Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

S100A1 and calmodulin compete for the same binding site on ryanodine receptor

In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca(2+) release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca(2+)-S100A1 competes with Ca(2+)-calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca(2+)-S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616-3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca(2+)-S100A1. These same residues were shown previously to be important for RyR1 binding to Ca(2+)-CaM. A model for regulating muscle contraction is presented in which Ca(2+)-S100A1 and Ca(2+)-CaM compete directly for the same binding site on the ryanodine receptor.     

Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12

The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes. The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide.     

Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation

S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two alpha subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca(2+)-titration curves of Trp90 fluorescence for both the native protein and its Glu32 --> Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 --> Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K(1) =4 +/- 2 x 10(3) m(-1) (C-loops) and K(2) approximately 10(2) m(-1) (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid - a fluorescent probe of protein-surface hydrophobicity. S-Glutathionylation of the single cysteine residue (85) of the alpha subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous - four orders of magnitude - increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding to DeltaDeltaG = -6 +/- 1 kcal.mol(-1). A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.     

S100A12 protein is a strong inducer of neurite outgrowth from primary hippocampal neurons

Several members of the S100 family of Ca(2+) binding proteins are at present known to be secreted and to have extracellular activities. We have investigated the neurite inducing potential of extracellularly added S100A12. Human recombinant S100A12 was found to dramatically induce neuritogenesis of hippocampal cells isolated from 17 to 19 days old rat embryos. The response to S100A12 was dependent on the dose in a bell-shaped manner. A 10-fold increase in neurite outgrowth was observed upon treatment with S100A12 in concentrations between 0.1 and 2.0 microM already after 24 h. Exposure to S100A12 for only 15 min was enough to induce neuritogenesis when measured after 24 h, but to obtain a maximal response, S100A12 had to be present in the culture for at least 4 h. The response to S100A12 was abolished by inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca(2+) flux, Ca(2+)/calmodulin dependent kinase II (CaMKII) or mitogen-activated protein kinase kinase (MEK). Therefore, we suggest that extracellular S100A12 triggers intracellular signal transduction in neurons, involving the classical mitogen-activated protein (MAP) kinase pathway and a phospholipase C-generated second messenger pathway leading to an increase in intracellular Ca(2+) and activation of PKC, ultimately resulting in neuronal differentiation.     

Eicosanoid activation of extracellular signal-regulated kinase1/2 in human epidermoid carcinoma cells

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase Cgamma1 and activity of protein kinase Calpha (PKCalpha). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nm) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.     

Evidence for differential S100 gene over-expression in psoriatic patients from genetically heterogeneous pedigrees

Psoriasis is an inflammatory skin disorder characterised by keratinocyte hyper-proliferation and altered differentiation. To date, linkage analyses have identified at least seven distinct disease susceptibility regions (PSORS1-7). The PSORS4 locus was mapped by our group to chromosome 1q21, within the Epidermal Differentiation Complex. This cluster contains 13 genes encoding S100 calcium-binding proteins, some of which ( S100A7, S100A8 and S100A9) are known to be up-regulated in individual patient keratinocytes. In this study, we analysed S100 gene expression in psoriatic individuals from families characterised by linkage studies. We first selected individuals from two large pedigrees, one of which was linked to the 1q21 locus, whereas the other was unlinked to that region. We studied the expression of 12 S100 genes, by semi-quantitative RT-PCR and Northern blot. These analyses demonstrated up-regulation of S100A8, S100A9 and, to a lesser extent, S100A7 and S100A12, only in the 1q21 linked family. We subsequently analysed S100A7, S100A8, S100 A9 and S100 A12 in three additional samples and were able to confirm S100A8/ S100A9-specific over-expression in 1q-linked pedigrees. Thus, our data provide preliminary evidence for a locus-specific molecular mechanism underlying psoriasis susceptibility.     

Solution Structure of S100A1 Bound to the CapZ Peptide (TRTK12)

As is typical for S100-target protein interactions, a Ca²⁺-dependent conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin-capping protein CapZ. In addition, the Ca²⁺-binding affinity of S100A1 is found to be tightened (greater than threefold) when TRTK12 is bound. To examine the biophysical basis for these observations, we determined the solution NMR structure of TRTK12 in a complex with Ca²⁺-loaded S100A1. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca²⁺-bound state. Next, the structure of S100A1-TRTK12 was compared to that of another S100A1-target complex (i.e., S100A1-RyRP12), which illustrated how the binding pocket in Ca²⁺-S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when the structures of S100A1-TRTK12 and S100B-TRTK12 were compared, providing insights regarding how more than one S100 protein can interact with the same peptide target. Such comparisons, including those with other S100-target and S100-drug complexes, provide the basis for designing novel small-molecule inhibitors that could be specific for blocking one or more S100-target protein interactions.     

S100A1 enhances the L-type Ca2+ current in embryonic mouse and neonatal rat ventricular cardiomyocytes

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p < 0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.     

Refined mapping of the psoriasin gene S100A7 to chromosome 1cen-q21

Psoriasin is a low molecular weight protein of the S100 family, which is highly upregulated in psoriatic epidermis, and whose function is related to skin inflammatory responses. We have applied a cDNA probe from the corresponding psoriasin gene S100A7 in a refined localisation analysis. S100A7 was mapped physically by human/rodent somatic cell hybrid analysis, and more precisely genetically by multilocus linkage analysis of 40 CEPH (Centre d'Etude du Polymorphisme Humain) families. The resulting 12-point linkage map was supported by odds of at least 1000:1, where S100A7 could be placed with a multipoint lodscore of 27.4 in an approximately 7cM interval. The order of the 12 loci was as follows (with the best estimates of recombination frequencies given in between): AMY2B_-0.091-D1S73_0.039-D1S11_-0.053-D 1 S189 _-0.017-D1S252_-0.017-D1S13_-0.078-DIZ5_-0.051-S100A7_-0.022- MUC1_-0.026-SPTA1_-0.066-ATP1A2_-0.014-APOA2. Furthermore, from this map S100A7 could be assigned to the regional position of chromosome 1cen-q21. The linkage information presented should be of great value in association and linkage studies of diseases where psoriasin, or some of the several other very closely linked and functionally related genes, are seen as candidate genes, e.g. in psoriasis.     

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.     

CacyBP/SIP,a calcyclin and Siah-1-interacting protein,binds EF-hand proteins of the S100 family

Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating beta-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915-926). In murine brain, CacyBP/SIP is expressed at a high level, but S100A6 is expressed at a very low level. Consequently we carried out experiments to determine if CacyBP/SIP binds to other S100 proteins in this tissue. Using CacyBP/SIP affinity chromatography, we found that S100B from the brain extract binds to CacyBP/SIP in a Ca2+-dependent manner. Using a nitrocellulose overlay assay with 125I-CacyBP/SIP and CacyBP/SIP affinity chromatography, we found that this protein binds purified S100A1, S100A6, S100A12, S100B, and S100P but not S100A4, calbindin D(9k), parvalbumin, and calmodulin. The interaction of S100 proteins with CacyBP/SIP occurs via its C-terminal fragment (residues 155-229). Co-immunoprecipitation of CacyBP/SIP with S100B from brain and with S100A6 from Ehrlich ascites tumor cells suggests that these interactions are physiologically relevant and that the ubiquitinylation complex involving CacyBP/SIP might be regulated by S100 proteins.     

FGF-2, IL-1beta and TGF-beta regulate fibroblast expression of S100A8

Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+-binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon gamma (IFNgamma), tumour-necrosis factor (TNF) and TGF-beta did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged by heparin. Interleukin-1beta (IL-1beta) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-2 and IL-1beta was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-2-responsive elements were distinct from the IL-1beta-responsive elements in the S100A8 gene promoter. FGF-2-/heparin-induced, but not IL-1beta-induced responses were significantly suppressed by TGF-beta, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-2/IL-1beta, down-regulation by TGF-beta, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.     

Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: identification of putative drug binding sites on S100A1 protein

S100 proteins are a multigenic family of low-molecular-weight Ca(2+)-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca(2+)-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca(2+)-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1. These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family.     

Expression, purification and fluorine-18 radiolabeling of recombinant S100 proteins--potential probes for molecular imaging of receptor for advanced glycation endproducts (RAGE) in vivo

Data concerning the pathophysiological role of the interaction of circulating S100 proteins, a multigenic family of Ca(2+)-modulated proteins, with the receptor for advanced glycation endproducts (RAGE) in cardiovascular diseases, inflammatory processes, and tumorigenesis in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A1, S100B, and S100A12 as potential probes for molecular imaging of this interaction. Therefore, human S100 proteins were cloned as GST fusion proteins in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100 proteins were radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radiolabeled recombinant S100 proteins ((18)F-S100) were used in biodistribution experiments and small animal positron emission tomography (PET) studies in rats. The tissue-specific distribution of (18)F-S100 proteins in vivo correlated well with the anatomical localization of RAGE, e.g., in lungs and in the vascular system. These findings indicate circulating S100A1, S100B, and S100A12 proteins to be ligands for RAGE in rats in vivo. The approach allows the use of small animal PET and provides novel probes to delineate functional expression of RAGE under normal and pathophysiological conditions in rodent models of disease.     

Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12.

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.     

Design of high‐affinity S100‐target hybrid proteins

S100B and S100A10 are dimeric, EF‐hand proteins. S100B undergoes a calcium‐dependant conformational change allowing it to interact with a short contiguous sequence from the actin‐capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N‐terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100‐target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1–15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by ¹H‐¹⁵N HSQC spectra showed the linker did not perturb the structures of the S100A10‐annexin A2 or S100B‐TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments (¹H, ¹⁵N, and ¹³C) showed that residues T102‐S108 of annexin A2 formed a well‐defined α‐helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N‐terminus with a single turn of α‐helix from D108‐K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.     

Characterization of the human S100A12 (calgranulin C, p6, CAAF1, CGRP) gene, a new member of the 5100 gene cluster on chromosome 1q21

Here, we report the characterization of a human cDNA coding for the recently published amino acid sequence of a calcium-binding S100 protein, S100A12 (CGRP, calgranulin C, CAAF1, p6). The exon/intron structure of the S100A12 gene is similar to most other S100 genes. It is composed of three exons which are divided by two introns of 900 by and 400 bp. The protein is encoded by sequences in exons 2 and 3, with exon 2 coding for the N-terminal 45 amino acids and exon 3 coding for the C-terminal 46 amino acids. So far, ten S100 genes are known to be located on human chromosome 1q21 in a clustered organization. Hence, we investigated whether S100A11 (S1000, calgizzarin) and S100A12 are also localized in the S100 gene cluster. We found both genes within the cluster, with S100A11 being close to S100A10 and S100A12 between the genes S100A8 and S100A9. Therefore, the S100 gene cluster now is composed of 12 differentially expressed family members.     

Mast cell and monocyte recruitment by S100A12 and its hinge domain

S100A12 is expressed at sites of acute, chronic, and allergic inflammation. S100 proteins have regions of high sequence homology, but the "hinge" region between the conserved calcium binding domains is structurally and functionally divergent. Because the murine S100A8 hinge domain (mS100A8(42-55)) is a monocyte chemoattractant whereas the human sequence (hS100A8(43-56)) is inactive, we postulated that common hydrophobic amino acids within the S100A12 hinge sequence may be functional. The hinge domain, S100A12(38-53), was chemotactic for human monocytes and murine mast cells in vitro. S100A12(38-53) provoked an acute inflammatory response similar to that elicited by S100A12 in vivo and caused edema and leukocyte and mast cell recruitment. Circular dichroism studies showed that S100A12(38-53) had increased helical structure in hydrophobic environments. Mutations in S100A12(38-53) produced using an alanine scan confirmed that specific hydrophobic residues (I44A, I47A, and I53A) on the same face of the helix were critical for monocyte chemotaxis in vitro and generation of edema in vivo. In a hydrophobic environment such as the cell membrane, these critical residues would likely align on one face of an alpha-helix to facilitate receptor interaction. Interaction is unlikely to occur via the receptor for advanced glycation end products but, rather, via a G-protein-coupled mechanism.     

IL-22 is induced by S100/calgranulin and impairs cholesterol efflux in macrophages by downregulating ABCG1

S100A8/9 and S100A12 are emerging biomarkers for disease activity of autoimmune and cardiovascular diseases. We demonstrated previously that S100A12 accelerates atherosclerosis accompanied by large cholesterol deposits in atherosclerotic lesions of apoE-null mice. The objective of this study was to ascertain whether S100/calgranulin influences cholesterol homeostasis in macrophages. Peritoneal macrophages from transgenic mice expressing human S100A8/9 and S100A12 in myeloid cells [human bacterial artificial chromosome (hBAC)/S100] have increased lipid content and reduced ABCG1 expression and [³H]cholesterol efflux compared with WT littermates. This was associated with a 6-fold increase in plasma interleukin (IL)-22 and increased IL-22 mRNA in splenic T cells. These findings are mediated by the receptor for advanced glycation endproducts (RAGE), because hBAC/S100 mice lacking RAGE had normal IL-22 expression and normal cholesterol efflux. In vitro, recombinant IL-22 reduced ABCG1 expression and [³H]cholesterol efflux in THP-1 macrophages, while recombinant S100A12 had no effect on ABCG1 expression. In conclusion, S100/calgranulin has no direct effect on cholesterol efflux in macrophages, but rather promotes the secretion of IL-22, which then directly reduces cholesterol efflux in macrophages by decreasing the expression of ABCG1.