Defining the mechanism of galectin-3-mediated TGF-ß1 activation and its role in lung fibrosis.

Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.

The adaptability of the ion-binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram-negative bacteria. Sil proteins also bind Cu(I) but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry, we show that binding of both ions is not only tighter than previously thought but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram-negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

Experimental support for reclassification of the light scattering second virial coefficient from macromolecular solutions as a hydrodynamic parameter.

This investigation examines the source of the disparity between experimental values of the light scattering second virial coefficient [Formula: see text] (mL.mol/g2) for proteins and those predicted on the statistical mechanical basis of excluded volume. A much better theoretical description of published results for lysozyme is obtained by considering the experimental parameters to monitor the difference between the thermodynamic excluded volume term and its hydrodynamic counterpart. This involves a combination of parameters quantifying concentration dependence of the translational diffusion coefficient obtained from dynamic light scattering measurements. That finding is shown to account for observations of a strong correlation between [Formula: see text] (mL/g), where M2 is the molar mass (molecular weight) of the macromolecule and the diffusion concentration parameter [Formula: see text] (mL/g). On the grounds that [Formula: see text] is regarded as a hydrodynamic parameter, the same status should be accorded the light scattering second virial coefficient rather than its current incorrect thermodynamic designation as [Formula: see text] (mL.mol/g2), or just B, the osmotic second virial coefficient for protein self-interaction.

Retrospective rationalization of disparities between the concentration dependence of diffusion coefficients obtained by boundary spreading and dynamic light scattering.

This study establishes the existence of substantial agreement between published results from traditional boundary spreading measurements (including synthetic boundary measurements in the analytical ultracenrifuge) on two globular proteins (bovine serum albumin, ovalbumin) and the concentration dependence of diffusion coefficient predicted for experiments conducted under the operative thermodynamic constraints of constant temperature and solvent chemical potential. Although slight negative concentration dependence of the translational diffusion coefficient is the experimentally observed as well as theoretically predicted, the extent of the concentration dependence is within the limits of experimental uncertainty inherent in diffusion coefficient measurement. Attention is then directed toward the ionic strength dependence of the concentration dependence coefficient ([Formula: see text]) describing diffusion coefficients obtained by dynamic light scattering, where, in principle, the operative thermodynamic constraints of constant temperature and pressure preclude consideration of results in terms of single-solute theory. Nevertheless, good agreement between predicted and published experimental ionic strength dependencies of [Formula: see text] for lysozyme and an immunoglobulin is observed by a minor adaptation of the theoretical treatment to accommodate the fact that thermodynamic activity is monitored on the molal concentration scale because of the constraint of constant pressure that pertains in dynamic light scattering experiments.

Thermodynamics of semi-specific ligand recognition: the binding of dipeptides to the E.coli dipeptide binding protein DppA.

This investigation of the temperature dependence of DppA interactions with a subset of three dipeptides (AA. AF and FA) by isothermal titration calorimetry has revealed the negative heat capacity ([Formula: see text]) that is a characteristic of hydrophobic interactions. The observation of enthalpy-entropy compensation is interpreted in terms of the increased structuring of water molecules trapped in a hydrophobic environment, the enthalpic energy gain from which is automatically countered by the entropy decrease associated with consequent loss of water structure flexibility. Specificity for dipeptides stems from appropriate spacing of designated DppA aspartate and arginine residues for electrostatic interaction with the terminal amino and carboxyl groups of a dipeptide, after which the binding pocket closes to become completely isolated from the aqueous environment. Any differences in chemical reactivity of the dipeptide sidechains are thereby modulated by their occurrence in a hydrophobic environment where changes in the structural state of entrapped water molecules give rise to the phenomenon of enthalpy-entropy compensation. The consequent minimization of differences in the value of ΔG0 for all DppA-dipeptide interactions thus provides thermodynamic insight into the biological role of DppA as a transporter of all dipeptides across the periplasmic membrane.

Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.

Many subsurface microorganisms couple their metabolism to the reduction or oxidation of extracellular substrates. For example, anaerobic mineral-respiring bacteria can use external metal oxides as terminal electron acceptors during respiration. Porin-cytochrome complexes facilitate the movement of electrons generated through intracellular catabolic processes across the bacterial outer membrane to these terminal electron acceptors. In the mineral-reducing model bacterium Shewanella oneidensis MR-1, this complex is composed of two decaheme cytochromes (MtrA and MtrC) and an outer-membrane ß-barrel (MtrB). However, the structures and mechanisms by which porin-cytochrome complexes transfer electrons are unknown. Here, we used small-angle neutron scattering (SANS) to study the molecular structure of the transmembrane complexes MtrAB and MtrCAB. Ab initio modeling of the scattering data yielded a molecular envelope with dimensions of ∼105 × 60 × 35 Å for MtrAB and ∼170 × 60 × 45 Å for MtrCAB. The shapes of these molecular envelopes suggested that MtrC interacts with the surface of MtrAB, extending ∼70 Å from the membrane surface and allowing the terminal hemes to interact with both MtrAB and an extracellular acceptor. The data also reveal that MtrA fully extends through the length of MtrB, with ∼30 Å being exposed into the periplasm. Proteoliposome models containing membrane-associated MtrCAB and internalized small tetraheme cytochrome (STC) indicate that MtrCAB could reduce Fe(III) citrate with STC as an electron donor, disclosing a direct interaction between MtrCAB and STC. Taken together, both structural and proteoliposome experiments support porin-cytochrome-mediated electron transfer via periplasmic cytochromes such as STC.

Use of molecular crowding for the detection of protein self-association by size-exclusion chromatography.

The feasibility of employing molecular crowding cosolutes to facilitate the detection of protein self-association by zonal size exclusion chromatography is investigated. Theoretical considerations have established that although the cosolute-induced displacement of a self-association equilibrium towards the oligomeric state invariably occurs in the mobile phase of the column, that displacement is only manifested as a decreased protein elution volume for cosolutes sufficiently small to partition between the mobile and stationary phases. Indeed, the use of a crowding agent sufficiently large to be confined to the mobile phase gives rise to an increased elution volume that could be misconstrued as evidence of cosolute-induced protein dissociation. Those theoretical considerations are reinforced by experimental studies of α-chymotrypsin (a reversibly dimerizing enzyme) on Superdex 200. The use of cosolutes such as sucrose and small polyethylene glycol fractions such as PEG-2000 is therefore recommended for the detection of protein self-association by molecular crowding effects in size exclusion chromatography.

STAT2 Is a Pervasive Cytokine Regulator due to Its Inhibition of STAT1 in Multiple Signaling Pathways.

STAT2 is the quintessential transcription factor for type 1 interferons (IFNs), where it functions as a heterodimer with STAT1. However, the human and murine STAT2-deficient phenotypes suggest important additional and currently unidentified type 1 IFN-independent activities. Here, we show that STAT2 constitutively bound to STAT1, but not STAT3, via a conserved interface. While this interaction was irrelevant for type 1 interferon signaling and STAT1 activation, it precluded the nuclear translocation specifically of STAT1 in response to IFN-γ, interleukin-6 (IL-6), and IL-27. This is explained by the dimerization between activated STAT1 and unphosphorylated STAT2, whereby the semiphosphorylated dimers adopted a conformation incapable of importin-α binding. This, in turn, substantially attenuated cardinal IFN-γ responses, including MHC expression, senescence, and antiparasitic immunity, and shifted the transcriptional output of IL-27 from STAT1 to STAT3. Our results uncover STAT2 as a pervasive cytokine regulator due to its inhibition of STAT1 in multiple signaling pathways and provide an understanding of the type 1 interferon-independent activities of this protein.

Experimental determination of second virial coefficients by small-angle X-ray scattering: a problem revisited.

This investigation examines the validity of employing single-solute theory to interpret SAXS measurements on buffered protein solutions-the current practice despite the necessity to regard the buffer components as additional non-scattering solutes rather than as part of the solvent. The present study of bovine serum albumin in phosphate-buffered saline supplemented with 20-100 g/L sucrose as small cosolute has certainly verified the prediction that the experimentally obtained second virial coefficient should contain protein-cosolute contributions. Nevertheless, the second virial coefficient determined for protein solutions supplemented with high cosolute concentrations on the basis of single-solute theory remains a valid means for identifying conditions conducive to protein crystallization, because the return of a slightly negative second virial coefficient based on single-solute theory [Formula: see text] still establishes the existence of slightly associative interactions between protein molecules, irrespective of the molecular source-protein self-interactions and/or protein-cosolute contributions.

The carbonic anhydrase of Clostridium autoethanogenum represents a new subclass of ß-carbonic anhydrases.

Carbonic anhydrase catalyses the interconversion of carbon dioxide and water to bicarbonate and protons. It was unknown if the industrial-relevant acetogen Clostridium autoethanogenum possesses these enzymes. We identified two putative carbonic anhydrase genes in its genome, one of the ß class and one of the γ class. Carbonic anhydrase activity was found for the purified ß class enzyme, but not the γ class candidate. Functional complementation of an Escherichia coli carbonic anhydrase knock-out mutant showed that the ß class carbonic anhydrase could complement this activity, but not the γ class candidate gene. Phylogenetic analysis showed that the ß class carbonic anhydrase of Clostridium autoethanogenum represents a novel sub-class of ß class carbonic anhydrases that form the F-clade. The members of this clade have the shortest primary structure of any known carbonic anhydrase.

Reconstructing promoter activity from Lux bioluminescent reporters.

The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens-the source of modern Lux reporters-while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.

Allowance for radial dilution in evaluating the concentration dependence of sedimentation coefficients for globular proteins.

The accuracy with which the concentration dependence of the sedimentation coefficient, s = s 0(1 - kc), can be quantified for globular proteins by commonly used procedures has been examined by subjecting simulated sedimentation velocity distributions for ovalbumin to c(s)‒s analysis. Because this procedure, as well as its g(s)‒s counterpart, is based on assumed constancy of s over the time course of sedimentation coefficient measurement in a given experiment, the best definition of the concentration coefficient k is obtained by associating the measured s with the mean of plateau concentrations for the initial and final distributions used for its determination. The return of a slightly underestimated k (by about 3%) is traced to minor mislocation of the air‒liquid meniscus position as the result of assuming time independence of s in a given experiment. Although more accurate quantification should result from later SEDFIT and SEDANAL programs incorporating the simultaneous evaluation of s 0 and k, the procedures based on assumed constancy of s suffice for determining the limiting sedimentation coefficient s 0-the objective of most s‒c dependence studies.

Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.

Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator.

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.

Effect of Accommodating Resistance on the Postactivation Potentiation Response in Rugby League Players.

Scott, DJ, Ditroilo, M, and Marshall, P. Effect of accommodating resistance on the post-activation potentiation response in rugby league players. J Strength Cond Res 32(9): 2510-2520, 2018-This study examined the postactivation potentiation (PAP) response of 2 conditioning activities (CA), the hex bar deadlift and back squat, combined with accommodating resistance; this adds a percentage of the total resistance during the exercise. Twenty amateur rugby league players performed 2 experimental trials and a control trial without a CA. Participants performed a countermovement jump (CMJ) before and 30, 90, and 180 seconds after 1 set of 3 repetitions of each CA at 70% 1 repetition maximum (RM), with up to an additional 23% 1RM from accommodating resistance. Peak power output (PPO), force at PPO, velocity at PPO, and jump height were calculated for each CMJ. Surface electromyography (EMG) of the vastus lateralis (VL), rectus femoris (BF), tibialis anterior (TA), and gastrocnemius medialis (GM) was also measured. Repeated-measures analysis of variance revealed no significant (p > 0.05) PAP response for either exercise condition when comparing CMJ variables with baseline values nor were there any significant (p > 0.05) differences between exercise conditions. However, individualized recovery intervals (baseline vs. maximum potentiation response) demonstrated significant (p ≤ 0.05) improvements in PPO (3.99 ± 4.99%), force at PPO (4.87 ± 6.41%), velocity at PPO (4.30 ± 5.86%), jump height (8.45 ± 10.08%), VL EMG (20.37 ± 34.48%), BF EMG (22.67 ± 27.98%), TA EMG (21.96 ± 37.76%), and GM EMG (21.89 ± 19.65%). Results from this study must be interpreted with caution; however, it is conceivable that athletic performance can be acutely enhanced when complex training variables are individualized.

SilE is an intrinsically disordered periplasmic "molecular sponge" involved in bacterial silver resistance.

Ag(+) resistance was initially found on the Salmonella enetrica serovar Typhimurium multi-resistance plasmid pMG101 from burns patients in 1975. The putative model of Ag(+) resistance, encoded by the sil operon from pMG101, involves export of Ag(+) via an ATPase (SilP), an effluxer complex (SilCFBA) and a periplasmic chaperon of Ag(+) (SilE). SilE is predicted to be intrinsically disordered. We tested this hypothesis using structural and biophysical studies and show that SilE is an intrinsically disordered protein in its free apo-form but folds to a compact structure upon optimal binding to six Ag(+) ions in its holo-form. Sequence analyses and site-directed mutagenesis established the importance of histidine and methionine containing motifs for Ag(+) -binding, and identified a nucleation core that initiates Ag(+) -mediated folding of SilE. We conclude that SilE is a molecular sponge for absorbing metal ions.

Analytical ultracentrifugation: A versatile tool for the characterisation of macromolecular complexes in solution.

Analytical ultracentrifugation, an early technique developed for characterizing quantitatively the solution properties of macromolecules, remains a powerful aid to structural biologists in their quest to understand the formation of biologically important protein complexes at the molecular level. Treatment of the basic tenets of the sedimentation velocity and sedimentation equilibrium variants of analytical ultracentrifugation is followed by considerations of the roles that it, in conjunction with other physicochemical procedures, has played in resolving problems encountered in the delineation of complex formation for three biological systems - the cytoplasmic dynein complex, mitogen-activated protein kinase (ERK2) self-interaction, and the terminal catalytic complex in selenocysteine synthesis.

Complex Training: The Effect of Exercise Selection and Training Status on Postactivation Potentiation in Rugby League Players.

This study compared the postactivation potentiation (PAP) response of the hex bar deadlift (HBD) and back squat (BS) exercises. The PAP response between different levels of athletes was also compared. Ten professional and 10 amateur rugby league players performed 2 experimental sessions. Participants performed a countermovement jump (CMJ) before and 2, 4, 6, 8, 10, 12, 14, and 16 minutes after a conditioning activity (CA) that contained 1 set of 3 repetitions at 93% 1 repetition maximum of either HBD or BS. A force platform determined peak power output (PPO), force at PPO, velocity at PPO, and jump height of each CMJ. Surface electromyography (EMG) of the vastus lasteralis, rectus femoris, tibialis anterior, and gastrocnemius medialis of each participant's dominant leg was recorded during each CMJ. A further 10 participants performed a control trial without a CA. The HBD expressed PAP between 2 and 6 minutes post-CA, whereas the BS did not. The HBD exhibited a significantly (p ≤ 0.05) greater PAP response than the BS for PPO. There were no significant (p > 0.05) differences between stronger and weaker players. There were no significant (p > 0.05) changes in the EMG variables. These results suggest that HBD is a suitable CA for eliciting PAP in stronger and weaker athletes. Strength and conditioning coaches should consider the CA and time frame between the CA and the plyometric exercise for optimal PAP responses.

Three-Dimensional Distribution of Phospholipids in Gram Negative Bacteria.

Exploration of the molecular structure of the bacterial cell envelope informs our understanding of its role in bacterial growth. This is crucial for research into both inhibiting and promoting bacterial growth as well as fundamental studies of cell cycle control. The spatial arrangement of the lipids in the cell envelope of Gram negative bacteria in particular has attracted considerable research attention in recent years. In this mini-review, we explore advances in understanding the spatial distribution of lipids in the model Gram negative prokaryote Escherichia coli. This includes the distribution of lipids in three dimensions, (a) lateral distribution within a monolayer, (b) asymmetry between bilayers and monolayers, and (c) distribution as a function of progress through membrane division (temporal shifts). We conclude that lipid distribution in E. coli and probably all bacteria is dynamic despite a narrow lipid profile and that the biophysical properties of the membrane are inhomogeneous as a result. Finally, we suggest that further work in this field may indicate how lipid distribution is controlled and what this means for bacterial growth and metabolism and even cell cycle control.

N-terminal domain of prion protein directs its oligomeric association.

The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar ß-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined ß-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23-90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91-231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein.