acceptor. Phr1p-2p used exclusively β-1,3-glucan or cell wall glucan as donor and laminarin-derived oligosaccharides as acceptor. The acceptor efficiency increased with the length of the oligosaccharide. The temperature optimum was 30°C. The pH optimum was 5.8 for Phr1p and 3 for Phr2p. Overall, adaptation to pH of C. albicans appears to involve a fine interplay among the pH-dependent activity of Phr1p and Phr2p, the pH-regulated expression of their genes and protein stability. Unexpectedly, Pga4p was inactive suggesting that it turned into a structural mannoprotein.

A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE).

The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction.

An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris.

The receptor for the advanced glycation end products (RAGE) is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily and binds a variety of unrelated ligands sharing a negative charge. Most ligands bind to the extracellular V or VC1 domains of the receptor. In this work, V and VC1 of human RAGE were produced in the methylotrophic yeast Pichia pastoris and directed to the secretory pathway. Fusions to a removable C-terminal His-tag evidenced proteolytic processing of the tag by extracellular proteases and also intracellular degradation of the N-terminal portion of V-His. Expression of untagged forms was attempted. While the V domain was retained intracellularly, VC1 was secreted into the medium and was functionally active in binding AGEs. The glycosylation state of VC1 was analyzed by mass spectrometry and peptide-N-glycosidase F digestion. Like RAGE isolated from mammalian sources, the degree of occupancy of the N-glycosylation sites was full at Asn25 and partial at Asn81 which was also subjected to non-enzymatic deamidation. A simple procedure for the purification to homogeneity of VC1 from the medium was developed. The folded state of the purified protein was assessed by thermal shift assays. Recombinant VC1 from P. pastoris showed a remarkably high thermal stability as compared to the protein expressed in bacteria. Our in vivo approach indicates that the V and C1 domains constitute a single folding unit. The stability and solubility of the yeast-secreted VC1 may be beneficial for future in vitro studies aimed to identify new ligands or inhibitors of RAGE.

The PHR Family: The Role of Extracellular Transglycosylases in Shaping Candida albicans Cells

Candida albicans is an opportunistic microorganism that can become a pathogen causing mild superficial mycosis or more severe invasive infections that can be life-threatening for debilitated patients. In the etiology of invasive infections, key factors are the adaptability of C. albicans to the different niches of the human body and the transition from a yeast form to hypha. Hyphal morphology confers high adhesiveness to the host cells, as well as the ability to penetrate into organs. The cell wall plays a crucial role in the morphological changes C. albicans undergoes in response to specific environmental cues. Among the different categories of enzymes involved in the formation of the fungal cell wall, the GH72 family of transglycosylases plays an important assembly role. These enzymes cut and religate β-(1,3)-glucan, the major determinant of cell shape. In C. albicans, the PHR family encodes GH72 enzymes, some of which work in specific environmental conditions. In this review, we will summarize the work from the initial discovery of PHR genes to the study of the pH-dependent expression of PHR1 and PHR2, from the characterization of the gene products to the recent findings concerning the stress response generated by the lack of GH72 activity in C. albicans hyphae.

Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms.

Identification and characterization of pro-inflammatory AGEs and ALEs by VC1-affinity chromatography and mass spectrometry

The Receptor for AGEs (RAGE), is a membrane receptor binding different ligands sharing a common pattern, including Advanced Glycation/Lipoxidation Endproducts (AGEs/ALEs). The precise mechanism and structural requirements by which AGEs/ALEs interact with RAGE still remain to be elucidated. We have set-up a selective enrichment procedure for AGEs/ALEs based on RAGE ectodomain (VC1) as a stationary phase. The technique was validated using in-vitro produced AGEs/ALEs: HSA as a model protein and reactive carbonyl species and reducing sugars as modifying agents. Generated AGEs/ALEs were successfully entrapped and then fully characterized by Mass Spectrometry (MS). A cyclic moiety was identified as a common structure of the retained AGEs and ALEs, and its involvement in RAGE binding was clarified by molecular modelling studies. Currently, the assay is used to enrich AGEs/ALEs present in different biological matrices, such as oxidized human plasma and clinical samples, for their characterization by MS. The advantage of the herein described technique is to entrap RAGE ligands, independently of their structures, and without the use of any covalent derivatization process. The pro-inflammatory activity of characterized AGEs/ALEs was then tested in a cellular model overexpressing RAGE.

Data from docking simulations to develop an efficient strategy able to evaluate the interactions between RAGE and MDA-induced albumin adducts

This data article contains the results of docking simulations performed in order to develop a suitable in silico strategy able to assess the stability of the putative complexes between RAGE and MDA induced adducts on human albumin as experimentally determined doi: 10.1016/j.redox.2016.12.017, (Degani et al., 2017) [1]. The docking simulations involved different approaches to give a simplified yet realistic representation of the protein adducts and their environment. With increasing complexity, simulations involved the corresponding albumin tripeptides and pentapeptides with the modified residue in the central position as well as pseudo-structures which were generated by collecting the albumin residues around the adducted residue within a sphere of 7.5 Å and 5 Å radius. The reliability of the tested approaches was assessed by monitoring the score differences between adducted and unmodified residues. The obtained results revealed the greater predictive power of the spherical pseudo-structures compared to the simple tri- or pentapeptidic sequences thus suggesting that RAGE recognition involves residues which are spatially close to the modified residue even though not necessarily adjacent in the primary sequence.