Pharmacoproteomic analysis of topical dapsone and prednisolone interventions in the aqueous humor of anterior uveitis.

Uveitis is the inflammation of uveal tract comprising of iris, ciliary body and choroid. Blood ocular barriers maintaining the homeostasis of eye breach during uveitis, leads to high risk for sight-threatening complications. The purpose of this study was to compare the anti-inflammatory activity enabled by two diverse pharmacological agents (prednisolone and dapsone) using their effect on aqueous humor proteome. Wistar rats of either sex (150-200g) were used and randomly divided into various groups. Normal group was injected with 0.1ml normal saline (NS), endotoxin (LPS) (200 µg/0.1ml NS) was injected into endotoxin induced inflammatory groups followed by 0.1% dapsone and 1% prednisolone treatment in endotoxin induced uveitis (EIU) groups, respectively. Aqueocentesis was performed post 24 hour inflammation and samples were subjected for clinical parameter evaluation, cytokine analysis as well as global proteomic analysis using High-resolution mass spectrometer. Following which spectrum analysis, production spectra of peptides were matched against R. Norvegicus Protein Database (Uniport) using Proteome Discoverer (v2.2). Upon clinical evaluation, the anterior segment images post dapsone and prednisolone treatment have shown marked decrease in hyperaemia, miosis and iridial vessels vasodilation in rat eyes as compared to inflammation group. The result of cytokine analysis revealed 0.1% dapsone and prednisolone both significantly decreased the TNF-α levels. HRMS studies analysis expressed 140, 160, 158 and 141 proteins unique to normal, EIU, Dapsone and prednisolone group respectively. To conclude aqueous humor pharmacoproteomic revealed the anti-inflammatory activity of the dapsone comparable to the prednisolone treatment in endotoxin induced uveitis. The topical dapsone may be used as an alternative therapeutic option in treating uveitis without elevating intraocular pressure.

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states.

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2'-deoxyadenosine triphosphate (2'-dATP) (F). These were determined at 1.67Å, 1.60Å, 2.20Å, 1.70Å, 2.07Å and 1.90Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2'-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ(1)=-66°and χ(2)=165° in structures (A) and (B); (ii) χ(1)=-95° and χ(2)=70° in structures (C), (D) and (E); and (iii) χ(1)=-163° and χ(2)=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.

Modulation of inhibitory activity of xylanase-α-amylase inhibitor protein (XAIP): binding studies and crystal structure determination of XAIP-II from Scadoxus multiflorus at 1.2 Å resolution.

BACKGROUND: Plants produce a wide range of proteinaceous inhibitors to protect themselves against hydrolytic enzymes. Recently a novel protein XAIP belonging to a new sub-family (GH18C) was reported to inhibit two structurally unrelated enzymes xylanase GH11 and α-amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of α-amylase GH13. A new form of XAIP (XAIP-II) that inhibits α-amylase GH13 with a greater potency than that of XAIP and xylanase GH11 with a lower potency than that of XAIP, has been identified in the extracts of underground bulbs of Scadoxus multiflorus. This kind of occurrence of isoforms of inhibitor proteins is a rare observation and offers new opportunities for understanding the principles of protein engineering by nature. RESULTS: In order to determine the structural basis of the enhanced potency of XAIP-II against α-amylase GH13 and its reduced potency against xylanase GH11 as compared to that of XAIP, we have purified XAIP-II to homogeneity and obtained its complete amino acid sequence using cloning procedure. It has been crystallized with 0.1 M ammonium sulphate as the precipitating agent and the three-dimensional structure has been determined at 1.2 Å resolution. The binding studies of XAIP-II with xylanase GH11 and α-amylase GH13 have been carried out with surface plasmon resonance (SPR). CONCLUSION: The structure determination revealed that XAIP-II adopts the well known TIM barrel fold. The xylanase GH11 binding site in XAIP-II is formed mainly with loop α3-ß3 (residues, 102 - 118) which has acquired a stereochemically less favorable conformation for binding to xylanase GH11 because of the addition of an extra residue, Ala105 and due to replacements of two important residues, His106 and Asn109 by Thr107 and Ser110. On the other hand, the α-amylase binding site, which consists of α-helices α6 (residues, 193 - 206), α7 (residues, 230 - 243) and loop ß6-α6 (residues, 180 - 192) adopts a stereochemically more favorable conformation due to replacements of residues, Ser190, Gly191 and Glu194 by Ala191, Ser192 and Ser195 respectively in α-helix α6, Glu231 and His236 by Thr232 and Ser237 respectively in α-helix α7. As a result, XAIP-II binds to xylanase GH11 less favorably while it interacts more strongly with α-amylase GH13 as compared to XAIP. These observations correlate well with the values of 4.2 × 10(-6) M and 3.4 × 10(-8) M for the dissociation constants of XAIP-II with xylanase GH11 and α-amylase GH13 respectively and those of 4.5 × 10(-7) M and 3.6 × 10(-6) M of XAIP with xylanase GH11 and α-amylase GH13 respectively.

Crystal structure determination and inhibition studies of a novel xylanase and alpha-amylase inhibitor protein (XAIP) from Scadoxus multiflorus.

A novel plant protein isolated from the underground bulbs of Scadoxus multiflorus, xylanase and alpha-amylase inhibitor protein (XAIP), inhibits two structurally and functionally unrelated enzymes: xylanase and alpha-amylase. The mature protein contains 272 amino acid residues which show sequence identities of 48% to the plant chitinase hevamine and 36% to xylanase inhibitor protein-I, a double-headed inhibitor of GH10 and GH11 xylanases. However, unlike hevamine, it is enzymatically inactive and, unlike xylanase inhibitor protein-I, it inhibits two functionally different classes of enzyme. The crystal structure of XAIP has been determined at 2.0 A resolution and refined to R(cryst) and R(free) factors of 15.2% and 18.6%, respectively. The polypeptide chain of XAIP adopts a modified triosephosphate isomerase barrel fold with eight beta-strands in the inner circle and nine alpha-helices forming the outer ring. The structure contains three cis peptide bonds: Gly33-Phe34, Tyr159-Pro160 and Trp253-Asp254. Although hevamine has a long accessible carbohydrate-binding channel, in XAIP this channel is almost completely filled with the side-chains of residues Phe13, Pro77, Lys78 and Trp253. Solution studies indicate that XAIP inhibits GH11 family xylanases and GH13 family alpha-amylases through two independent binding sites located on opposite surfaces of the protein. Comparison of the structure of XAIP with that of xylanase inhibitor protein-I, and docking studies, suggest that loops alpha3-beta4 and alpha4-beta5 may be involved in the binding of GH11 xylanase, and that helix alpha7 and loop beta6-alpha6 are suitable for the interaction with alpha-amylase.

Structural evidence of substrate specificity in mammalian peroxidases: structure of the thiocyanate complex with lactoperoxidase and its interactions at 2.4 A resolution.

The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN(-)) has been determined at 2.4A resolution. It revealed that the SCN(-) ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN(-) ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN(-) forms a hydrogen bond with a water (Wat) molecule at position 6'. This water molecule is stabilized by two hydrogen bonds with Gln(423) N(epsilon2) and Phe(422) oxygen. In contrast, the placement of the SCN(-) ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln(423), Phe(422) oxygen, and Wat(6)' in LPO is occupied primarily by the side chain of Phe(407) in MPO due to an entirely different conformation of the loop corresponding to the segment Arg(418)-Phe(431) of LPO. This arrangement in MPO does not favor a similar orientation of the SCN(-) ion. The orientation of the catalytic product OSCN(-) as reported in the structure of LPO.OSCN(-) is similar to the orientation of SCN(-) in the structure of LPO.SCN(-). Similarly, in the structure of LPO.SCN(-).CN(-), in which CN(-) binds at Wat(1), the position and orientation of the SCN(-) ion are also identical to that observed in the structure of LPO.SCN.

Sequence and stability of the goat cytochrome c.

We have determined the sequence of mitochondrial cytochrome c (cyt-c) from the goat heart, and it was found to have a unique amino acid sequence among all amino acid sequences of cyt-c reported till date. Its sequence alignment with the bovine cytochrome c (b-cyt-c) led us to conclude that the goat cytochrome c (g-cyt-c) differs in amino acid sequence from b-cyt-c at only one position, i.e., Pro44(bovine) --> Ala44(goat). It has been observed that guanidinium chloride (GdmCl) induces a two-state transition between the native (N) and denatured (D) states of g-cyt-c. This conclusion is reached from the coincidence of GdmCl-induced transition curves monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. Analysis of denaturation curves for the Gibbs energy of stabilization suggests that the stability of g-cyt-c is, within experimental errors, identical to that of b-cyt-c. We have also measured the effect of temperature on the equilibrium, N state <--> D state of g-cyt-c in the presence of different GdmCl concentrations. These measurements gave values of transition temperature (T(m)), changes in enthalpy (DeltaH(m)) and heat capacity (DeltaC(p)) of g-cyt-c in the absence of GdmCl, which are compared with those of b-cyt-c. We have used crystal structure coordinates of b-cyt-c to predict the structure and stability of g-cyt-c, which are compared with those of the bovine protein.

Crystal structure of lactoperoxidase at 2.4 A resolution.

Lactoperoxidase (LPO) is a member of the mammalian peroxidase superfamily. It catalyzes the oxidation of thiocyanate and halides. Freshly isolated and purified samples of caprine LPO were saturated with ammonium iodide and crystallized using 20% polyethylene glycol 3350 in a hanging drop vapor diffusion setup. The structure has been determined using X-ray crystallographic method and refined to R(cryst) and R(free) factors of 0.196 and 0.203, respectively. The structure determination revealed an unexpected phosphorylation of Ser198 in LPO, which is also confirmed by anti-phosphoserine antibody binding studies. The structure is also notable for observing densities for glycan chains at all the four potential glycosylation sites. Caprine LPO consists of a single polypeptide chain of 595 amino acid residues and folds into an oval-shaped structure. The structure contains 20 well-defined alpha-helices of varying lengths including a helix, H(2a), unique to LPO, and two short antiparallel beta-strands. The structure confirms that the heme group is covalently linked to the protein through two ester linkages involving carboxylic groups of Glu258 and Asp108 and modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is slightly distorted from planarity, but pyrrole ring B is distorted considerably. However, an iron atom is displaced only by 0.1 A from the plane of the heme group toward the proximal site. The substrate diffusing channel in LPO is cylindrical in shape with a diameter of approximately 6 A. Two histidine residues and six buried water molecules are connected through a hydrogen-bonded chain from the distal heme cavity to the surface of protein molecule and seemingly form the basis of proton relay for catalytic action. Ten iodide ions have been observed in the structure. Out of these, only one iodide ion is located in the distal heme cavity and is hydrogen bonded to the water molecule W1. W1 is also hydrogen bonded to the heme iron as well as to distal His109. The structure contains a calcium ion that is coordinated to seven oxygen atoms and forms a typical pentagonal bipyramidal coordination geometry.

Structure of a bovine secretory signalling glycoprotein (SPC-40) at 2.1 Angstrom resolution.

A recently discovered new class of 40 kDa glycoproteins forms a major component of the secretory proteins in the dry secretions of non-lactating animals. These proteins are implicated as protective signalling factors that determine which cells are to survive during the processes of drastic tissue remodelling. In order to understand its role in the remodelling of mammary glands, the detailed three-dimensional structure of the bovine signalling glycoprotein (SPC-40) has been determined using X-ray crystallography. SPC-40 was purified from bovine dry secretions and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.6, b = 67.4, c = 106.9 Angstrom. The protein was also cloned in order to determine its complete amino-acid sequence. Its three-dimensional structure has been determined using data to 2.1 Angstrom resolution. The amino-acid sequence determination of SPC-40 reveals two potential N-glycosylation sites at Asn39 and Asn345, but electron density for a glycan chain was only present at Asn39. The protein adopts a conformation with the classical (beta/alpha)(8)-barrel fold of triosephosphate isomerase (TIM barrel; residues 1-237 and 310-360) with the insertion of a small alpha+beta domain (residues 240-307) similar to that observed in chitinases. However, the substitution of Leu for Glu in the consensus catalytic sequence in SPC-40 caused a loss of chitinase activity. Furthermore, the chitin-binding groove in SPC-40 is considerably distorted owing to unfavourable conformations of several residues, including Trp78, Tyr120, Asp186 and Arg242. Three surface loops, His188-His197, Phe202-Arg212 and Tyr244-Pro260, have exceptionally high B factors, suggesting large-scale flexibility. Fluorescence studies indicate that various sugars bind to SPC-40 with low affinities.