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SummaryInterleukin-1 (IL-1)-mediated signaling in T cells is essential for T helper 17 (Th17) cell differentiation. We showed here that SIGIRR, a negative regulator of IL-1 receptor and Toll-like receptor signaling, was induced during Th17 cell lineage commitment and governed Th17 cell differentiation and expansion through its inhibitory effects on IL-1 signaling. The absence of SIGIRR in T cells resulted in increased Th17 cell polarization in vivo upon myelin oligodendrocyte glycoprotein (MOG35-55) peptide immunization. Recombinant IL-1 promoted a marked increase in the proliferation of SIGIRR-deficient T cells under an in vitro Th17 cell-polarization condition. Importantly, we detected increased IL-1-induced phosphorylation of JNK and mTOR kinase in SIGIRR-deficient Th17 cells compared to wild-type Th17 cells. IL-1-induced proliferation was abolished in mTOR-deficient Th17 cells, indicating the essential role of mTOR activation. Our results demonstrate an important mechanism by which SIGIRR controls Th17 cell expansion and effector function through the IL-1-induced mTOR signaling pathway.

Prostaglandins, particularly prostaglandin E2 (PGE2), play an important role during inflammation. This is exemplified by the clinical use of cyclooxygenase 2 inhibitors, which interfere with PGE2 synthesis, as effective antiinflammatory drugs. Here, we show that PGE2 directly promotes differentiation and proinflammatory functions of human and murine IL-17–producing T helper (Th17) cells. In human purified naive T cells, PGE2 acts via prostaglandin receptor EP2- and EP4-mediated signaling and cyclic AMP pathways to up-regulate IL-23 and IL-1 receptor expression. Furthermore, PGE2 synergizes with IL-1β and IL-23 to drive retinoic acid receptor–related orphan receptor (ROR)-γt, IL-17, IL-17F, CCL20, and CCR6 expression, which is consistent with the reported Th17 phenotype. While enhancing Th17 cytokine expression mainly through EP2, PGE2 differentially regulates interferon (IFN)-γ production and inhibits production of the antiinflammatory cytokine IL-10 in Th17 cells predominantly through EP4. Furthermore, PGE2 is required for IL-17 production in the presence of antigen-presenting cells. Hence, the combination of inflammatory cytokines and noncytokine immunomodulators, such as PGE2, during differentiation and activation determines the ultimate phenotype of Th17 cells. These findings, together with the altered IL-12/IL-23 balance induced by PGE2 in dendritic cells, further highlight the crucial role of the inflammatory microenvironment in Th17 cell development and regulation.

Thirteen peroxidase spots on two-dimensional gels were identified by comprehensive proteome analysis of the barley seed. Mass spectrometry tracked multiple forms of three different peroxidase isozymes: barley seed peroxidase 1, barley seed-specific peroxidase BP1 and a not previously identified putative barley peroxidase. The presence of multiple spots for each of the isozymes reflected variations in post-translational glycosylation and protein truncation. Complete sequence coverage was achieved by using a series of proteases and chromatographic resins for sample preparation prior to mass spectrometric analysis. Distinct peroxidase spot patterns divided the 16 cultivars tested into two groups. The distribution of the three isozymes in different seed tissues (endosperm, embryo, and aleurone layer) suggested the peroxidases to play individual albeit partially overlapping roles during germination. In summary, a subset of three peroxidase isozymes was found to occur in the seed, whereas products of four other barley peroxidase genes were not detected. The present analysis documents the selective expression profiles and post-translational modifications of isozymes from a large plant gene family.

Studies have shown that transforming growth factor-beta (TGF-beta) and interleukin 6 (IL-6) are required for the lineage commitment of pathogenic IL-17-producing T helper cells (T(H)-17 cells). Unexpectedly, here we found that stimulation of myelin-reactive T cells with TGF-beta plus IL-6 completely abrogated their pathogenic function despite upregulation of IL-17 production. Cells stimulated with TGF-beta plus IL-6 were present in the spleen as well as the central nervous system, but they failed to upregulate the proinflammatory chemokines crucial for central nervous system inflammation. In addition, these cells produced IL-10, which has potent anti-inflammatory activities. In contrast, stimulation with IL-23 promoted expression of IL-17 and proinflammatory chemokines but not IL-10. Hence, TGF-beta and IL-6 'drive' initial lineage commitment but also 'restrain' the pathogenic potential of T(H)-17 cells. Our findings suggest that full acquisition of pathogenic function by effector T(H)-17 cells is mediated by IL-23 rather than by TGF-beta and IL-6.

Ten genes from two multigene families encode barley α-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the α-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis, western blotting, and mass spectrometry. Mass spectrometric analysis confirmed that the 29 α-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding α-amylase 1 and 2, respectively, but lacked products from seven other genes. Eleven spots were identified only by immunostaining. Mass spectrometry identified 12 full-length forms and 12 fragments from the cultivar Barke. Products of both α-amylase 2 entries co-migrated in five full-length and one fragment spot. The α-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that α-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (βα)8-barrel between β-strand 3 and α-helix 3, followed by cleavage on the C-terminal side of domain B and near the C-terminus. Only two shorter fragments were identified of the other α-amylase 2 (gi|166985). The 2D gels of dissected tissues showed α-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length α-amylase forms. While only products of the above three genes appeared by germination also of 15 other barley cultivars, the cultivars had distinct repertoires of charge and molecular mass variant forms. These patterns appeared not to be correlated with malt quality.

Proteome analysis of mature barley (Hordeum vulgare subsp. vulgare) seeds has led to the identification of proteins in about 450 spots on 2D-gels. To shed light on the role of some of these proteins, their temporal appearance was monitored over 5 weeks during grain-filling and maturation of field-grown barley. Appearance profiles are described for 105 proteins identified in 185 2D-gel spots in the overlapping pI ranges 4–7 and 6–11. Grouping of proteins according to appearance across functional categories revealed instances of differential regulation of protein forms. Thus, a single 1-cys-peroxiredoxin isoform was identified in three spots, one present throughout grain filling, one appearing during desiccation and one observed only in mature seeds. This suggested post-translational modification of the protein to different degrees during seed maturation. Distinct isoforms of several proteins were identified in spots with individual appearance profiles, indicating differential expression of isoforms. Three isoforms of β-1,3 endoglucanase, including one not previously observed, each had a different temporal appearance pattern probably reflecting involvement in diverse processes such as cell wall modification or defence against fungal pathogens. Comparison of two cultivars, Barke and Morex, led to identification of protein spots appearing earlier in Morex than Barke, reflecting the faster maturation of Morex seeds.

Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites.

Protein engineering of barley α-amylase addressed the roles of Ca2+ in activity and inhibition by barley α-amylase/subtilisin inhibitor (BASI), multiple attach in polysaccharide hydrolysis, secondary starch binding sites, and BASI hot spots in AMY2 recognition. AMY1/AMY2 isozyme chimeras faciliatated assignment of function to specific regions of the structure. An AMY1 fusion with starch binding domain and AMY1 mutants in the substrate binding cleft gave degree of multiple attack of 0.9–3.3, compared to 1.9 for wild-type. About 40% of the secondary attacks, succeeding the initial endo-attack, produced DP5-10 maltooligosaccharides in similar proportion for all enzyme variants, whereas shorter products, comprising about 25%, varied depending on the mutation. Secondary binding sites were important in both multiple attack and starch granule hydrolysis. Surface plasmon resonance and inhibition analyses indicated the importance of fully hydrated Ca2+ at the AMY2/BASI interface to strengthen the complex. ...

Proteomes of barley seeds were described by 2-D gel electrophoresis and spots selected for proteinidentification by mass spectrometry and database searches. Proteins were categorised according to temporalappearance during seed development and maturation. Fragments of β-amylases appeared transiently at midgrain filling and during germination. The α-amylase/trypsin inhibitors increased during grain filling andtypical housekeeping enzymes were present throughout the period. Germination altered the proteome and dissection of micromalted seeds enabled localization of selected proteins to specific tissues. The embryo was particularly rich in soluble proteins. Barley α-amylase/subtilisin inhibitor (BASI) and its target α-amylaseisozyme 2 occurred in endosperm and aleurone. Immunoblotting showed how the α-amylase changed duringgermination. Mutational analysis in a-amylase and BASI was guided by the three-dimensional structures.Mutants along the 10 binding subsites in barley a-amylase 1 addressed roles of individual residues in thepreference for starch over oligosaccharide substrates and vice versa, action patterns on oligosaccharides, andmultiple attack on amylose. Although the wild-type enzyme was proficient in transglycosylation, mutants ofthe catalytic nucleophile did not act as glycosynthase. C-terminal fusion of a starch binding domain from Aspergillus niger glucoamylase to barley α-amylase isozyme 1 enhanced degradation of starch granules. Recent heterologous expression of BASI allowed mutation of residues critical in enzyme binding as monitored by activity inhibition and surface plasmon resonance assays. A fully hydrated Ca2+ at the protein interface secured contact between BASI and the catalytic residues in the α-amylase.

Met53 in barley α-amylase 1 (AMY1) is situated at the high-affinity subsite -2. While Met53 is unique to plant α-amylases, the adjacent Tyr52 stacks onto substrate at subsite - I and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in β→α loop 2 of the catalytic (β/α) 8 -barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/ Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP 17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl β-D-maltoheptaoside the activity (k c a t /K m ) of all mutants was reduced to 5.5-0.01 and 1.7-0.02% of wild-type, respectively. K m increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl α-D-maltohexaoside (PNPG 6 ) and PNPG 5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite -2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG 2 from PNPG 6 and similar amounts of PNPG 2 and PNPG 3 accounting for 85% of the products from PNPG 5 , wild-type released 4-nitrophenol from PNPG 6 and PNPG and PNPG 2 in equal amounts from PNPG 5 . Met53Trp affected the action pattern on PNPG 7 . which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation - promoted probably by slow substrate hydrolysis to produce up to maltoundecaose from PNPG 6 .

Enzymatic properties of barley α-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites −5/−6 (Cys95Ala/Thr) and +1/+2 (Met298Ala/Asn/Ser) as well as in the double mutants, Cys95Ala/Met298Ala/Asn/Ser. Cys95Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl β-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95Ala and wild-type. Met298Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are ≤ 30% and ≤ 10% of wild-type, respectively. The activity of Cys95Ala/Met298Ala/Asn/Ser is 100–180% towards starch, and the kcat/Km is 15–30%, and 0.4–1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites −5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6′′′-maltotriosyl-maltohexaose thus covering subsites −1 to +5, while productive binding of unbranched substrate involves subsites −3 to +3.

Analysis of the water-soluble barley seed proteome has led to the identification of proteins by MS in the major spots on two-dimensional gels covering the pI ranges 4–7 and 6–11. This provides the basis for in-depth studies of proteome changes during seed development and germination, tissue-specific proteomes, cultivar differences related to quality parameters, analysis of the genetic basis for spot variations and targeted investigations of specific proteins.

The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites -6 and +4 (substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved in plant alpha-amylases whereas Thr(212) varies in these and related enzymes. Compared with wild-type AMY1, the subsite -6 mutant Y105A has 140, 15, and

Met53 in barley alpha-amylase 1 (AMY1) is situated at the high-affinity subsite -2. While Met53 is unique to plant alpha-amylases, the adjacent Tyr52 stacks onto substrate at subsite -1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in beta--alpha loop 2 of the catalytic (beta/alpha)8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5-0.01 and 1.7-0.02% of wild-type, respectively. Km increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl alpha-d-maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite -2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild-type released 4-nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5. Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation - promoted probably by slow substrate hydrolysis - to produce up to maltoundecaose from PNPG6.

During the past few years great progress has been made in the understanding of the function of starch-degrading enzymes. Combining mutational analysis, multiple sequence alignment, and three dimensional structures of enzyme substrate analogue complexes formed the basis for developing amylolytic and related enzymes through protein engineering. In the case of barley a-amylase this has involved rational site-directed mutagenesis, regional random mutagenesis and isozyme sequence exchange as well as generation of isozyme chimeras. The approach has enabled modulation of enzyme specificity and identification of the functional roles of distinct residues and regions. The mutational analysis included the complex between barley α-amylase 2 and a proteinaceous inhibitor from barley seeds, BASI (barley a-amylase/subtilisin inhibitor). The interplay between the catalytic and the starch binding domains in glucoamylase from Aspergillus niger was investigated. The two domains are connected by a highly O-glycosylated linker and bind with 1:1 stoichiometry double-headed heterobifunctional synthetic analogues targeted to each domain. Glucoamylase is an inverting hydrolase and replacement of the catalytic base, a glutamic acid, by a cysteinesulfinic acid, gave rise to activity surpassing that of the wild-type enzyme, indicating a most surprising flexibility for the catalytic base.