Our data suggest divergent effects of tenofovir and AZT on proinflammatory responses in monocytes (CCL3 and IL-8) and PBMCs (CCL3). Moreover, tenofovir shifts the IL-10/IL-12 balance after cell stimulation with TLR ligands or infection with live bacteria, thus suggesting that the choice of nucleoside reverse transcriptase inhibitor affects overall inflammation and early immune responses against secondary pathogens.

Purpose In this study, we investigated whether the phenotypic difference observed between two lattice corneal dystrophy type 1 (LCD type 1) cases caused by either a single A546D substitution or a A546D/P551Q double substitution in TGFBIp, can be ascribed to (I) a difference in the proteomes of corneal amyloid deposits, (II) altered proteolysis of TGFBIp or (III) structural changes of TGFBIp introduced by the P551Q amino acid substitution. Experimental design Amyloid deposits were isolated from the corneas of two siblings with LCD type 1 resulting from A546D/P551Q mutations in TGFBI using laser capture microdissection and a subsequently analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteolytic processing of TGFBIp was addressed by counting peptide spectra. Lastly, to study the possible effect of the P551Q substitution, recombinant FAS1-4 domain variants were subjected to in vitro stability assays. Results The amyloid proteomes and TGFBIp processing of the two A546D/P551Q LCD type 1 cases were similar to each other as well as to the A546D amyloid proteome previously reported by us. The stability assays revealed a minor destabilization of the FAS1-4 domain upon the addition of the P551Q mutation, moreover, it resulted in different accessibility to tryptic cleavage sites between the A546D and A546D/P551Q mutant FAS1-4 domain variants. Conclusion The difference in A546D and A546D/P551Q LCD type 1 phenotypes cannot be ascribed to altered corneal amyloid composition or altered in vivo proteolytic processing of TGFBIp. Instead, a small difference in thermodynamic stability introduced by the P551Q mutation most likely causes structural changes of TGFBIp.

Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified “invisible” folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å−3.

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Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T 1 relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T 1 values across the entire sequence of the intrinsically disordered protein α-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in 'proton-less' NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from α1-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.