Unbiased descriptor and parameter selection confirms the potential of proteochemometric modelling

Background  

Proteochemometrics is a new methodology that allows prediction of protein function directly from real interaction measurement data without the need of 3D structure information. Several reported proteochemometric models of ligand-receptor interactions have already yielded significant insights into various forms of bio-molecular interactions. The proteochemometric models are multivariate regression models that predict binding affinity for a particular combination of features of the ligand and protein. Although proteochemometric models have already offered interesting results in various studies, no detailed statistical evaluation of their average predictive power has been performed. In particular, variable subset selection performed to date has always relied on using all available examples, a situation also encountered in microarray gene expression data analysis.     

Identification of the binding pocket for the TRH peptide in the melanocortin 1 receptor

We found recently that thyrotropin-releasing hormone (TRH) acts as a selective agonist on the melanocortin MC₁ receptor. While the TRH was capable of fully activating the MC₁ receptor, it did not interact with any of the other MSH peptide binding G-protein coupled melanocortin receptor subtypes MC_3-5. The MC₁ receptor is a promising target for the development of anti-inflammatory and immuno-modulatory drugs, and it was of wide interest to explore the binding site of the TRH in this receptor. Here we have investigated the binding of TRH to MC₁/MC₃ chimeric receptors and used a partial least squares (PLS) modelling approach for the data evaluation. Statistically valid PLS models (R² = 0.80; Q² = 0.66) were obtained explaining the contribution of the amino acid sequence parts of the receptor chimeras for the binding of TRH. By using the variable importances in the projection (VIPs) deduced from the PLS-model, it was revealed that the transmembrane (TM) regions TM1 and TM2/TM3 contribute the most to the TRH binding. The TM6/TM7 also had a significant influence on the binding. Moreover, it was revealed that an interaction between TM1 and TM6/TM7 of the receptor contributed to the binding of TRH. The data are in full agreement with a 3D model of a TRH peptide and MC₁ receptor complex and validates the location of the TRH ligand binding pocket between the TM1, TM2 and TM7 of the receptor.     

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3′ containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.     

Dietary vitamin D3 supplements reduce demyelination in the cuprizone model

Vitamin D is emerging as a probably important environmental risk factor in multiple sclerosis, affecting both susceptibility and disease progression. It is not known to what extent this effect is due to a modulation of peripheral lymphocyte function, or to intrathecal effects of vitamin D. We investigated the effect of dietary vitamin D3 content on de/remyelination in the cuprizone model, which is a well established toxic model of demyelination, with no associated lymphocyte infiltration. The mice received diets either deficient of (<50 IU/kg), or supplemented with low (500 IU/kg), high (6200 IU/kg) or very high (12500 IU/kg) amounts of vit D3. Cuprizone (0.2%) was added to the diet for six weeks, starting two weeks after onset of the experimental diets. Mouse brain tissue was histopathologically evaluated for myelin and oligodendrocyte loss, microglia/macrophage activation, and lymphocyte infiltration after six weeks of cuprizone exposure, and two weeks after discontinuation of cuprizone exposure. High and very high doses of vitamin D3 significantly reduced the extent of white matter demyelination (p = 0.004) and attenuated microglia activation (p = 0.001). No differences in the density of oligodendrocytes were observed between the diet groups. Two weeks after discontinuation of cuprizone exposure, remyelination was only detectable in the white matter of mice receiving diets deficient of or with low vitamin D3 content. In conclusion, high dietary doses of vitamin D3 reduce the extent of demyelination, and attenuate microglia activation and macrophage infiltration in a toxic model of demyelination, independent of lymphocyte infiltration.     

SDS-facilitated in vitro formation of a transmembrane B-type cytochrome is mediated by changes in local pH

The folding and stabilization of α-helical transmembrane proteins are still not well understood. Following cofactor binding to a membrane protein provides a convenient method to monitor the formation of appropriate native structures. We have analyzed the assembly and stability of the transmembrane cytochrome b₅₅₉′, which can be efficiently assembled in vitro from a heme-binding PsbF homo-dimer by combining free heme with the apo-cytochrome b₅₅₉′. Unfolding of the protein dissolved in the mild detergent dodecyl maltoside may be induced by addition of SDS, which at high concentrations leads to dimer dissociation. Surprisingly, absorption spectroscopy reveals that heme binding and cytochrome formation at pH 8.0 are optimal at intermediate SDS concentrations. Stopped-flow kinetics revealed that genuine conformational changes are involved in heme binding at these SDS concentrations. GPS (Global Protein folding State mapping) NMR measurements showed that optimal heme binding is intimately related to a change in the degree of histidine protonation. In the absence of SDS, the pH curve for heme binding is bell-shaped with an optimum at around pH 6-7. At alkaline pH values, the negative electrostatic potential of SDS lowers the local pH sufficiently to restore efficient heme binding, provided the amount of SDS needed for this does not denature the protein. Accordingly, the higher the pH value above 6-7, the more SDS is needed to improve heme binding, and this competes with the inherent tendency of SDS to dissociate cytochrome b₅₅₉′. Our work highlights that, in addition to its denaturing properties, SDS can affect protein functions by lowering the local pH.     

Factors affecting germline mutations in a hypervariable microsatellite: a comparative analysis of six species of swallows (Aves: Hirundinidae)

Microsatellites mutate frequently by replication slippage. Empirical evidence shows that the probability of such slippage mutations may increase with the length of the repeat region as well as exposure to environmental mutagens, but the mutation rate can also differ between the male and female germline. It has been hypothesized that more intense sexual selection or sperm competition can also lead to elevated mutation rates, but the empirical evidence is inconclusive. Here, we analyzed the occurrence of germline slippage mutations in the hypervariable pentanucleotide microsatellite locus HrU10 across six species of swallow (Aves: Hirundinidae). These species exhibit marked differences in the length range of the microsatellite, as well as differences in the intensity of sperm competition. We found a strong effect of microsatellite length on the probability of mutation, but no residual effect of species or their level of sperm competition when the length effect was accounted for. Neither could we detect any difference in mutation rate between tree swallows (Tachycineta bicolor) breeding in Hamilton Harbour, Ontario, an industrial site with previous documentation of elevated mutation rates for minisatellite DNA, and a rural reference population. However, our cross-species analysis revealed two significant patterns of sex differences in HrU10 germline mutations: (1) mutations in longer alleles occurred typically in the male germline, those in shorter alleles in the female germline, and (2) male germline mutations were more often expansions than contractions, whereas no directional bias was evident in the female germline. These results indicate some fundamental differences in male and female gametogenesis affecting the probability of slippage mutations. Our study also reflects the value of a comparative, multi-species approach for locus-specific mutation analyses, through which a wider range of influential factors can be assessed than in single-species studies.     

Sex Hormones Selectively Impact the Endocervical Mucosal Microenvironment: Implications for HIV Transmission

Several studies suggest that progesterone and estrogens may affect HIV transmission in different, possibly opposing ways. Nonetheless, a direct comparison of their effects on the mucosal immune system has never been done. We hypothesize that sex hormones might impact the availability of cells and immune factors important in early stages of mucosal transmission, and, in doing so influence the risk of HIV acquisition. To test this hypothesis, we employed 15 ovarectomized rhesus macaques: 5 were treated with Depot Medroxy Progesterone Acetate (DMPA), 6 with 17-β estradiol (E2) and 4 were left untreated. All animals were euthanized 5 weeks after the initiation of hormone treatment, a time post-DMPA injection associated with high susceptibility to SIV infection. We found that DMPA-treated macaques exhibited higher expression of integrin α₄β₇ (α₄β₇) on CD4⁺ T cells, the gut homing receptor and a marker of cells highly susceptible to HIV, in the endocervix than did the E2-treated animals. In contrast, the frequency of CCR5⁺ CD4⁺ T cells in DMPA-treated macaques was higher than in the E2-treated group in vaginal tissue, but lower in endocervix. α₄β₇ expression on dendritic cells (DCs) was higher in the DMPA-treated group in the endocervical tissue, but lower in vaginal tissue and on blood DCs compared with the E2-treated animals. Soluble MAdCAM-1, the α₄β₇ ligand, was present in the vaginal fluids of the control and E2-treated groups, but absent in the fluids from DMPA-treated animals. Both hormones modulated the expression and release of inflammatory factors and modified the distribution of sialomucins in the endocervix. In summary, we found that sex hormones profoundly impact mucosal immune factors that are directly implicated in HIV transmission. The effect is particularly significant in the endocervix. This may increase our understanding of the potential hormone-driven modulation of HIV susceptibility and potentially guide contraceptive policies in high-risk settings.