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There are known associations between opioids, obesity, and the gut microbiome, but the molecular connection/mediation of these relationships is not understood. To better clarify the interplay of physiological, genetic, and microbial factors, this study investigated the microbiome and host inflammatory responses to chronic opioid administration in genetically obese, diet-induced obese, and lean mice. Samples of feces, urine, colon tissue, and plasma were analyzed using targeted LC-MS/MS quantification of metabolites, immunoassays of inflammatory cytokine levels, genome-resolved metagenomics, and metaproteomics. Genetic obesity, diet-induced obesity, and morphine treatment in lean mice each showed increases in distinct inflammatory cytokines. Metagenomic assembly and binning uncovered over 400 novel gut bacterial genomes and species. Morphine administration impacted the microbiome's composition and function, with the strongest effect observed in lean mice. This microbiome effect was less pronounced than either diet or genetically driven obesity. Based on inferred microbial physiology from the metaproteome datasets, a high-fat diet transitioned constituent microbes away from harvesting diet-derived nutrients and towards nutrients present in the host mucosal layer. Considered together, these results identified novel host-dependent phenotypes, differentiated the effects of genetic obesity versus diet induced obesity on gut microbiome composition and function, and showed that chronic morphine administration altered the gut microbiome., (© 2022. UT-Battele, LLC.)

Components of the fibrinolytic system are subjected to stringent control to maintain proper hemostasis. Central to this regulation is the serpin plasminogen activator inhibitor-1 (PAI-1), which is responsible for specific and rapid inhibition of fibrinolytic proteases. Active PAI-1 is inherently unstable and readily converts to a latent, inactive form. The binding of vitronectin and other ligands influences stability of active PAI-1. Our laboratory recently observed reciprocal effects on the stability of active PAI-1 in the presence of transition metals, such as copper, depending on the whether vitronectin was also present (Thompson et al. Protein Sci 20:353-365, 2011). To better understand the molecular basis for these copper effects on PAI-1, we have developed a gel-based copper sensitivity assay that can be used to assess the copper concentrations that accelerate the conversion of active PAI-1 to a latent form. The copper sensitivity of wild-type PAI-1 was compared with variants lacking N-terminal histidine residues hypothesized to be involved in copper binding. In these PAI-1 variants, we observed significant differences in copper sensitivity, and these data were corroborated by latency conversion kinetics and thermodynamics of copper binding by isothermal titration calorimetry. These studies identified a copper-binding site involving histidines at positions 2 and 3 that confers a remarkable stabilization of PAI-1 beyond what is observed with vitronectin alone. A second site, independent from the two histidines, binds metal and increases the rate of the latency conversion.

Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function.

Unlabelled: Plasminogen activator inhibitor-1 (PAI-1) is a biologically important serine protease inhibitor (serpin) that, when overexpressed, is associated with a high risk for cardiovascular disease and cancer metastasis. Several of its ligands, including vitronectin, tissue-type and urokinase-type plasminogen activator (tPA, uPA), affect the fate of PAI-1. Here, we measured changes in the solvent accessibility and dynamics of an important unresolved functional region, the reactive center loop (RCL), upon binding of these ligands. Binding of the catalytically inactive S195A variant of tPA to the RCL causes an increase in fluorescence, indicating greater solvent protection, at its C-terminus, while mobility along the loop remains relatively unchanged. In contrast, a fluorescence increase and large decrease in mobility at the N-terminal RCL is observed upon binding of S195A-uPA to PAI-1. At a site distant from the RCL, binding of vitronectin results in a modest decrease in fluorescence at its proximal end without restricting overall loop dynamics. These results provide the new evidence for ligand effects on RCL conformation and dynamics and differences in the Michaelis complex with plasminogen activators that can be used for the development of more specific inhibitors to PAI-1. This study is also the first to use electron paramagnetic resonance (EPR) spectroscopy to investigate PAI-1 dynamics., Significance: Balanced blood homeostasis and controlled cell migration requires coordination between serine proteases, serpins, and cofactors. These ligands form noncovalent complexes, which influence the outcome of protease inhibition and associated physiological processes. This study reveals differences in binding via changes in solvent accessibility and dynamics within these complexes that can be exploited to develop more specific drugs in the treatment of diseases associated with unbalanced serpin activity., (© 2015 The Protein Society.)

Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans 3 orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptide measurements. Peptide mass spectral data were searched by the "hybrid" software packages Inspect and Byonic, which incorporate de novo elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than 40 oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive "hotspot" sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues.

Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent (i.e., hydroxyl radicals) for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate the oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins.

This paper reports on a Leptospira isolate of bovine origin and its identification as belonging to a previously unknown serovar, for which the name Topaz is proposed. The isolate (94-79970/3) was cultured from bovine urine from a north Queensland dairy farm in Australia. Strain 94-79970/3 grew at 30 degrees C in Ellinghausen McCullough Johnson Harris (EMJH) medium but failed to grow at 13 degrees C in EMJH medium or in the presence of 8-azaguanine. Serologically, strain 94-79970/3 produced titres against the Leptospira borgpetersenii serovar Tarassovi, the reference strain for the Tarassovi serogroup; however, no significant titres to any other serovars within the serogroup were obtained. Using 16S rRNA and DNA gyrase subunit B gene analysis, strain 94-79970/3 was identified as a member of the species Leptospira weilii. We propose that the serovar be named Topaz, after the location where the original isolate was obtained. The reference strain for this serovar is 94-79970/3 (=KIT 94-79970/3=LT722).