Delineation of solution burst-phase protein folding events by encapsulating the proteins in silica gels.

Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine ß-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.

Quantification of individual proteins in silicone hydrogel contact lens deposits.

PURPOSE: The aim of this study was to quantify specific proteins deposited on daily wear silicone hydrogel lenses used in combination with multipurpose disinfecting solutions (MPDSs) by applying multiple-reaction-monitoring mass spectrometry (MRM-MS). METHODS: Balafilcon A or senofilcon A contact lenses used with different MPDSs on a daily wear schedule were collected. Each worn lens was extracted and then digested with trypsin. MRM-MS was applied to quantify the amounts of lysozyme, lactoferrin, lipocalin-1, proline-rich protein-4, and keratin-1 in the extracts. RESULTS: The amount of protein extracted from the contact lenses was affected by the individual wearers, lens material, and type of care system used. Higher amounts of proteins were extracted from lenses after wear when they were used with an MPDS containing polyhexamethylene biguanide (PHMB) and poloxamer 407 compared with MPDSs containing polyquaternium-1 (PQ-1)/alexidine dihydrochloride with Tetronic 904 or PQ-1/ PHMB with poloxamine and sulfobetaine (p < 0.05). There was a correlation between the amount of lipocalin-1 or keratin-1 extracted from lenses and symptoms of ocular dryness. CONCLUSIONS: The MRM-MS technique is a promising approach that could be used to reveal associations of individual proteins deposited on lenses with performance of contact lenses during wear.

The conserved disulfide bond of human tear lipocalin modulates conformation and lipid binding in a ligand selective manner.

The primary aim of this study is the elucidation of the mechanism of disulfide induced alteration of ligand binding in human tear lipocalin (TL). Disulfide bonds may act as dynamic scaffolds to regulate conformational changes that alter protein function including receptor-ligand interactions. A single disulfide bond, (Cys61-Cys153), exists in TL that is highly conserved in the lipocalin superfamily. Circular dichroism and fluorescence spectroscopies were applied to investigate the mechanism by which disulfide bond removal effects protein stability, dynamics and ligand binding properties. Although the secondary structure is not altered by disulfide elimination, TL shows decreased stability against urea denaturation. Free energy change (ΔG(0)) decreases from 4.9±0.2 to 2.1±0.3kcal/mol with removal of the disulfide bond. Furthermore, ligand binding properties of TL without the disulfide vary according to the type of ligand. The binding of a bulky ligand, NBD-cholesterol, has a decreased time constant (from 11.8±0.2 to 3.3s). In contrast, the NBD-labeled phospholipid shows a moderate decrease in the time constant for binding, from 33.2±0.2 to 22.2±0.4s. FRET experiments indicate that the hairpin CD is directly involved in modulation of both ligand binding and flexibility of TL. In TL complexed with palmitic acid (PA-TL), the distance between the residues 62 of strand D and 81 of loop EF is decreased by disulfide bond reduction. Consequently, removal of the disulfide bond boosts flexibility of the protein to reach a CD-EF loop distance (24.3Å, between residues 62 and 81), which is not accessible for the protein with an intact disulfide bond (26.2Å). The results suggest that enhanced flexibility of the protein promotes a faster accommodation of the ligand inside the cavity and an energetically favorable ligand-protein complex.

The evolutionary history of the SAL1 gene family in eutherian mammals.

BACKGROUND: SAL1 (salivary lipocalin) is a member of the OBP (Odorant Binding Protein) family and is involved in chemical sexual communication in pig. SAL1 and its relatives may be involved in pheromone and olfactory receptor binding and in pre-mating behaviour. The evolutionary history and the selective pressures acting on SAL1 and its orthologous genes have not yet been exhaustively described. The aim of the present work was to study the evolution of these genes, to elucidate the role of selective pressures in their evolution and the consequences for their functions. RESULTS: Here, we present the evolutionary history of SAL1 gene and its orthologous genes in mammals. We found that (1) SAL1 and its related genes arose in eutherian mammals with lineage-specific duplications in rodents, horse and cow and are lost in human, mouse lemur, bushbaby and orangutan, (2) the evolution of duplicated genes of horse, rat, mouse and guinea pig is driven by concerted evolution with extensive gene conversion events in mouse and guinea pig and by positive selection mainly acting on paralogous genes in horse and guinea pig, (3) positive selection was detected for amino acids involved in pheromone binding and amino acids putatively involved in olfactory receptor binding, (4) positive selection was also found for lineage, indicating a species-specific strategy for amino acid selection. CONCLUSIONS: This work provides new insights into the evolutionary history of SAL1 and its orthologs. On one hand, some genes are subject to concerted evolution and to an increase in dosage, suggesting the need for homogeneity of sequence and function in certain species. On the other hand, positive selection plays a role in the diversification of the functions of the family and in lineage, suggesting adaptive evolution, with possible consequences for speciation and for the reinforcement of prezygotic barriers.

Two newly identified cat allergens: the von Ebner gland protein Fel d 7 and the latherin-like protein Fel d 8.

INTRODUCTION: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. MATERIALS AND METHODS: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. RESULTS: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. DISCUSSION: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

pH-Dependent conformational changes in tear lipocalin by site-directed tryptophan fluorescence.

Tear lipocalin (TL), a major protein of human tears, binds a broad array of endogenous ligands. pH-dependent ligand binding in TL may have functional implications in tears. Previously, conformational selections of the AB and GH loops have been implicated in ligand binding by site-directed tryptophan fluorescence (SDTF). In this study, SDTF was applied to the AB and GH loops to investigate pH-driven conformational changes relevant to ligand binding. Both loops demonstrate significant but distinct conformational rearrangements over a wide pH range. In the low-pH transition, from 7.3 to 3.0, residues of the GH loop exhibit decreased solvent accessibilities. In acrylamide quenching experiments, the average quenching rate constant (k(q), accessibility parameter) of the residues in the GH loop is decreased approximately 38%, from 2.1 x 10(9) to 1.3 x 10(9) M(-1) s(-1). However, despite the significant changes in accessibilities for some residues in the AB loop, the average accessibility per residue remained unchanged (average k(q) = 1.2 M(-1) s(-1)). Accordingly, the low-pH transition induces conformational changes that reshuffle the accessibility profiles of the residues in the AB loop. A significant difference in the titration curves between the holo and apo forms of the W28 mutant suggests that the protonation states of the residues around position 28 modulate conformational switches of the AB loop relevant to ligand binding.

Intracavitary ligand distribution in tear lipocalin by site-directed tryptophan fluorescence.

Site-directed tryptophan fluorescence has been successfully used to determine the solution structure of tear lipocalin. Here, the technique is extended to measure the binding energy landscape. Single Trp mutants of tear lipocalin are bound to the native ligand and an analogue tagged with a quencher group to both populate and discriminate the excited protein states. Steady-state and time-resolved fluorescence quenching data reveal the intracavitary state of the ligand. The static components of fluorescence quenching identify the residues where nonfluorescence complexes form. An asymmetric distribution of the ligand within the cavity reflects the complex energy landscape of the excited protein states. These findings suggest that the excited protein states are not unique but consist of many substates. The roughness of the binding energy landscape is about 2.5kBT. The excited protein states originate primarily from conformational selections of loops AB and GH, a portal region. In contrast to static quenching, the dynamic components of fluorescence quenching by the ligand are relevant to both local side chain and ligand dynamics. Apparent bimolecular rate constants for collisional quenching of Trp by the nitroxide moiety are approximately 1 / 5 x 10(12) M(-1) s(-1). Estimations made for effective ligand concentrations establish actual rate constants on the order of 12 x 10(9) M(-1) s(-1). Prior to exit from the cavity of the protein, ligands explore binding sites in nanoseconds. Although microsecond fluctuations are rate-limiting processes in ligand binding for many proteins, accompanying nanosecond motion may be necessary for propagation of ligand binding.

Non-native alpha-helix formation is not necessary for folding of lipocalin: comparison of burst-phase folding between tear lipocalin and beta-lactoglobulin.

Tear lipocalin and beta-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of beta-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence.

Identification of LCN1 as a Potential Biomarker for Breast Cancer by Bioinformatic Analysis.

The biological functions of lipocalin-1 (LCN1) are involved in innate immune responses and act as a physiological scavenger of potentially harmful lipophilic molecules. However, the relevance of LCN1 with cancer is rarely concerned currently. The aim of this study is to address the relevance of LCN1 with BRCA by bioinformatics. In this study, we found that the expressions of LCN1 increased significantly in various cancerous tissues, including BRCA, compared with their adjacent normal tissues through the TIMER database. Furthermore, UALCAN database analysis showed that the expression of LCN1 increased gradually from stage 1 to stage 4 and was upregulated in BRCA patients with different races and subtypes compared with that in the normal. In addition, those patients with perimenopause and postmenopause status displayed higher LCN1 expression. Importantly, LCN1 genetic alterations, including copy number amplification, deep deletion, and missense mutation, could be found, and the alteration frequency showed difference in various invasive BRCA through cBioPortal database. Moreover, a positive correlation between LCN1 somatic copy number alterations and immune cell enrichments was revealed in basal like BRCA by GISTIC 2.0. Finally, analysis on prognostic value of LCN1 by Kaplan-Meier plotter showed that low LCN1 expression correlated with poor prognosis for relapse-free survival in all types of BRCA, overall survival in luminal B BRCA, distant metastasis free survival in human epithelial growth factor receptor-2 (HER2) positive BRCA, and postprogression survival (PPS) in luminal A BRCA. But high LCN1 expression also displayed poor prognosis for PPS in HER2 positive BRCA. The results together verified the significance of LCN1 in BRCA, suggesting that it may be a potential biomarker for BRCA diagnosis.

Sex hormone regulation of tear lipocalin in the rabbit lacrimal gland.

Tear lipocalin (TL) (approximately 18 kDa), a member of the lipocalin superfamily, has been identified as one of the major proteins present in rabbit lacrimal fluid. The concentration of TL has been found to be decreased in the tears of patients with dry eye disease. Lacrimal gland insufficiency, one of the major causes of dry eye disease, is known to affect mainly postmenopausal women, where there is a significant decrease in the production of androgen and estrogen. These observations suggest that sex hormones might influence dry eye indirectly by regulating the expression of TL. The purpose of this study was to determine: (1) the effect of sexual maturation on the expression of TL; and (2) if the expression of TL is regulated by the estrogen, 17beta-estradiol, and/or the androgen, dihydrotestosterone, in sexually mature female rabbits. Lacrimal fluid (LF) and lacrimal gland soluble fraction (Si) was collected from juvenile (2 kg) and sexually mature (4 kg) male and female New Zealand white (NZW) rabbits. In addition, LF and Si were collected from 4 kg rabbits, 7 days after being either sham operated (control), ovariectomized (OVX), ovariectomized treated with estrogen (OVX+E) or ovariectomized treated with dihydrotestosterone (OVX+DHT). Samples were analyzed for protein levels of TL by SDS-PAGE and Western blotting using a polyclonal rat anti-rabbit TL antibody. Densitometry analysis showed that TL protein levels in both LF and Si increased with age in male and female rabbits. In addition, TL protein levels were significantly higher in the sexually mature 4 kg male compared with the 4 kg female, while no significant difference in TL protein levels were seen among the juvenile male and female rabbits. Furthermore, ovariectomy decreased the protein levels of TL in LF and Si fraction by 50% and 20% respectively, compared with control values. Estrogen treatment increased TL protein levels by 30% and 50% in the LF and Si fraction respectively, compared with the sham operated group. DHT treatment also increased TL protein levels by approximately 150% in both LF and Si fraction compared with control values. These results support the hypothesis that sex hormones influence TL protein levels in rabbit lacrimal glands. The possibility of a role of TL in dry eye needs to be further investigated.

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin.

The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the (1)L(a) and (1)L(b) components. The relative intensities of the 0-0 transitions of the (1)L(b) bands correlate linearly with the solvent polarity values (E(T)(N)). A novel strategy that uses a set of the experimental (1)L(b) bands was employed to resolve the near-UV circular dichroism (CD) spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence and structural data of TL. Analysis of the (1)L(b) bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The dimethyl sulfoxide (DMSO)-like (1)L(b) band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method, which we call site-directed circular dichroism, is broadly applicable to various proteins to obtain the position-specific data.

Double tryptophan exciton probe to gauge proximal side chains in proteins: augmentation at low temperature.

The circular dichroic (CD) exciton couplet between tryptophans and/or tyrosines offers the potential to probe distances within 10 Å in proteins. The exciton effect has been used with native chromophores in critical positions in a few proteins. Here, site-directed mutagenesis created double tryptophan probes for key sites of a protein (tear lipocalin). For tear lipocalin, the crystal and solution structures are concordant in both apo- and holo-forms. Double tryptophan substitutions were performed at sites that could probe conformation and were likely within 10 Å. Far-UV CD spectra of double Trp mutants were performed with controls that had noninteracting substituted tryptophans. Low temperature (77 K) was tested for augmentation of the exciton signal. Exciton coupling appeared with tryptophan substitutions at positions within loop A-B (28 and 31, 33), between loop A-B (28) and strand G (103 and 105), as well as between the strands B (35) and C (56). The CD exciton couplet signals were amplified 3-5-fold at 77 K. The results were concordant with close distances in crystal and solution structures. The exciton couplets had functional significance and correctly assigned the holo-conformation. The methodology creates an effective probe to identify proximal amino acids in a variety of motifs.

Excited protein states of human tear lipocalin for low- and high-affinity ligand binding revealed by functional AB loop motion.

Human tear lipocalin (TL), a prominent member of lipocalin family, exhibits functional and structural promiscuity. The plasticity of loop regions modulates entry to the ligand pocket at the "open" end of the eight-stranded beta-barrel. Site-directed multi-distance measurements using fluorescence resonance energy transfer between functional loops register two excited protein states for low- and high-affinity ligand binding. At low pH, the longest loop AB adopts the conformation of the low-affinity excited protein state that matches the crystal structure of holo-TL at pH 8. A "crankshaft" like movement is detected for the loop AB in a low pH transition. At pH 7.3 the holo-protein assumes a high-affinity excited protein state, in which the loop AB is more compact (RMS=3.1A). In the apo-holo transition, the reporter Trp 28 moves about 4.5A that reflects a decrease in distance between Glu27 and Lys108. This interaction fixes the loop AB conformation for the high-affinity mode. No such movement is detected at low pH, where Glu27 is protonated. Data strongly indicate that the protonation state of Glu27 modulates the conformation of the loop AB for high- and low-affinity binding.

Studies on the pathogenesis of avascular retina and neovascularization into the vitreous in peripheral severe retinopathy of prematurity (an american ophthalmological society thesis).

PURPOSE: To study vascular endothelial growth factor (VEGF) regulation in the development of intravitreous neovascularization and peripheral avascular retina in peripheral severe retinopathy of prematurity (ROP). METHODS: The rat 50/10 model of ROP mimics zone II, stage 3 severe ROP and recreates fluctuations in transcutaneous oxygen levels in preterm infants. On postnatal (p) day ages p0, p8, p11-p14, and p18, retinas from the model or room-air (RA) age-matched pups were analyzed for mRNA of VEGF splice variants and receptors using real-time polymerase chain reaction or VEGF protein using enzyme-linked immunosorbent assay. RESULTS: On p14, when retinas were only 70% vascularized in the model but fully vascularized in RA, VEGF164 expression was threefold greater in the model compared to RA. On p18, intravitreous neovascularization was associated with a 5-fold increase in VEGF164 mRNA in the model compared to RA. By analysis of variance, VEGF164 and VEGFR2 mRNAs were up-regulated in association with increasing developmental age (P<.0001 for both comparisons) or exposure to the model compared to RA (P<.0001 and P=.0247, respectively), whereas increasing developmental age was associated only with up-regulated VEGF120 (P=.0006), VEGF188 (P=.0256), and VEGFR1 (P<.0001) mRNAs. VEGF protein increased significantly in the model and on p14 and p18 compared to RA (P<.0001). CONCLUSIONS: The model mimics contemporary severe ROP in the United States unlike other models of oxygen-induced retinopathy. Compared to RA retinas, VEGF significantly increased in association with avascular retina and intravitreous neovascularization. A hypothesis is proposed that VEGF up-regulation plays a role in the development of both important features.

Fatty acids bound to recombinant tear lipocalin and their role in structural stabilization.

A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analysed by gas chromatography, mass spectroscopy and nuclear magnetic resonance spectroscopy. Five major fatty acids were identified as hexadecanoic acid (palmitic acid, PA), cis-9-hexadecenoic acid (palmitoleic acid), 9,10-methylenehexadecanoic acid, cis-11-octadecenoic acid (vaccenic acid) and 11,12-methyleneoctadecanoic acid (lactobacillic acid). The composition of the bound fatty acids was similar to the fatty acid composition of E. coli extract, suggesting that the binding affinities are similar for these fatty acids. The urea-induced and thermal-unfolding transitions of the holoprotein (nondelipidated), apoprotein (delipidated) and PA-bound protein were observed by circular dichroism. Holoproteins and PA-bound proteins showed the same stability against urea and heat, and were more stable than apoprotein. These results show that each bound fatty acid stabilizes recombinant tear lipocalin to a similar extent.