Association study of TRAF1-C5 polymorphisms with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese.

OBJECTIVE: The primary aim of this study was to investigate the association of polymorphisms of TRAF1-C5, a newly identified rheumatoid arthritis (RA) risk locus in Caucasians, with susceptibility to RA and systemic lupus erythematosus (SLE) in Japanese populations. Gene expression levels of TRAF1 and C5 to assess the functional significance of genotypes were also analysed. METHODS: A multicentre association study consisting of 4 RA case-control series (4397 cases and 2857 controls) and 3 SLE case-control series (591 cases and 2199 shared controls) was conducted. Genotyping was performed using TaqMan genotyping assay for two single nucleotide polymorphisms (SNPs) that showed the best evidence of association in the previous Caucasian studies. Quantifications of TRAF1 and C5 expression were performed with TaqMan expression assay. RESULTS: Significant differences in allele frequency for both SNPs were observed between RA and control subjects (combined odds ratio = 1.09), while no significant difference was detected between patients with SLE and controls. Interestingly, alleles rs3761847 A and rs10818488 G had increased the risk for RA in the present study, while they decreased the risk in the original studies. A significant difference was found between risk allele carriers and non-carriers of rs10818488 for the expression level of TRAF1 in phorbol myristate acetate-stimulated lymphoblastoid cell lines (p = 0.04). CONCLUSION: Association of TRAF1-C5 locus with RA susceptibility was detected in the Japanese populations with modest magnitude, while no significant association was observed for SLE. Significant positive effect of genotype on the expression of TRAF1 might support the genetic association between TRAF1 and RA.

Intraocular Lymphoma or Infection? Subretinal Aspirate Confirms the Diagnosis.

PURPOSE: To demonstrate the importance of subretinal biopsy to reach a diagnosis when vitreous biopsy is negative or inconclusive. METHODS: A 54-year-old Caucasian gentleman presented with bilateral anterior uveitis at JCUH. He initially responded to topical steroids and dilating agents. Subsequently he developed bilateral panuveitis and cataract with poor response to treatment. Detailed workup had been done to rule out infectious etiology. A suspicion of lymphoma was considered and vitreous biopsy sample was taken from one eye, which was inconclusive. Then, to help with definitive diagnosis vitreous sample, subretinal aspirate and retinal biopsy were taken. RESULTS: Subretinal aspirate revealed Aspergillus niger. Treatment was initiated accordingly. CONCLUSIONS: Subretinal aspirate and retinal biopsy can help with diagnosis of unusual clinical panuveitis like presentation.

Nanoscale Dynamics of Protein Assembly Networks in Supersaturated Solutions.

Proteins in solution are conventionally considered macromolecules. Dynamic microscopic structures in supersaturated protein solutions have received increasing attention in the study of protein crystallisation and the formation of misfolded aggregates. Here, we present a method for observing rotational dynamic structures that can detect the interaction of nanoscale lysozyme protein networks via diffracted X-ray tracking (DXT). Our DXT analysis demonstrated that the rearrangement behaviours of lysozyme networks or clusters, which are driven by local density and concentration fluctuations, generate force fields on the femtonewton to attonewton (fN - aN) scale. This quantitative parameter was previously observed in our experiments on supersaturated inorganic solutions. This commonality provides a way to clarify the solution structures of a variety of supersaturated solutions as well as to control nucleation and crystallisation in supersaturated solutions.

CT-based morphological assessment of the hip joint in Japanese patients: association with radiographic predictors of femoroacetabular impingement.

AIMS: Femoroacetabular impingement (FAI) has been highlighted and well documented primarily in Western countries and there are few large studies focused on FAI-related morphological assessment in Asian patients. We chose to investigate this subject. PATIENTS AND METHODS: We assessed the morphology of the hip and the prevalence of radiographic FAI in Japanese patients by measuring predictors of FAI. We reviewed a total of 1178 hips in 695 men and 483 women with a mean age of 58.2 years (20 to 89) using CT images that had been obtained for reasons unrelated to symptoms from the hip. We measured the lateral centre edge angle, acetabular index, crossover sign, alpha angle and anterior femoral head-neck offset ratio. RESULTS: A total of 441 hips (37.4%) had pincer-type deformity (41.7% men, 31.3% women) and 534 (45.3%) had cam-type deformity (54.4% men, 32.3% women). Moreover, 773 hips (65.6%) had at least one parameter that predisposes to FAI (74.0% men, 53.6% women) and 424 hips (36.0%) had two or more parameters (43.6% men, 25.0% women). CONCLUSION: The prevalence of radiographic FAI was common in Japanese patients who are generally considered to have dysplastic hips. Cite this article: Bone Joint J 2016;98-B:1167-74.

The equilibrium intermediate of beta-lactoglobulin with non-native alpha-helical structure.

It is generally considered that intermediates of protein folding contain partially formed native-like secondary structures. In contrast, we recently reported that the kinetic folding intermediate of bovine beta-lactoglobulin contains non-native alpha-helical structures. To understand the mechanism that stabilizes the non-native intermediate, we characterized by circular dichroism (CD) the equilibrium unfolding transition of beta-lactoglobulin induced by guanidine hydrochloride (Gdn-HCl) at pH 2 and 4 degrees C. The unfolding transition measured by near-UV CD preceded the transition measured by far-UV CD, indicating the accumulation of the intermediate state. The far-UV CD spectrum of the intermediate, obtained by global fitting analysis of the CD spectra in the presence of various concentrations of Gdn-HCl, was similar to the burst-phase intermediate observed in the refolding kinetics, and contained non-native alpha-helical structures. Addition of 10% (v/v) 2,2,2-trifluoroethanol (TFE) increased the helical content of the equilibrium intermediate, although the protein still assumed the native structure in the absence of Gdn-HCl. A phase diagram of the conformational states, i.e. the alpha-helical intermediate, unfolded and native states, against the concentration of TFE and Gdn-HCl was constructed. This indicated that, because of the high helical preference of the amino acid sequence of beta-lactoglobulin, the helical region protrudes into the boundary between the native and unfolded states, resulting in non-monotonic accumulation of the helical intermediate upon equilibrium unfolding of the native beta-sheet structure. This is the first observation to indicate that a non-native alpha-helical intermediate accumulates during equilibrium unfolding of a predominantly beta-sheet protein.

High helical propensity of the peptide fragments derived from beta-lactoglobulin, a predominantly beta-sheet protein.

It is generally assumed that peptide fragments derived from globular proteins are either unfolded, or adopt native-like secondary structures, in particular alpha-helix, which are similar to those occurring in the early stages of protein folding. Since the structured conformations of short peptides are unstable, 2,2,2-trifluoroethanol (TFE) is often used to stabilize them. To examine the folding process of beta-proteins, we synthesized three fragments of beta-lactoglobulin corresponding to two beta-strands and one helix region, and examined their conformation by circular dichroism and nuclear magnetic resonance. These regions were chosen because, according to secondary structure prediction, all three should have high helix propensities. In aqueous solution, the three peptides had only a little ordered structure, but when TFE was added, they exhibited marked helical propensities, as also observed for the whole molecule of beta-lactoglobulin. These results indicate that the intrinsic helical propensity of a peptide fragment elucidated by the addition of TFE is not necessarily related to the secondary structure in the native state. The results further suggest a case of non-hierarchical protein folding model, in which non-native structures may be involved in the early stage of folding.

Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c.

One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. Its stability against heat or denaturants was much less than that of the intact protein, and similar to that of the acidic molten globule state of the holoprotein. These results indicate that, at neutral pH, the ligation of His18 of the iron is important for the maintenance of the native structure whereas the Met80 ligation is not essential, and that porphyrin-cytochrome c assumes a molten globule-like state. Porphyrin-cytochrome c was largely unfolded at pH 2.0 in the absence of salt, but assumed another molten globule-like structure in the presence of anions. The salt-induced stabilization of the molten globule-like state was the same as that of apocytochrome c, requiring a much higher salt concentration than holocytochrome c. These results indicate that, at acidic pH, the His18 ligation is important, although not essential, for stabilizing the molten globule state. Taken together, both specific (i.e. the His18 axial ligand, as observed at acidic pH) and nonspecific interactions (the hydrophobic effects of the heme, as observed at neutral pH) contribute to stabilizing the molten globule state.

Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization.

The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53-Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly a-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53-Gly69 of cAcP indicate that only the native-like trans isomer of the Gly-Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an approximately 1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53-Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.

Trifluoroethanol-induced conformational transition of hen egg-white lysozyme studied by small-angle X-ray scattering.

The trifluoroethanol (TFE)-induced conformational transition of hen lysozyme was studied with the combined use of far-UV circular dichroism (CD) and small-angle X-ray scattering. At pH 2.0 and 20 degrees C, the addition of TFE to the native lysozyme induced a cooperative transition to an intermediate state with an increased helical content (TFE state). Small-angle X-ray scattering measurements indicated that the TFE state has a radius of gyration which is 20% larger than that of the native state and assumes a chain-like conformation with some remaining globularity. The TFE-induced transition curves obtained by CD and the small-angle X-ray scattering measurements agreed well, consistent with a two-state transition mechanism. A singular value decomposition analysis of Kratky plots of the small-angle X-ray scattering profiles indicated that two basic scattering functions reproduce the observed spectra, further confirming the validity of a two-state approximation.

The effects of enriching laboratory cages using various physical structures on multiple measures of welfare in singly-housed rats.

The single housing of laboratory rats may be recommended in some situations such as hypothesis-driven or test-specific studies, during electroencephalogram recording of phases of sleep and after surgical procedures. However, as single housing of laboratory rats has been shown to be stressful, modification of the housing environment is needed to improve the welfare of these animals. This experiment was carried out to investigate the long-term effects of environmental enrichment on some behavioural, physiological, pathological and psychological measures of welfare. With two batches of animals, 24 rats were housed singly in either enriched cages (EC) (n = 12 cages) or unenriched cages (UC) (n = 12 cages). Behaviour was sampled every week and so was body weight and weight gain over a six-week observation period. Behaviours of the rats in the elevated plus-maze were recorded on the seventh week, whereas organ weights were recorded postmortem. The results revealed that long-term single housing of rats in super-enriched cages increased levels of indicators of good welfare including sleep, exploration, movement and feeding behaviour, body weights, weight gains and the relative weights of the thymus gland and spleen, and decreased levels of indicators of poor welfare such as stationary behaviour and the relative weight of adrenal glands. Thus, enrichment of conventional cages of newly weaned singly-housed laboratory rats with multiple physical structures appeared to improve their ability to control the environment and to promote their species-specific behaviour; changes that can ultimately result in good welfare.

Non-native alpha-helical intermediate in the refolding of beta-lactoglobulin, a predominantly beta-sheet protein.

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. We have studied the refolding kinetics of bovine beta-lactoglobulin using stopped-flow circular dichroism and find that a partly alpha-helical intermediate accumulates transiently before formation of the native beta-sheets. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism, in which non-native alpha-helical structures play important roles.

High helicity of peptide fragments corresponding to beta-strand regions of beta-lactoglobulin observed by 2D-NMR spectroscopy.

BACKGROUND: Whereas protein fragments, when they are structured, adopt conformations similar to that found in the native state, the high helical propensity of beta-lactoglobulin, a predominantly beta-sheet protein, suggested that the fragments of beta-lactoglobulin can assume the non-native helical conformation. In order to assess this possibility, we synthesized four 17-18-residue peptides corresponding to three beta-strand regions and one helical region (as a control) of beta-lactoglobulin and examined their conformation. RESULTS: We observed residual helicities of up to 17% in water, by far-UV CD, for all four peptide fragments. The helices could be significantly stabilized by the addition of TFE, and the NMR analyses in a mixture of 50% water/TFE indicated that helical structures are formed in the central region whereas both termini are frayed. Thus, the very same residues that form strands in the native beta-lactoglobulin showed high helical preferences. CONCLUSIONS: These results stand out from the current general view that peptide fragments isolated from proteins either are unfolded or adopt native-like secondary structures. The implications of the results in the mechanism of protein folding and in designing proteins and peptides are significant.

Evidence concerning rate-limiting steps in protein folding from the effects of trifluoroethanol.

The refolding kinetics of 13 proteins have been studied in the presence of 2,2,2-trifluoroethanol (TFE). Low concentrations of TFE increased the folding rates of all the proteins, whereas higher concentrations have the opposite effect. The extent of deceleration of folding correlates closely with similar effects of guanidine hydrochloride and can be related to the burial of accessible surface area during folding. For those proteins folding in a two-state manner, the extent of acceleration of folding correlates closely with the number of local backbone hydrogen bonds in the native structure. For those proteins that fold in a multistate manner, however, the extent of acceleration is much smaller than that predicted from the data for two-state proteins. These results support the concept that for two-state proteins the search for native-like contacts is a key aspect of the folding reaction, whereas the rate-determining steps for folding of multistate proteins are associated with the reorganization of stable structure within a collapsed state or with the search for native-like interactions within less structured regions.

Acid-induced unfolding and refolding transitions of cytochrome c: a three-state mechanism in H2O and D2O.

Whereas the salt-dependent conformational transition of acid-denatured horse ferricytochrome c at pH 2 is approximated by a two-state mechanism from the acid-unfolded state to the molten globule state [Kataoka, M., Hagihara, Y., Mihara, K., & Goto, Y. (1993) J. Mol. Biol. 229, 591-596], the corresponding transition in D2O has been proposed to involve a noncompact, alpha-helical intermediate state (the pre-molten globule state) [Jeng, M.-F., & Englander, S. W. (1991) J. Mol. Biol. 221, 1045-1061]. To examine the proposed difference in the conformational transitions, we carried out the HCl and DCl titrations of cytochrome c in H2O and D2O, respectively, measured by far-UV circular dichroism, tryptophan fluorescence, and Soret absorption. In both D2O and H2O, unfolding from the native state to the acid-unfolded state and subsequent refolding to the molten globule state were observed. In either solvent, the conformational transitions were well approximated by a minimal three-state mechanism consisting of the native, molten globule, and acid-unfolded states. Thus, our results did not substantiate the presence of a pre-molten globule state in D2O. Acetylation of amino groups of cytochrome c is known to stabilize the molten globule state at pH 2. On the basis of the three-state mechanism, we constructed a conformational phase diagram for the effect of pH and the degree of acetylation. This phase diagram was similar to that of the pH- and salt-dependent conformational transition of cytochrome c, suggesting that the effects of acetylation on the conformational states are similar to those of salt.

Salt-induced formation of the molten globule state of cytochrome c studied by isothermal titration calorimetry.

Although the molten globule state has been proposed as a major intermediate of protein folding, it has proven difficult to obtain thermodynamic data characterizing this state. To explore another approach for characterizing the molten globule state, salt-induced formation of the molten globule state of horse cytochrome c at pH 1.8 was studied by isothermal titration calorimetry. By titrating the acid-unfolded cytochrome c with sodium perchlorate, an exothermic reaction was observed. The titration curve obtained from the heat was cooperative and agreed well with the conformational transition curve measured by CD at 222 nm. This result indicated that the salt-induced conformation change is well approximated by a two-state transition between the acid-unfolded and molten globule states. The heat for formation of the molten globule state estimated by isothermal titration calorimetry was consistent with the enthalpy change for unfolding of the sodium perchlorate-stabilized molten globule state at pH 1.8, which was measured by differential scanning calorimetry and CD. These results indicate that the heat of titration largely reflects the enthalpy change of the conformational transition. From these results, we consider that isothermal titration calorimetry will become a useful approach for investigating the molten globule state.

Intermediate conformational states of apocytochrome c.

Horse apocytochrome c has been assumed to be a typical unfolded protein. At low ionic strength, the far- and near-UV circular dichroism spectra are typical of an unfolded protein at all pH values between 2 and 9. On the other hand, in the presence of high concentrations of salt, substantial secondary structure is present at both neutral and acidic pH. At low pH, perchlorate anion, either from salt or from acid, stabilizes an intermediate state (the A state) with secondary structure similar to that previously observed in the molten globule state of holocytochrome c. To further characterize the conformational states of apocytochrome c as a function of pH and salt, a fluorescence-labeled derivative was prepared, in which the two cysteine residues were labeled with N-(iodoacetyl)-N'-(5-sulfo-1- naphthyl)ethylenediamine(IAEDANS). The conformational transitions of the fluorescence-labeled apocytochrome c measured by circular dichroism were similar to those of unmodified apocytochrome c, indicating that the effects of the modification on the conformation and stability are small. Fluorescence energy transfer from tryptophan to the fluorescence label revealed several salt- and pH-dependent transitions. At very low ionic strength, apocytochrome c became compact as the pH was increased with a transition midpoint at pH 4.5. At acidic pH, increasing concentration of perchlorate induced a more compact state with a transition midpoint similar to that observed by circular dichroism. In contrast, at neutral pH, increasing perchlorate concentration had little effect on the compactness, as determined by a lack of change in the energy-transfer efficiency (but did increase the amount of secondary structure).(ABSTRACT TRUNCATED AT 250 WORDS)

Chain-like conformation of heat-denatured ribonuclease A and cytochrome c as evidenced by solution X-ray scattering.

BACKGROUND: Although the characterization of heat-denatured proteins is essential for understanding the thermodynamic mechanism of protein folding, their structural features are still unclear and controversial. In order to address this problem, we studied the size and shape of the heat-denatured states of bovine ribonuclease A (RNase A) and horse ferricytochrome c (cytochrome c) by solution X-ray scattering. RESULTS: RNase A has four disulfide bonds, whereas cytochrome c, with a covalently bound heme group, has no disulfide bond. Guinier plots show that the heat-denatured RNase A is relatively compact, but the heat-denatured cytochrome c is expanded. On the other hand, the Kratky plots of the two proteins are similar, indicating that the heat-denatured proteins assume a chain-like disordered conformation. The X-ray scattering of RNase A and cytochrome c at various temperatures confirmed that their thermal transitions from a globular native state to a chain-like extended conformation can be approximated well by a two-state transition. CONCLUSIONS: These results indicate that the heat-denatured RNase A and cytochrome c are substantially unfolded according to the criteria of solution X-ray scattering, although the heat-denatured RNase A remains compact because of the presence of the disulfide bonds. The results also confirm that the thermal denaturation occurs cooperatively with the breakdown of secondary and tertiary structure.

Acceleration of the folding of acylphosphatase by stabilization of local secondary structure.

The addition of trifluoroethanol or hexafluoroisopropanol converts the apparent two-state folding of acylphosphatase, a small alpha/beta protein, into a multistate mechanism where secondary structure accumulates significantly in the denatured state before folding to the native state. This results in a marked acceleration of folding as revealed by following the intrinsic fluorescence and circular dichroism changes upon folding. The folding rate is at a maximum when the secondary-structure content of the denatured state corresponds to that of the native state, while further stabilization of secondary structure decreases the folding rate. These findings indicate that stabilization of intermediate structure can either enhance or retard folding depending on its nature and content of native-like interactions.