Peptide inhibitors of HIV-1 integrase dissociate the enzyme oligomers.

Integration of HIV-1 genome into host cell chromosome is mediated by viral integrase (IN). The IN catalytic core (CC, IN(50-212)) dimerizes through mutual interactions of its alpha1 and alpha5 helices. Peptides INH1 and INH5 reproducing these helix sequences strongly inhibited IN. For instance, an IC(50) of 80 nM was determined for INH5 in integration assays using wild-type IN (wtIN). In size exclusion chromatography, INH1 and INH5 perturbed the association-dissociation equilibrium of both dmIN (IN(1-288)/F185K/C280S) and CC, leading to monomers as surviving species, while in circular dichroism, binding of peptides to dmIN altered the protein conformation. Thus, enzyme deactivation, subunit dissociation, and protein unfolding are events which parallel one another. The target of INH5 in the enzyme was then identified. In fluorescence spectroscopy, C(0.5) values of 168 and 44 nM were determined for the binding affinity of INH5 to IN and CC, respectively, at 115 nM subunit concentration, while interaction of INH5 with INH1 was found stronger than interaction of INH5 with itself (23 times larger in term of dissociation constants). These results strongly suggested that the alpha1 helix is the privileged target of INH5. The latter could serve as a lead for the development of new chemotherapeutic agents against HIV-1.     

Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the concentration-dependent equilibrium between U-shaped and coiled-coil conformations of a peptide derived from the catalytic core of HIV-1 integrase

The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147-175). In the crystal structure of CC, the corresponding segment belongs to the alpha4 helix (residues 148-168, including residues Glu 152, Lys 156 and Lys 159, crucial for enzyme activity and DNA recognition), a loop (residues 169-171) and a part of the alpha5 helix (171-175), involved in enzyme dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence) concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159 and on the local orientation and environment of the residue. The propensity of TFE to stabilize alpha-helical conformations in peptides was confirmed in CD and fluorescence experiments at relatively high (20-160 microM) and low (2-16 microM) concentrations, respectively. At relatively high concentration, stabilization of the peptide into alpha-helical conformation favored its auto-association likely in parallel coiled-coil dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern-Volmer analysis with positively and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme.     

General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

Summary ¹³C, ¹⁵N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments.The first two authors contributed equally to this work.     

Self-association and DNA binding properties of the human topoisomerase IIA alpha2HTH module

The eukaryotic topoisomerase II is an ubiquitous nuclear enzyme involved in vital cellular functions. It is also the target for some of the most active anticancer drugs. In the various crystal structures of yeast topoisomerase II, the 701-748 segment homologous to the human topoisomerase II alpha 724-771 segment folds into a compact alpha(2)beta(1)alpha(3)talpha(4) conformation, hereafter termed alpha(2)HTH module (helix turn helix (HTH), alpha(3)talpha(4)). The crystal structure of gyrase A has suggested a model wherein HTH is involved in both the enzyme dimerization and the binding to DNA. These two properties were investigated in solution, using the recombinant alpha(2)HTH module of human topoisomerase II alpha and its synthetic components HTH, alpha(4), alpha(3) and turn. The homology-based structure model of human alpha(2)HTH superposed that of yeast in the crystal structure with a rmsd of 1.03 A. Circular dichroism spectra showed that the helical content of human alpha(2)HTH in solution is similar to that of its counterpart within yeast topoisomerase II in the solid state. The chemical cross-linking data indicated that alpha(2)HTH self-associated into dimers while gel mobility shift assays and anisotropy fluorescence titrations demonstrated that alpha(2)HTH, HTH and alpha(4), but not alpha(3), bind efficiently to DNA (dissociation constants of 3.10(-7) M for alpha(2)HTH and alpha(4), of 3.10(-6) M for HTH and of only 1.10(-5) M for alpha(3)). Correlatively, alpha(2)HTH, alpha(4) and HTH, but not alpha(3), were able to inhibit topoisomerase II in DNA relaxation assays, stipulating that alpha(4) is the DNA recognition helix. All suggests that the alpha(2)HTH module once separated from the whole protein conserves a compact conformation, integral to specific dimerization and DNA recognition. The module may thus be used for the search of drugs efficient in hindering topoisomerase II dimerization or binding to DNA.     

The intrinsic mechanics of B-DNA in solution characterized by NMR

Experimental characterization of the structural couplings in free B-DNA in solution has been elusive, because of subtle effects that are challenging to tackle. Here, the exploitation of the NMR measurements collected on four dodecamers containing a substantial set of dinucleotide sequences provides new, consistent correlations revealing the DNA intrinsic mechanics. The difference between two successive residual dipolar couplings (ΔRDCs) involving C6/8-H6/8, C3′-H3′ and C4′-H4′ vectors are correlated to the ³¹P chemical shifts (δP), which reflect the populations of the BI and BII backbone states. The δPs are also correlated to the internucleotide distances (D_inter) involving H6/8, H2′ and H2″ protons. Calculations of NMR quantities on high resolution X-ray structures and controlled models of DNA enable to interpret these couplings: the studied ΔRDCs depend mostly on roll, while D_inter are mainly sensitive to twist or slide. Overall, these relations demonstrate how δP measurements inform on key inter base parameters, in addition to probe the BI↔BII backbone equilibrium, and shed new light into coordinated motions of phosphate groups and bases in free B-DNA in solution. Inspection of the 5′ and 3′ ends of the dodecamers also supplies new information on the fraying events, otherwise neglected.     

Proteomic analysis of necroptotic extracellular vesicles

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.Subject terms: Necroptosis, Cell death and immune response     

Silymarin, a natural antioxidant, protects cerebral cortex against manganese-induced neurotoxicity in adult rats

Manganese (Mn) is an essential element for biological systems, nevertheless occupational exposure to high levels of Mn can lead to neurodegenerative disorders, characterized by serious oxidative and neurotoxic effects with similarities to Parkinson’s disease. The aim of this study was to investigate the potential effects of silymarin (SIL), an antioxidant flavonoid, against manganese chloride induced neurotoxicity both in vivo (cerebral cortex of rats) and in vitro (Neuro2a cells). Twenty-eight male Wistar rats were randomly divided into four groups: the first group (C) received vehicle solution (i.p.) served as controls. The second group (Mn) received orally manganese chloride (20 mg/ml). The third group (Mn + SIL) received both Mn and SIL. The fourth group (SIL) received only SIL (100 mg/kg/day, i.p.). Animals exposed to Manganese chloride showed a significant increase in TBARS, NO, AOPP and PCO levels in cerebral cortex. These changes were accompanied by a decrease of enzymatic (SOD, CAT, GPx) and non-enzymatic (GSH, NpSH, Vit C) antioxidants. Co-administration of silymarin to Mn-treated rats significantly improved antioxidant enzyme activities and attenuated oxidative damages observed in brain tissue. The potential effect of SIL to prevent Mn induced neurotoxicity was also reflected by the microscopic study, indicative of its neuroprotective effects. We concluded that silymarin possesses neuroprotective potential, thus validating its use in alleviating manganese-induced neurodegenerative effects.     

In planta production of indole-3-acetic acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.