A genome-wide association study suggests that MAPK14 is associated with diabetic foot ulcers.

BACKGROUND: Diabetic foot ulcers (DFUs) are a devastating complication of diabetes. OBJECTIVES: To identify genetic contributors to the development of DFUs in the presence of peripheral neuropathy in a Scottish cohort with diabetes using a genome-wide association study. METHODS: A genome-wide association approach was applied. A case was defined as a person with diabetes (type 1 or type 2) who had ever had a foot ulcer (current or previous) in at least one foot, as well as a positive monofilament test result (i.e. evidence of peripheral neuropathy) recorded in their longitudinal e-health records. A control was defined as an individual with diabetes (type 1 or type 2) who has never been recorded as having a foot ulcer in either foot but who had a positive monofilament test result recorded in either foot in their longitudinal e-health records. RESULTS: There were 699 DFU cases and 2695 controls in the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) dataset. The single-nucleotide polymorphism rs80028505 (Chr6p21·31) in MAPK14 reached genome-wide significance with a lowest P-value of 2·45 × 10-8 . The narrow-sense heritability of this phenotype is 0·06. CONCLUSIONS: We suggest that MAPK14 is associated with DFUs.

Association of Toll-like receptor 4 (TLR4) with chronic plaque type psoriasis and psoriatic arthritis.

Family studies have provided overwhelming evidence for an underlying genetic component to psoriasis. Toll-like receptors (TLRs) are key transmembrane proteins in both the innate and adaptive immune responses which are known to be integral processes in psoriasis. Recent functional studies support this notion having suggested a role for TLR4 in the pathogenesis of psoriasis. Furthermore a missense polymorphism in the TLR4 gene has been associated with a number of autoimmune conditions, including Crohn diseases, making TLR4 a viable candidate gene for investigation. The aim of this study was to investigate polymorphisms across the TLR4 region with a high-density single nucleotide polymorphism (SNP) panel in a large cohort of patients with chronic plaque type psoriasis. Twenty SNPs were successfully genotyped using Sequenom iPLEX Gold platform in 2826 UK chronic plaque type psoriasis patients including subgroup data on presence of confirmed psoriatic arthritis (n = 1839) and early-onset psoriasis (n = 1466) was available. Allele frequencies for psoriasis patients were compared against imputed Wellcome Trust Case Control Consortium controls (n = 4861). Significant association was observed between a missense variant rs4986790 of TLR4 (Asp229Gly) and plaque type psoriasis (p = 2 × 10(-4)) which was also notable in those with psoriatic arthritis (p = 2 × 10(-4)) and early-onset psoriasis (p = 8 × 10(-4)). We present data suggestive of an association between a functional variant and an intronic variant of TLR4 and chronic plaque type psoriasis and psoriatic arthritis. However, validation of this association in independent cohorts will be necessary.

Identification of loci associated with late-onset psoriasis using dense genotyping of immune-related regions.

BACKGROUND: Chronic plaque psoriasis can be subdivided into two groups according to the age of onset: type 1 (early onset, before 40 years) and type 2 (late onset, at or beyond 40 years). So far, 36 genetic loci have been associated with early-onset psoriasis in genome-wide association studies of white populations, while few studies have investigated genetic susceptibility to late-onset psoriasis. OBJECTIVES: To characterize the genetics underpinning late-onset psoriasis. METHODS: We genotyped 543 cases of late-onset psoriasis and 4373 healthy controls using the Immunochip array, a dense genotyping chip containing single-nucleotide polymorphisms previously associated with autoimmune diseases. Imputation using SNP2HLA and stepwise logistic regression analysis was performed for markers spanning the human leucocyte antigen gene region. RESULTS: Two loci (HLA-C and IL12B) previously associated with early-onset psoriasis showed significant association at a genome-wide threshold in the current study (P < 5 × 10(-8)). Six more loci (TRAF3IP2, IL23R, RNF114, IFIH1, IL23A and HLA-A) showed study-wide significant association (P < 2·3 × 10(-5); calculated using Genetic type 1 error calculator). Additionally, we identified an association at IL1R1 on chromosome 2q13, which is not associated with early-onset disease. CONCLUSIONS: This is the largest study to date of genetic loci in late-onset psoriasis, and demonstrates the overlap that exists with early-onset psoriasis. It also suggests that some loci are associated exclusively with late-onset psoriasis.

Genetic susceptibility to psoriasis and psoriatic arthritis: implications for therapy.

The era of genome-wide association studies has revolutionized the search for genetic susceptibility loci in complex genetic conditions such as psoriasis. There are currently 16 loci with confirmed evidence for association with psoriasis susceptibility but there is the potential for more to be discovered as the genetic heritability of the disease has not yet been fully explained. Many of the associated loci overlap with those for psoriatic arthritis. In contrast to psoriasis susceptibility, few studies have been performed to identify predictors of drug response in psoriasis. As large-scale collaborations and registries for psoriasis and psoriatic arthritis are established, it is likely that a genome-wide approach may be used as a more effective method of searching for genetic predictors of treatment response. However, candidate gene studies will still have a role; for example, it is likely that some disease susceptibility genes will also be markers of treatment response, based on evidence from other diseases. This review summarizes recent advances in investigating the role genetics plays in psoriasis susceptibility and contrasts these to advances made in psoriatic arthritis. Further, it describes the genetics of treatment response in the two diseases and indicates how susceptibility loci could be used to identify drug response in the future.

Self-help cessation programs for smokeless tobacco users: long-term follow-up of a randomized trial.

This paper presents long-term outcomes of the largest clinical trial of smokeless tobacco (SLT) cessation reported to date. SLT users in five northwestern states were recruited to call a toll-free number, and 1,069 users were randomized to one of two self-help conditions: either a manual-only condition or an assisted self-help condition, which included the manual, a targeted video, and two support phone calls. Significant between-group differences were not found for either the 12- or 18-month point-prevalence measure of abstinence from either SLT only or all tobacco products using outcomes based on either the responder or intention-to-treat outcomes. However, using a repeated point-prevalence measure across all three assessment points, we found that significantly more assisted self-help participants reported abstinence, compared with manual-only participants. Compared with manual-only participants, those in the assisted self-help condition were significantly more likely to use recommended cessation techniques. Results demonstrate that low-cost, minimal interventions delivered by mail and phone can help a sizable proportion of individuals quit using SLT.

Association of renal Na,K-ATPase alpha-subunit with the beta- and gamma-subunits based on cryoelectron microscopy.

Na,K-ATPase transports Na(+) and K(+) across cell membranes and consists of alpha- and beta-subunits. Na,K-ATPase also associates with small FXYD proteins that regulate the activity of the pump. We have used cryoelectron microscopy of two-dimensional crystals including data to 8 A resolution to determine the three-dimensional (3-D) structure of renal Na,K-ATPase containing FXYD2, the gamma-subunit. A homology model for the alpha-subunit was calculated from a Ca(2+)-ATPase structure and used to locate the additional beta- and gamma-subunits present in the 3-D map of Na,K-ATPase. Based on the 3-D map, the beta-subunit is located close to transmembrane helices M8 and M10 and the gamma-subunit is adjacent to helices M2 and M9 of the alpha-subunit.

Structure analysis of soluble proteins using electron crystallography.

Electron crystallography as a structural determination technique has grown dramatically in use over recent years. Improvements in microscopes, equipment, practical techniques, computation facilities and image processing methods are reflected in the increasing number of near-atomic resolution structures that have been published. In this review we shall summarize the techniques involved in structure determination of soluble proteins using electron crystallography. Many soluble protein structures have been investigated in this manner over the past two decades. Here we present several examples where a variety of approaches have been used to gradually increase the information obtained.

Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals.

The structure of Na, K-ATPase was determined by electron crystallography at 9.5 A from multiple small 2-D crystals induced in purified membranes isolated from the outer medulla of pig kidney. The density map shows a protomer stabilized in the E(2) conformation which extends approximately 65 A x 75 A x 150 A in the asymmetric unit of the P2 type unit cell. The alpha, beta, and gamma subunits were demonstrated in the membrane crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains, which are similar in overall shape to the domains of the calcium pump of the sarcoplasmic reticulum. One of these domains gives rise to a characteristic elongated projection onto the membrane plane while the putative nucleotide binding and phosphorylation domains form comparatively compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit and is located as an extension of the transmembrane region perpendicular to the membrane plane. The structure of the lipid bilayer spanning part suggests the positions for the transmembrane helix from the beta subunit as well as the small gamma subunit present in this Na,K-ATPase. Two groups of ten helices from the catalytic alpha subunit corresponds to the remaining density in the transmembrane region. The present results demonstrate distinct similarities between the structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy and the reported X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmatic domains.

The three-dimensional map of microsomal glutathione transferase 1 at 6 A resolution.

Microsomal glutathione transferase 1 (MGST1) is representative of a superfamily of membrane proteins where different members display distinct or overlapping physiological functions, including detoxication of reactive electrophiles (glutathione transferase), reduction of lipid hydroperoxides (glutathione peroxidase), and production of leukotrienes and prostaglandin E. It follows that members of this superfamily constitute important drug targets regarding asthma, inflammation and the febrile response. Here we propose that this superfamily consists of a new class of membrane proteins built on a common left-handed four-helix bundle motif within the membrane, as determined by electron crystallography of MGST1 at 6 A resolution. Based on the 3D map and biochemical data we discuss a model for the membrane topology. The 3D structure differs significantly from that of soluble glutathione transferases, which display overlapping substrate specificity with MGST1.

Evaluation of scanners and CCD cameras for high-resolution TEM of protein crystals and single particles.

The modulation transfer function (MTF) and the geometric errors of two flatbed scanners, a slow-scan CCD (SSC) camera and film, have been measured and compared. The geometric errors of the SSC camera and film have been measured using diffraction spots from a lipid crystal. The SSC camera was shown to have the smallest geometric errors while film had the best MTF. Even though film had the best MTF, this is significantly reduced when scanning the film, so that the MTF of the film and scanner combined are comparable to the MTF of the SSC camera.

The projection structure of the membrane protein microsomal glutathione transferase at 3 A resolution as determined from two-dimensional hexagonal crystals.

The formation of two-dimensional crystals of the membrane-bound enzyme microsomal glutathione transferase is sensitive to fractional changes in the lipid-to-protein ratio. Variation of this parameter results in crystal polymorphism. The projection structure of a p6 crystal form of the enzyme has been determined by the use of electron crystallography. The unit cell at 3 A resolution is comprised of two trimers. The hexagonal p6 and the orthorhombic p21212 crystal types have common elements in the packing arrangement which imply dominant crystal contacts. An overall structural similarity between the protein molecules in the two crystal forms is suggested by the projection maps. Furthermore, a comparison of the p6 and p21212 projection maps identifies additional corresponding protein densities which could not be assigned to the microsomal glutathione transferase trimer previously. Surprisingly, an ambiguity of the rotational orientation was found for trimers interspersed at certain positions within the crystal lattice.

Parameters for the two-dimensional crystallization of the membrane protein microsomal glutathione transferase.

Various crystallization parameters were investigated to obtain two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase for structural analysis by electron crystallography. The protein was crystallized by reconstitution of the solubilized trimer into proteoliposomes. Crystallization occurs when minimal amounts of lipid in the range of three lipid molecules per protein trimer are added to the dialysate. Once crystals were obtained, the effect of several parameters on the crystallization was determined. The temperature and initial detergent concentration were found to be crucial parameters in influencing the size of the crystals, and conclusions could be drawn about the rate dependence of the crystallization process. Two highly ordered crystal forms, which are suitable for structural analysis by electron crystallography, were obtained under the two-dimensional crystallization conditions described here.

Two-dimensional crystallization of the chaperonin TF55 from the hyperthermophilic archaeon Sulfolobus solfataricus.

Oligomers of the chaperonin TF55 from Sulfolobus solfataricus have been successfully crystallized in two dimensions via their interaction with a phospholipid monolayer at the air/liquid interface. Oligomer orientation was dependent upon the lipid headgroup used. A neutral lipid monolayer gave rise to small paracrystalline areas of TF55 side views, whereas a negatively charged lipid monolayer resulted in large coherent crystalline areas of the chaperonin in an end-on orientation. These 2D crystals had p312 symmetry (a = b = 162 A, gamma = 60 degrees). Two-dimensional projection structures of the end-on arrays were produced by electron microscopy and image processing techniques. Under the conditions used to grow the crystals, the protein formed complexes of two stacked nine-subunit rings with threefold symmetry.

Two-dimensional crystals of reconstituted beta-subunits of the chaperonin TF55 from Sulfolobus shibatae.

We have obtained 2-dimensional crystals of the beta-subunits of the chaperonin TF55 from Sulfolobus shibatae reconstituted into oligomers in the absence of alpha-subunits. The subunits form rings with 9-fold rotational symmetry which arrange themselves in a trigonal lattice. From electron micrographs of negatively stained specimens we have calculated a projection map in plane group p312 showing the rings in top-view.

The 3.0 A projection structure of microsomal glutathione transferase as determined by electron crystallography of p 21212 two-dimensional crystals.

Two-dimensional crystals of rat microsomal glutathione transferase were grown during dialysis of detergent-solubilized enzyme after addition of a small amount of phospholipid. The crystals had two-sided plane group symmetry p21212 with a calibrated unit cell size of a=91.90 A, b=90.83 A. Electron diffraction patterns were recorded showing significant reflections extending to 3.0 A. A combination of these structure factor amplitudes with phases from high-resolution images following image processing was used to calculate a projection map of the protein. The asymmetric unit of the structure consists of three microsomal glutathione transferase molecules. The local 3-fold axis at the center of the trimer is delineated by six parallel alpha-helices, two from each monomer. The two helices differ significantly in their respective projection structure. The inner helical core of the trimer is partly surrounded by elongated domains with extensions towards the helices and which contain resolved density maxima at a spacing of 4 to 5 A. A well-defined strong peak is localized close to the elongated domain and at a distance of about 9.5 A from two of the inner helices.

Three-dimensional structure of the porcine gastric H,K-ATPase from negatively stained crystals.

A low-resolution three-dimensional model of membrane-bound H,K-ATPase from pig gastric mucosa has been reconstructed by electron microscopy and image processing of two-dimensional crystals in negative stain. The crystal formation is induced by magnesium and vanadate, which stabilize the E2 conformation of the enzyme. The unit cell, with a size of a = b = 123 A, gamma = 90 degrees, has tetragonal p4 symmetry. There are four separate alpha beta protomers within each unit cell. The high-contrast region is limited to the cytoplasmic part of the protein. The total volume of the observed asymmetric protein domain corresponds to a molecular mass of 80-90 kDa. It consists mainly of a large pear-shaped domain measuring 60 x 45 A2, with a height of 50 A as measured perpendicular to the membrane plane. A small stalk segment, 20 A in length, forms a connection to the transmembrane region.

Staphylococcus aureus alpha-toxin: characterization of protein/lipid interactions, 2D crystallization on lipid monolayers, and 3D structure.

Staphylococcus aureus alpha-toxin was characterized with respect to surface activity and its interaction with lipid monolayers. The protein alone had a detergent-like behavior at the air/water interface. Its affinity was higher for negatively charged than for neutral phospholipids. The interaction was pH dependent, showing a maximum increase at pH 7.0. Only a small part of the protein oligomer appeared to be inserted into the monolayers. Crystalline sheets of alpha-toxin were formed using negatively charged phospholipids. Electron microscopy of such areas, at different tilt angles, allowed reconstruction of a three-dimensional model following image processing. The sheets analyzed consisted of two protein layers arranged on a tetragonal lattice. Under the conditions used to grow the crystals the toxin formed 90-A-wide cylinders with a height of 70 A. One of the imposed fourfold axes running perpendicular to the plane of the crystalline layer is positioned at a protein-deficient region which forms a 25-A-wide pore through the oligomer.

The projection structure of microsomal glutathione transferase.

Through the use of electron crystallography, it has been possible to obtain high resolution structural information regarding a mammalian protein that spans the lipid bilayer. Two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase were induced by slow detergent removal from a mixture containing low amounts of phospholipid. Images of specimens stabilized in tannin were collected using electron cryomicroscopy. The projection structure at 4 A shows tightly packed trimers of the protein. Each of them contains an inner core of six parallel alpha-helices delineating a central low density region. The helical bundle is partly surrounded by elongated domains.

The molecular chaperonin TF55 from the Thermophilic archaeon Sulfolobus solfataricus. A biochemical and structural characterization.

The purification and characterization of a new type of thermostable chaperonin from the archaebacterium Sulfolobus solfataricus is described. The chaperonin forms a hetero-oligomeric complex of two different, but closely related, subunits, which we have assigned TF55-alpha and TF55-beta. Their N-terminal sequences and amino acid residue compositions are reported. Two-dimensional projections of the chaperonin have been reconstructed from electron microscopy images, showing a 9-fold symmetrical complex, about 17.5 nm in height and 16 nm in diameter, with a central cavity of 4.5 nm. The complex is resistant to denaturing agents at room temperature and only pH values lower than 2 lead to dissociation. The separated subunits do not reassemble spontaneously but require Mg2+ and ATP for complex formation. Both subunits are necessary for formation of the TF55 oligomer. Significant structural changes have been observed after phosphorylation, thus providing evidence for a structural mobility during the chaperonin-assisted folding process of a protein. The phosphorylation reaction is modulated by potassium and magnesium ions. Magnesium seems to have an inhibitory effect, whereas potassium enhances this reaction.