Thermal unfolding of Apo and Holo Desulfovibrio desulfuricans flavodoxin: cofactor stabilizes folded and intermediate states

We here compare thermal unfolding of the apo and holo forms of Desulfovibrio desulfuricans flavodoxin, which noncovalently binds a flavin mononucleotide (FMN) cofactor. In the case of the apo form, fluorescence and far-UV circular dichroism (CD) detected transitions are reversible but do not overlap (T(m) of 50 and 60 degrees C, respectively, pH 7). The thermal transitions for the holo form follow the same pattern but occur at higher temperatures (T(m) of 60 and 67 degrees C for fluorescence and CD transitions, respectively, pH 7). The holoprotein transitions are also reversible and exhibit no protein concentration dependence (above 10 microM), indicating that the FMN remains bound to the polypeptide throughout. Global analysis shows that the thermal reactions for both apo and holo forms proceed via an equilibrium intermediate that has approximately 90% nativelike secondary structure and significant enthalpic stabilization relative to the unfolded states. Incubation of unfolded holoflavodoxin at high temperatures results in FMN dissociation. Rebinding of FMN at these conditions is nominal, and therefore, cooling of holoprotein heated to 95 degrees C follows the refolding pathway of the apo form. However, FMN readily rebinds to the apoprotein at lower temperatures. We conclude that (1) a three-state thermal unfolding behavior appears to be conserved among long- and short-chain, as well as apo and holo forms of, flavodoxins and (2) flavodoxin's thermal stability (in both native and intermediate states) is augmented by the presence of the FMN cofactor.     

IL-22RA2 associates with multiple sclerosis and macrophage effector mechanisms in experimental neuroinflammation

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the CNS. Recent advances in whole-genome screening tools have enabled discovery of several MS risk genes, the majority of which have known immune-related functions. However, disease heterogeneity and low tissue accessibility hinder functional studies of established MS risk genes. For this reason, the MS model experimental autoimmune encephalomyelitis (EAE) is often used to study neuroinflammatory disease mechanisms. In this study, we performed high-resolution linkage analysis in a rat advanced intercross line to identify an EAE-regulating quantitative trait locus, Eae29, on rat chromosome 1. Eae29 alleles from the resistant strain both conferred milder EAE and lower production of proinflammatory molecules in macrophages, as demonstrated by the congenic line, DA.PVG-Eae29 (Dc1P). The soluble IL-22R α2 gene (Il-22ra2) lies within the Eae29 locus, and its expression was reduced in Dc1P, both in activated macrophages and splenocytes from immunized rats. Moreover, a single nucleotide polymorphism located at the end of IL-22RA2 associated with MS risk in a combined Swedish and Norwegian cohort comprising 5019 subjects, displaying an odds ratio of 1.26 (p = 8.0 × 10(-4)). IL-22 and its receptors have been implicated in chronic inflammation, suggesting that IL-22RA2 regulates a central immune pathway. Through a combined approach including genetic and immunological investigation in an animal model and large-scale association studies of MS patients, we establish IL-22RA2 as an MS risk gene.     

Folding and assembly pathways of co-chaperonin proteins 10: Origin of bacterial thermostability

To compare folding/assembly processes of heptameric co-chaperonin proteins 10 (cpn10) from different species and search for the origin of thermostability in hyper-thermostable Aquifex aeolicus cpn10 (Aacpn10), we have studied two bacterial variants-Aacpn10 and Escherichia coli cpn10 (GroES)-and compared the results to data on Homo sapiens cpn10 (hmcpn10). Equilibrium denaturation of GroES by urea, guanidine hydrochloride (GuHCl) and temperature results in coupled heptamer-to-monomer transitions in all cases. This is similar to the behavior of Aacpn10 but differs from hmcpn10 denaturation in urea. Time-resolved experiments reveal that GroES unfolds before heptamer dissociation, whereas refolding/reassembly begins with folding of individual monomers; these assemble in a slower step. The sequential folding/assembly mechanism for GroES is rather similar to that observed for Aacpn10 but contradicts the parallel paths of hmcpn10. We reveal that Aacpn10's stability profile is shifted upwards, broadened, and also moved horizontally to higher temperatures, as compared to that of GroES.     

S100A9 interaction with TLR4 promotes tumor growth

By breeding TRAMP mice with S100A9 knock-out (S100A9(-/-)) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+) S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+) macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/-) and TLR4(-/-), but not in RAGE(-/-) animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b(+) cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.     

Development of a model system to identify differences in spring and winter oat

Our long-term goal is to develop a Swedish winter oat (Avena sativa). To identify molecular differences that correlate with winter hardiness, a winter oat model comprising of both non-hardy spring lines and winter hardy lines is needed. To achieve this, we selected 294 oat breeding lines, originating from various Russian, German, and American winter oat breeding programs and tested them in the field in south- and western Sweden. By assaying for winter survival and agricultural properties during four consecutive seasons, we identified 14 breeding lines of different origins that not only survived the winter but also were agronomically better than the rest. Laboratory tests including electrolytic leakage, controlled crown freezing assay, expression analysis of the AsVrn1 gene and monitoring of flowering time suggested that the American lines had the highest freezing tolerance, although the German lines performed better in the field. Finally, six lines constituting the two most freezing tolerant lines, two intermediate lines and two spring cultivars were chosen to build a winter oat model system. Metabolic profiling of non-acclimated and cold acclimated leaf tissue samples isolated from the six selected lines revealed differential expression patterns of 245 metabolites including several sugars, amino acids, organic acids and 181 hitherto unknown metabolites. The expression patterns of 107 metabolites showed significant interactions with either a cultivar or a time-point. Further identification, characterisation and validation of these metabolites will lead to an increased understanding of the cold acclimation process in oats. Furthermore, by using the winter oat model system, differential sequencing of crown mRNA populations would lead to identification of various biomarkers to facilitate winter oat breeding.     

A SEL1L mutation links a canine progressive early-onset cerebellar ataxia to the endoplasmic reticulum-associated protein degradation (ERAD) machinery

Inherited ataxias are characterized by degeneration of the cerebellar structures, which results in progressive motor incoordination. Hereditary ataxias occur in many species, including humans and dogs. Several mutations have been found in humans, but the genetic background has remained elusive in dogs. The Finnish Hound suffers from an early-onset progressive cerebellar ataxia. We have performed clinical, pathological, and genetic studies to describe the disease phenotype and to identify its genetic cause. Neurological examinations on ten affected dogs revealed rapidly progressing generalized cerebellar ataxia, tremors, and failure to thrive. Clinical signs were present by the age of 3 months, and cerebellar shrinkage was detectable through MRI. Pathological and histological examinations indicated cerebellum-restricted neurodegeneration. Marked loss of Purkinje cells was detected in the cerebellar cortex with secondary changes in other cortical layers. A genome-wide association study in a cohort of 31 dogs mapped the ataxia gene to a 1.5 Mb locus on canine chromosome 8 (p_raw = 1.1×10⁻⁷, p_genome = 7.5×10⁻⁴). Sequencing of a functional candidate gene, sel-1 suppressor of lin-12-like (SEL1L), revealed a homozygous missense mutation, c.1972T>C; p.Ser658Pro, in a highly conserved protein domain. The mutation segregated fully in the recessive pedigree, and a 10% carrier frequency was indicated in a population cohort. SEL1L is a component of the endoplasmic reticulum (ER)–associated protein degradation (ERAD) machinery and has not been previously associated to inherited ataxias. Dysfunctional protein degradation is known to cause ER stress, and we found a significant increase in expression of nine ER stress responsive genes in the cerebellar cortex of affected dogs, supporting the pathogenicity of the mutation. Our study describes the first early-onset neurodegenerative ataxia mutation in dogs, establishes an ERAD–mediated neurodegenerative disease model, and proposes SEL1L as a new candidate gene in progressive childhood ataxias. Furthermore, our results have enabled the development of a genetic test for breeders.     

Role of cations in stability of acidic protein Desulfovibrio desulfuricans apoflavodoxin

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)). A linear correlation of stabilizing effects with cation hydration properties suggests an important role of dehydration in efficient cation interaction with the protein. The effects on stability are due to preferential binding of one cation to native apoflavodoxin and results in an increase in thermal midpoint of 20 degrees C and the free energy of unfolding (at 20 degrees C) increases fivefold. Tuning of biophysical properties (such as folding and ligand/cofactor binding) of acidic proteins by cation binding may be important in vivo.     

Probing copper ligands in denatured <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> azurin: unfolding His117Gly and His46Gly mutants

Azurin is a single-domain beta-barrel protein with a redox-active copper cofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one histidine imidazole, and a third, unknown ligand. In order to identify which histidine (histidine-117 and histidine-46 both coordinate copper in native azurin) is involved in copper coordination in denatured azurin, two single-site (histidine to glycine) mutants, His117Gly and His46Gly azurin, are investigated here. Equilibrium denaturation experiments of His46Gly azurin loaded with copper demonstrate that copper remains bound to this mutant in high urea concentrations where the protein's secondary structure is lost. In contrast, for copper-loaded His117Gly azurin, copper does not stay coordinated upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corresponding to residues 111-123 in azurin, suggesting similar metal coordination. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximity of histidine-117 to cysteine-112 in the primary sequence.     

Can landscape properties predict occurrence of grey-sided voles?

There has been a long-term decline in spring and fall numbers of Clethrionomys rufocanus in boreal Sweden in 1971–2005. Previous studies on permanent sampling plots in the centre of 2.5 × 2.5 km landscapes suggested that habitat fragmentation (sensu destruction) could have contributed to the decline. Therefore, we tested these findings in a field study and compared trapping results on the central sampling plots of landscapes with a low degree of fragmentation (LDF) and of “hot spot” type with trapping results in managed forest landscapes with a high degree of fragmentation (HDF). We predicted that C. rufocanus would be more common on the LDF plots. We used our permanent plots supplemented with a new sample of plots, mainly of the rare LDF type, inside or just outside the long-term study area. Very few voles were trapped on both plot types, and no difference was found. However, a subsequent pilot study with trapping in a national park with large areas of pristine, unfragmented forest yielded more voles than in the managed, more fragmented, areas. Consequently, the initial field study data and some other recent data were also re-analysed from a “local patch quality” perspective. This alternative approach revealed the positive importance of large focal patches of forest >60 years old and their content of old-growth (pine) forest (>100 years). Interestingly, at the landscape level, the frequency distribution of patches of forest >60 years old, old-growth (>100 years), and especially of old-growth pine forest (>100 years), relative to the properties of plots with C. rufocanus, suggested that there are few forest patches left that are suitable for C. rufocanus. Our current results suggest that habitat fragmentation cannot be excluded as a contributing cause to the long-term decline of C. rufocanus in boreal Sweden.