Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH.

Background: Tissue factor (TF) generates proteases that can signal through PAR-1 and PAR-2. We have previously demonstrated PAR-1 signalling primes innate myeloid cells to be exquisitely sensitive to interferon-gamma (IFNγ). In this work we explored how TF mediated PAR-2 signalling modulated responsiveness to IFNγ and investigated the interplay between PAR-1/-2 signalling on macrophages. Methodology: We characterised how TF through PAR-2 influenced IFNγ sensitivity in vitro using PCR and flow cytometry. and how it influenced oxazolone-induced delayed type hypersensitivity (DTH) responses in vivo. We investigated how basal signalling through PAR-2 influenced PAR-1 signalling using a combination of TF-inhibitors and PAR-1 &-2 agonists and antagonists. Finally, we investigated whether this system could be targeted therapeutically using 3-mercaptopropionyl-F-Cha-Cha-RKPNDK (3-MP), which has actions on both PAR-1 and -2. Results: TF delivered a basal signal through PAR-2 that upregulated SOCS3 expression and blunted M1 polarisation after IFNγ stimulation, opposing the priming achieved by signalling through PAR-1. PAR-1 and -2 agonists or antagonists could be used in combination to modify this basal signal in vitro and in vivo. 3-MP, by virtue of its PAR-2 agonist properties was superior to agents with only PAR-1 antagonist properties at reducing M1 polarisation induced by IFNγ and suppressing DTH. Tethering a myristoyl electrostatic switch almost completely abolished the DTH response. Conclusions: TF-mediated signalling through PARs-1 and -2 act in a homeostatic way to determine how myeloid cells respond to IFNγ. 3-MP, an agent that simultaneously inhibits PAR-1 whilst delivering a PAR-2 signal, can almost completely abolish immune responses dependent on M1 polarisation, particularly if potency is enhanced by targeting to cell membranes; this has potential therapeutic potential in multiple diseases.

PAR-1 signaling on macrophages is required for effective in vivo delayed-type hypersensitivity responses.

Delayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNγ receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNγ, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNγ in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses.

Thermodynamic and Evolutionary Coupling between the Native and Amyloid State of Globular Proteins.

The amyloid-like aggregation propensity present in most globular proteins is generally considered to be a secondary side effect resulting from the requirements of protein stability. Here, we demonstrate, however, that mutations in the globular and amyloid state are thermodynamically correlated rather than simply associated. In addition, we show that the standard genetic code couples this structural correlation into a tight evolutionary relationship. We illustrate the extent of this evolutionary entanglement of amyloid propensity and globular protein stability. Suppressing a 600-Ma-conserved amyloidogenic segment in the p53 core domain fold is structurally feasible but requires 7-bp substitutions to concomitantly introduce two aggregation-suppressing and three stabilizing amino acid mutations. We speculate that, rather than being a corollary of protein evolution, it is equally plausible that positive selection for amyloid structure could have been a driver for the emergence of globular protein structure.

Chronic histological changes in deceased donor kidneys at implantation do not predict graft survival: a single-centre retrospective analysis.

The use of preimplantation kidney biopsies (PIKBs) to aid deceased donor kidney utilization decisions is controversial. Outcomes of transplants that had been biopsied after the decision had been made to implant were analysed, in order to determine the association between chronic histological changes at implantation and graft outcomes. A retrospective analysis of transplants between the year range 2006-2015 was performed. Karpinski scores on biopsies were collected, and graft outcomes were analysed using univariate and multivariable techniques. Also, Karpinski scores from single and dual kidney transplants from older donors were examined to determine if knowledge of the score preoperatively would have altered utilization. Four hundred and eight single kidneys were transplanted. Although kidneys with scores >4 had lower 1- and 3-year median (IQR) estimated glomerular filtration rates (eGFRs) than those scoring 0-4 (51 (37-66) vs. 35 (26-52) ml/min/1.73 m2 , P < 0.001, and 52 (34-64) vs. 35 (24-52) ml/min/1.73 m2 , P < 0.001, respectively), there was no significant association between Karpinski score and death-censored graft survival on univariate or multivariable analyses. The utilization analysis (75 single and 25 dual kidney transplant recipients) suggested that systematic use of PIKBs would have resulted in 29% fewer patients being transplanted. This analysis does not support the systematic use of PIKBs to determine deceased donor kidney utilization.

Graft dysfunction in chronic antibody-mediated rejection correlates with B-cell-dependent indirect antidonor alloresponses and autocrine regulation of interferon-γ production by Th1 cells.

Chronic antibody-mediated rejection, a common cause of renal transplant failure, has a variable clinical phenotype. Understanding why some with chronic antibody-mediated rejection progress slowly may help develop more effective therapies. B lymphocytes act as antigen-presenting cells for in vitro indirect antidonor interferon-γ production in chronic antibody-mediated rejection, but many patients retain the ability to regulate these responses. Here we test whether particular patterns of T and B cell antidonor response associate with the variability of graft dysfunction in chronic antibody-mediated rejection. Our results confirm that dynamic changes in indirect antidonor CD4+ T-cell responses correlate with changes in estimated glomerular filtration rates, independent of other factors. Graft dysfunction progressed rapidly in patients who developed unregulated B-cell-driven interferon-γ production. However, conversion to a regulated or nonreactive pattern, which could be achieved by optimization of immunosuppression, associated with stabilization of graft function. Functional regulation by B cells appeared to activate an interleukin-10 autocrine pathway in CD4+ T cells that, in turn, impacted on antigen-specific responses. Thus, our data significantly enhance the understanding of graft dysfunction associated with chronic antibody-mediated rejection and provide the foundation for strategies to prolong renal allograft survival, based on regulation of interferon-γ production.

Solubis: a webserver to reduce protein aggregation through mutation.

Protein aggregation is a major factor limiting the biotechnological and therapeutic application of many proteins, including enzymes and monoclonal antibodies. The molecular principles underlying aggregation are by now sufficiently understood to allow rational redesign of natural polypeptide sequences for decreased aggregation tendency, and hence potentially increased expression and solubility. Given that aggregation-prone regions (APRs) tend to contribute to the stability of the hydrophobic core or to functional sites of the protein, mutations in these regions have to be carefully selected in order not to disrupt protein structure or function. Therefore, we here provide access to an automated pipeline to identify mutations that reduce protein aggregation by reducing the intrinsic aggregation propensity of the sequence (using the TANGO algorithm), while taking care not to disrupt the thermodynamic stability of the native structure (using the empirical force-field FoldX). Moreover, by providing a plot of the intrinsic aggregation propensity score of APRs corrected by the local stability of that region in the folded structure, we allow users to prioritize those regions in the protein that are most in need of improvement through protein engineering. The method can be accessed at http://solubis.switchlab.org/.

Structural hot spots for the solubility of globular proteins.

Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function.

Selectivity of aggregation-determining interactions.

Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein ß-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichia coli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.

Accurate prediction of DnaK-peptide binding via homology modelling and experimental data.

Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.