Terbium chelation, a specific fluorescent tagging of human transferrin. Optimization of conditions in view of its application to the HPLC analysis of carbohydrate-deficient transferrin (CDT).

Transferrin (Tf) is the major iron-transporting protein in the human body and, for this reason, has been extensively studied in biomedicine. This protein undergoes a complex glycosylation process leading to several glycoforms, some of which are important in the diagnosis of alcohol abuse and of congenital glycosylation defects under the collective name of carbohydrate-deficient transferrin (CDT). Exploiting the Tf ability to bind not only iron but also other ions, specific attention has been devoted to binding activity towards Tb3+, which was reported to greatly enhance its intrinsic fluorescence upon the interaction with Tf. However, the structural properties of the Tb3+-Tf complex have not been described so far. In the present work, the formation of the Tf-Tb3+ complex has been investigated by the employment of several biophysical techniques, such as fluorescence resonance energy transfer (FRET), "native" mass spectrometry (MS), and near-UV circular dichroism (CD). Each method allowed the detection of the Tf-Tb3+ complex, yielding a specific signature. The interaction of Tb3+ with Fe3+-free Tf (apoTf) has been described in terms of stoichiometry, affinity, and structural effects in comparison with Fe3+. These experiments led to the first direct detection of the Tf-Tb3+ complex by MS, indicating a 1:2 stoichiometry and allowing the investigation of structural effects of metal binding. Either Tb3+ or Fe3+ binding affected protein conformation, inducing structural compaction to a similar extent. Nevertheless, near-UV CD and pH-dependence profiles suggested subtle differences in the coordination of the two metals by Tf side chains. Experimental conditions that promote complex formation have been identified, highlighting the importance of alkaline pH and synergistic ions, such as carbonate. On the basis of these studies, sample pretreatment, separation, and detection conditions of a high-performance liquid chromatographic method for CDT analysis are optimized, achieving relevant increase (by a factor of ∼3) of analytical sensitivity. Graphical abstract Schematic representation of HPLC-separated transferrin glycoforms detected by fluorescence emission of the terbium ions bound to the protein.

Positive Selection Drives Evolution at the Host-Filovirus Interaction Surface.

Filovirus infection is mediated by engagement of the surface-exposed glycoprotein (GP) by its cellular receptor, NPC1 (Niemann-Pick C1). Two loops in the C domain of NPC1 (NPC1-C) bind filovirus GP. Herein, we show that filovirus GP and NPC1-C evolve under mutual selective pressure. Analysis of a large mammalian phylogeny indicated that strong functional/structural constraints limit the NPC1 sequence space available for adaptive change and most sites at the contact interface with GP are under negative selection. These constraints notwithstanding, we detected positive selection at NPC1-C in all mammalian orders, from Primates to Xenarthra. Different codons evolved adaptively in distinct mammals, and most selected sites are located within the two NPC1-C loops that engage GP, or at their anchor points. In Homininae, NPC1-C was a preferential selection target, and the T419I variant possibly represents a human-specific adaptation to filovirus infection. On the other side of the arms-race, GP evolved adaptively during filovirus speciation. One of the selected sites (S142Q) establishes several atom-to-atom contacts with NPC1-C. Additional selected sites are located within epitopes recognized by neutralizing antibodies, including the 14G7 epitope, where sites selected during the recent EBOV epidemic also map. Finally, pairs of co-evolving sites in Marburgviruses and Ebolaviruses were found to involve antigenic determinants. These findings suggest that the host humoral immune response was a major selective pressure during filovirus speciation. The S142Q variant may contribute to determine Ebolavirus host range in the wild. If this were the case, EBOV/BDBV (S142) and SUDV (Q142) may not share the same reservoir(s).

Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a ß-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-ß-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

The mammalian complement system as an epitome of host-pathogen genetic conflicts.

The complement system is an innate immunity effector mechanism; its action is antagonized by a wide array of pathogens and complement evasion determines the virulence of several infections. We investigated the evolutionary history of the complement system and of bacterial-encoded complement-interacting proteins. Complement components targeted by several pathogens evolved under strong selective pressure in primates, with selection acting on residues at the contact interface with microbial/viral proteins. Positively selected sites in CFH and C4BPA account for the human specificity of gonococcal infection. Bacterial interactors, evolved adaptively as well, with selected sites located at interaction surfaces with primate complement proteins. These results epitomize the expectation under a genetic conflict scenario whereby the host's and the pathogen's genes evolve within binding avoidance-binding seeking dynamics. In silico mutagenesis and protein-protein docking analyses supported this by showing that positively selected sites, both in the host's and in the pathogen's interacting partner, modulate binding.

DFT dissection of the reduction step in H2 catalytic production by [FeFe]-hydrogenase-inspired models: can the bridging hydride become more reactive than the terminal isomer?

Density functional theory has been used to study diiron dithiolates [HFe2(xdt)(PR3)n(CO)5-nX] (n = 0, 2, 4; R = H, Me, Et; X = CH3S(-), PMe3, NHC = 1,3-dimethylimidazol-2-ylidene; xdt = adt, pdt; adt = azadithiolate; pdt = propanedithiolate). These species are related to the [FeFe]-hydrogenases catalyzing the 2H(+) + 2e(-) ↔ H2 reaction. Our study is focused on the reduction step following protonation of the Fe2(SR)2 core. Fe(H)s detected in solution are terminal (t-H) and bridging (µ-H) hydrides. Although unstable versus µ-Hs, synthetic t-Hs feature milder reduction potentials than µ-Hs. Accordingly, attempts were previously made to hinder the isomerization of t-H to µ-H. Herein, we present another strategy: in place of preventing isomerization, µ-H could be made a stronger oxidant than t-H (E°µ-H > E°t-H). The nature and number of PR3 unusually affect ΔE°t-H-µ-H: 4PEt3 models feature a µ-H with a milder E° than t-H, whereas the 4PMe3 analogues behave oppositely. The correlation ΔE°t-H-µ-H ↔ stereoelectronic features arises from the steric strain induced by bulky Et groups in 4PEt3 derivatives. One-electron reduction alleviates intramolecular repulsions only in µ-H species, which is reflected in the loss of bridging coordination. Conversely, in t-H, the strain is retained because a bridging CO holds together the Fe2 core. That implies that E°µ-H > E°t-H in 4-PEt3 species but not in 4PMe3 analogues. Also determinant to observe E°µ-H > E°t-H is the presence of a Fe apical σ-donor because its replacement with a CO yields E°µ-H < E°t-H even in 4PEt3 species. Variants with neutral NHC and PMe3 in place of CH3S(-) still feature E°µ-H > E°t-H. Replacing pdt with (Hadt)(+) lowers E° but yields E°µ-H < E°t-H, indicating that µ-H activation can occur to the detriment of the overpotential increase. In conclusion, our results indicate that the electron richness of the Fe2 core influences ΔE°t-H-µ-H, provided that (i) the R size of PR3 must be greater than that of Me and (ii) an electron donor must be bound to Fe apically.

The heptad repeat region is a major selection target in MERS-CoV and related coronaviruses.

Middle East respiratory syndrome coronavirus (MERS-CoV) originated in bats and spread to humans via zoonotic transmission from camels. We analyzed the evolution of the spike (S) gene in betacoronaviruses (betaCoVs) isolated from different mammals, in bat coronavirus populations, as well as in MERS-CoV strains from the current outbreak. Results indicated several positively selected sites located in the region comprising the two heptad repeats (HR1 and HR2) and their linker. Two sites (R652 and V1060) were positively selected in the betaCoVs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. During the most recent evolution of MERS-CoV, adaptive mutations in the HR1 (Q/R/H1020) arose in camels or in a previous host and spread to humans. We determined that different residues at position 1020 establish distinct inter- and intra-helical interactions and affect the stability of the six-helix bundle formed by the HRs. A similar effect on stability was observed for a nearby mutation (T1015N) that increases MERS-CoV infection efficiency in vitro. Data herein indicate that the heptad repeat region was a major target of adaptive evolution in MERS-CoV-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function.

Evolutionary analysis identifies an MX2 haplotype associated with natural resistance to HIV-1 infection.

The protein product of the myxovirus resistance 2 (MX2) gene restricts HIV-1 and simian retroviruses. We demonstrate that MX2 evolved adaptively in mammals with distinct sites representing selection targets in distinct branches; selection mainly involved residues in loop 4, previously shown to carry antiviral determinants. Modeling data indicated that positively selected sites form a continuous surface on loop 4, which folds into two antiparallel α-helices protruding from the stalk domain. A population genetics-phylogenetics approach indicated that the coding region of MX2 mainly evolved under negative selection in the human lineage. Nonetheless, population genetic analyses demonstrated that natural selection operated on MX2 during the recent history of human populations: distinct selective events drove the frequency increase of two haplotypes in the populations of Asian and European ancestry. The Asian haplotype carries a susceptibility allele for melanoma; the European haplotype is tagged by rs2074560, an intronic variant. Analyses performed on three independent European cohorts of HIV-1-exposed seronegative individuals with different geographic origin and distinct exposure route showed that the ancestral (G) allele of rs2074560 protects from HIV-1 infection with a recessive effect (combined P = 1.55 × 10(-4)). The same allele is associated with lower in vitro HIV-1 replication and increases MX2 expression levels in response to IFN-α. Data herein exploit evolutionary information to identify a novel host determinant of HIV-1 infection susceptibility.

An evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection.

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.

RIG-I-like receptors evolved adaptively in mammals, with parallel evolution at LGP2 and RIG-I.

RIG-I-like receptors (RLRs) are nucleic acid sensors that activate antiviral innate immune response. These molecules recognize diverse non-self RNA substrates and are antagonized by several viral inhibitors. We performed an evolutionary analysis of RLR genes (RIG-I, MDA5, and LGP2) in mammals. Results indicated that purifying selection had a dominant role in driving the evolution of RLRs. However, application of maximum-likelihood analyses identified several positions that evolved adaptively. Positively selected sites are located in all domains of MDA5 and RIG-I, whereas in LGP2 they are confined to the helicase domain. In both MDA5 and RIG-I, the linkers separating the caspase activation and recruitment domain and the helicase domain represented preferential targets of positive selection. Independent selective events in RIG-I and LGP2 targeted the corresponding site (Asp421 and Asp179, respectively) within a protruding α-helix that grips the V-shaped structure formed by the pincer. Most of the positively selected sites in MDA5 are in regions unique to this RLR, including a characteristic insertion within the helicase domain. Additional selected sites are located at the contact interface between MDA5 monomers, in spatial proximity to a positively selected human polymorphism (Arg843His) and immediately external to the parainfluenza virus 5 V protein binding region. Structural analyses suggested that the positively selected His834 residue is involved in parainfluenza virus 5 V protein binding. Data herein suggest that RLRs have been engaged in host-virus genetic conflict leading to diversifying selection and indicate parallel evolution at the same site in RIG-I and LGP2, a position likely to be of central importance in antiviral responses.

A 175 million year history of T cell regulatory molecules reveals widespread selection, with adaptive evolution of disease alleles.

T cell activation plays a central role in immune response and in the maintenance of self-tolerance. We analyzed the evolutionary history of T cell regulatory molecules. Nine genes involved in triggering T cell activation or in regulating the ensuing response evolved adaptively in mammals. Several positively selected sites overlap with positions interacting with the binding partner or with cellular components. Population genetic analysis in humans revealed a complex scenario of local (FASLG, CD40LG, HAVCR2) and worldwide (FAS, ICOSLG) adaptation and H. sapiens-to-Neandertal gene flow (gene transfer between populations). Disease variants in these genes are preferential targets of pathogen-driven selection, and a Crohn's disease risk polymorphism targeted by bacterial-driven selection modulates the expression of ICOSLG in response to a bacterial superantigen. Therefore, we used evolutionary information to generate experimentally testable hypotheses concerning the function of specific genetic variants and indicate that adaptation to infection underlies the maintenance of autoimmune risk alleles.