S100B inhibition reduces behavioral and pathologic changes in experimental traumatic brain injury.

Neuroinflammation following traumatic brain injury (TBI) is increasingly recognized to contribute to chronic tissue loss and neurologic dysfunction. Circulating levels of S100B increase after TBI and have been used as a biomarker. S100B is produced by activated astrocytes and can promote microglial activation; signaling by S100B through interaction with the multiligand advanced glycation end product-specific receptor (AGER) has been implicated in brain injury and microglial activation during chronic neurodegeneration. We examined the effects of S100B inhibition in a controlled cortical impact model, using S100B knockout mice or administration of neutralizing S100B antibody. Both interventions significantly reduced TBI-induced lesion volume, improved retention memory function, and attenuated microglial activation. The neutralizing antibody also significantly reduced sensorimotor deficits and improved neuronal survival in the cortex. However, S100B did not alter microglial activation in BV2 cells or primary microglial cultures stimulated by lipopolysaccharide or interferon gamma. Further, proximity ligation assays did not support direct interaction in the brain between S100B and AGER following TBI. Future studies are needed to elucidate specific pathways underlying S100B-mediated neuroinflammatory actions after TBI. Our results strongly implicate S100B in TBI-induced neuroinflammation, cell loss, and neurologic dysfunction, thereby indicating that it is a potential therapeutic target for TBI.

S100 proteins in cancer.

In humans, the S100 protein family is composed of 21 members that exhibit a high degree of structural similarity, but are not functionally interchangeable. This family of proteins modulates cellular responses by functioning both as intracellular Ca(2+) sensors and as extracellular factors. Dysregulated expression of multiple members of the S100 family is a common feature of human cancers, with each type of cancer showing a unique S100 protein profile or signature. Emerging in vivo evidence indicates that the biology of most S100 proteins is complex and multifactorial, and that these proteins actively contribute to tumorigenic processes such as cell proliferation, metastasis, angiogenesis and immune evasion. Drug discovery efforts have identified leads for inhibiting several S100 family members, and two of the identified inhibitors have progressed to clinical trials in patients with cancer. This Review highlights new findings regarding the role of S100 family members in cancer diagnosis and treatment, the contribution of S100 signalling to tumour biology, and the discovery and development of S100 inhibitors for treating cancer.

The Ca2+ sensor S100A1 modulates neuroinflammation, histopathology and Akt activity in the PSAPP Alzheimer's disease mouse model.

The contribution of the Ca(2+) sensor S100A1 to in vivo Alzheimer's disease (AD) pathobiology has not been elucidated although S100A1 regulates numerous cellular processes linked to AD. This study uses genetic ablation to ascertain the effects of S100A1 on neuroinflammation, beta-amyloid (Aß) plaque deposition and Akt activity in the PSAPP AD mouse model. PSAPP/S100A1(-/-) mice exhibited decreases in astrocytosis (GFAP burden), microgliosis (Iba1 burden) and plaque load/number when compared to PSAPP/S100A1(+/+) mice at six and twelve months of age. The presence of detectable S100A1 staining in human AD specimens is consistent with a detrimental gain of S100A1 function in AD. S100A1 ablation also reduced plaque associated and increased non-plaque associated PO4-Akt and PO4-GSK3ß staining. S100A1·Akt complexes were undetectable in PC12 cells and AD brain tissue suggesting that S100A1 indirectly modulates Akt activity. In contrast, S100A1·RyR (ryanodine receptor) complexes were present in human/mouse AD brain and exhibited Ca(2+)-dependent formation in neuronal cells. This is the first direct demonstration of an S100A1· target protein complex in tissue/cells and identifies the RyR as a primary S100A1 target protein in the brain. Collectively, these data suggest that S100A1 inhibition may be a novel strategy for normalizing aberrant Ca(2+) signaling in AD.

Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

In vivo screening of S100B inhibitors for melanoma therapy.

S100 proteins are markers for numerous cancers, and in many cases high S100 protein levels are a prognostic indicator for poor survival. One such case is S100B, which is overproduced in a very large percentage of malignant melanoma cases. Elevated S100B protein was more recently validated to have causative effects towards cancer progression via down-regulating the tumor suppressor protein, p53. Towards eliminating this problem in melanoma, targeting S100B with small molecule inhibitors was initiated. This work relies on numerous chemical biology technologies including structural biology, computer-aided drug design, compound screening, and medicinal chemistry approaches. Another important component of drug development is the ability to test compounds and various molecular scaffolds for their efficacy in vivo. This chapter briefly describes the development of S100B inhibitors, termed SBiXs, for melanoma therapy with a focus on the inclusion of in vivo screening at an early stage in the drug discovery process.

Evolution of the S100 family of calcium sensor proteins.

The S100s are a large group of Ca(2+) sensors found exclusively in vertebrates. Transcriptomic and genomic data from the major radiations of mammals were used to derive the evolution of the mammalian S100s genes. In human and mouse, S100s and S100 fused-type proteins are in a separate clade from other Ca(2+) sensor proteins, indicating that an ancient bifurcation between these two gene lineages has occurred. Furthermore, the five genomic loci containing S100 genes have remained largely intact during the past 165 million years since the shared ancestor of egg-laying and placental mammals. Nonetheless, interesting births and deaths of S100 genes have occurred during mammalian evolution. The S100A7 loci exhibited the most plasticity and phylogenetic analyses clarified relationships between the S100A7 proteins encoded in the various mammalian genomes. Phylogenetic analyses also identified four conserved subgroups of S100s that predate the rise of warm-blooded vertebrates: A2/A3/A4/A5/A6, A1/A10/A11/B/P/Z, A13/A14/A16, and A7s/A8/A9/A12/G. The similarity between genomic location and phylogenetic clades suggest that these subfamilies arose by a series of tandem gene duplication events. Examination of annotated S100s in lower vertebrates suggests that the ancestral S100 was a member of the A1/A10/A11/B/P/Z subgroup and arose near the emergence of vertebrates approximately 500 million years ago.

PSAPP mice exhibit regionally selective reductions in gliosis and plaque deposition in response to S100B ablation.

BACKGROUND: Numerous studies have reported that increased expression of S100B, an intracellular Ca2+ receptor protein and secreted neuropeptide, exacerbates Alzheimer's disease (AD) pathology. However, the ability of S100B inhibitors to prevent/reverse AD histopathology remains controversial. This study examines the effect of S100B ablation on in vivo plaque load, gliosis and dystrophic neurons. METHODS: Because S100B-specific inhibitors are not available, genetic ablation was used to inhibit S100B function in the PSAPP AD mouse model. The PSAPP/S100B-/- line was generated by crossing PSAPP double transgenic males with S100B-/- females and maintained as PSAPP/S100B+/- crosses. Congo red staining was used to quantify plaque load, plaque number and plaque size in 6 month old PSAPP and PSAPP/S100B-/- littermates. The microglial marker Iba1 and astrocytic marker glial fibrillary acidic protein (GFAP) were used to quantify gliosis. Dystrophic neurons were detected with the phospho-tau antibody AT8. S100B immunohistochemistry was used to assess the spatial distribution of S100B in the PSAPP line. RESULTS: PSAPP/S100B-/- mice exhibited a regionally selective decrease in cortical but not hippocampal plaque load when compared to PSAPP littermates. This regionally selective reduction in plaque load was accompanied by decreases in plaque number, GFAP-positive astrocytes, Iba1-positive microglia and phospho-tau positive dystrophic neurons. These effects were not attributable to regional variability in the distribution of S100B. Hippocampal and cortical S100B immunoreactivity in PSAPP mice was associated with plaques and co-localized with astrocytes and microglia. CONCLUSIONS: Collectively, these data support S100B inhibition as a novel strategy for reducing cortical plaque load, gliosis and neuronal dysfunction in AD and suggest that both extracellular as well as intracellular S100B contribute to AD histopathology.

The Calcium-Dependent Interaction of S100B with Its Protein Targets.

S100B is a calcium signaling protein that is a member of the S100 protein family. An important feature of S100B and most other S100 proteins (S100s) is that they often bind Ca(2+) ions relatively weakly in the absence of a protein target; upon binding their target proteins, Ca(2+)-binding then increases by as much as from 200- to 400-fold. This manuscript reviews the structural basis and physiological significance of increased Ca(2+)-binding affinity in the presence of protein targets. New information regarding redundancy among family members and the structural domains that mediate the interaction of S100B, and other S100s, with their targets is also presented. It is the diversity among individual S100s, the protein targets that they interact with, and the Ca(2+) dependency of these protein-protein interactions that allow S100s to transduce changes in [Ca(2+)](intracellular) levels into spatially and temporally unique biological responses.

S100A1 promotes action potential-initiated calcium release flux and force production in skeletal muscle.

The role of S100A1 in skeletal muscle is just beginning to be elucidated. We have previously shown that skeletal muscle fibers from S100A1 knockout (KO) mice exhibit decreased action potential (AP)-evoked Ca(2+) transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulin-binding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that Ca(2+) release was suppressed at all test membrane potentials in S100A1(-/-) fibers. Here we examine the role of S100A1 during physiological AP-induced muscle activity, using an integrative approach spanning AP propagation to muscle force production. With the voltage-sensitive indicator di-8-aminonaphthylethenylpyridinium, we first demonstrate that the AP waveform is not altered in flexor digitorum brevis muscle fibers isolated from S100A1 KO mice. We then use a model for myoplasmic Ca(2+) binding and transport processes to calculate sarcoplasmic reticulum Ca(2+) release flux initiated by APs and demonstrate decreased release flux and greater inactivation of flux in KO fibers. Using in vivo stimulation of tibialis anterior muscles in anesthetized mice, we show that the maximal isometric force response to twitch and tetanic stimulation is decreased in S100A1(-/-) muscles. KO muscles also fatigue more rapidly upon repetitive stimulation than those of wild-type counterparts. We additionally show that fiber diameter, type, and expression of key excitation-contraction coupling proteins are unchanged in S100A1 KO muscle. We conclude that the absence of S100A1 suppresses physiological AP-induced Ca(2+) release flux, resulting in impaired contractile activation and force production in skeletal muscle.

S100A1: Structure, Function, and Therapeutic Potential.

S100A1 is a member of the S100 family of calcium-binding proteins. As with most S100 proteins, S100A1 undergoes a large conformational change upon binding calcium as necessary to interact with numerous protein targets. Targets of S100A1 include proteins involved in calcium signaling (ryanidine receptors 1 & 2, Serca2a, phopholamban), neurotransmitter release (synapsins I & II), cytoskeletal and filament associated proteins (CapZ, microtubules, intermediate filaments, tau, mocrofilaments, desmin, tubulin, F-actin, titin, and the glial fibrillary acidic protein GFAP), transcription factors and their regulators (e.g. myoD, p53), enzymes (e.g. aldolase, phosphoglucomutase, malate dehydrogenase, glycogen phosphorylase, photoreceptor guanyl cyclases, adenylate cyclases, glyceraldehydes-3-phosphate dehydrogenase, twitchin kinase, Ndr kinase, and F1 ATP synthase), and other Ca2+-activated proteins (annexins V & VI, S100B, S100A4, S100P, and other S100 proteins). There is also a growing interest in developing inhibitors of S100A1 since they may be beneficial for treating a variety of human diseases including neurological diseases, diabetes mellitus, heart failure, and several types of cancer. The absence of significant phenotypes in S100A1 knockout mice provides some early indication that an S100A1 antagonist could have minimal side effects in normal tissues. However, development of S100A1-mediated therapies is complicated by S100A1's unusual ability to function as both an intracellular signaling molecule and as a secreted protein. Additionally, many S100A1 protein targets have only recently been identified, and so fully characterizing both these S100A1-target complexes and their resulting functions is a necessary prerequisite.

Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1-/- muscle fibres.

In the preceding paper, we reported that flexor digitorum brevis (FDB) muscle fibres from S100A1 knock-out (KO) mice exhibit a selective suppression of the delayed, steeply voltage-dependent component of intra-membrane charge movement current termed Q(gamma). Here, we use 50 microm of the Ca(2+) indicator fluo-4 in the whole cell patch clamp pipette, in addition to 20 mM EGTA and other constituents included for the charge movement studies, and calculate the SR Ca(2+) release flux from the fluo-4 signals during voltage clamp depolarizations. Ca(2+) release flux is decreased in amplitude by the same fraction at all voltages in fibres from S100A1 KO mice compared to fibres from wild-type (WT) littermates, but unchanged in time course at each pulse membrane potential. There is a strong correlation between the time course and magnitude of release flux and the development of Q(gamma). The decreased Ca(2+) release in KO fibres is likely to account for the suppression of Q(gamma) in these fibres. Consistent with this interpretation, 4-chloro-m-cresol (4-CMC; 100 microm) increases the rate of Ca(2+) release and restores Q(gamma) at intermediate depolarizations in fibres from KO mice, but does not increase Ca(2+) release or restore Q(gamma) at large depolarizations. Our findings are consistent with similar activation kinetics for SR Ca(2+) channels in both WT and KO fibres, but decreased Ca(2+) release in the KO fibres possibly due to shorter SR channel open times. The decreased Ca(2+) release at each voltage is insufficient to activate Q(gamma) in fibres lacking S100A1.

The Qgamma component of intra-membrane charge movement is present in mammalian muscle fibres, but suppressed in the absence of S100A1.

S100A1 is a Ca(2+) binding protein that modulates excitation-contraction (EC) coupling in skeletal and cardiac muscle. S100A1 competes with calmodulin for binding to the skeletal muscle SR Ca(2+) release channel (the ryanodine receptor type 1, RyR1) at a site that also interacts with the C-terminal tail of the voltage sensor of EC coupling, the dihydropyridine receptor. Ablation of S100A1 leads to delayed and decreased action potential evoked Ca(2+) transients, possibly linked to altered voltage sensor activation. Here we investigate the effects of S100A1 on voltage sensor activation in skeletal muscle utilizing whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in isolated flexor digitorum brevis (FDB) muscle fibres from wild-type and S100A1 knock-out (KO) mice. In contrast to recent reports, we found that FDB fibres exhibit two distinct components of intra-membrane charge movement, an initial rapid component (Q(beta)), and a delayed, steeply voltage dependent 'hump' component (Q(gamma)) previously recorded primarily in amphibian but not mammalian fibres. Surprisingly, we found that Q(gamma) was selectively suppressed in S100A1 KO fibres, while the Q(beta) component of charge movement was unaffected. This result was specific to S100A1 and not a compensatory result of genetic manipulation, as transient intracellular application of S100A1 restored Q(gamma). Furthermore, we found that exposure to the RyR1 inhibitor dantrolene suppressed a similar component of charge movement in FDB fibres. These results shed light on voltage sensor activation in mammalian muscle, and support S100A1 as a positive regulator of the voltage sensor and Ca(2+) release channel in skeletal muscle EC coupling.

Solution structure of S100A1 bound to the CapZ peptide (TRTK12).

As is typical for S100-target protein interactions, a Ca 2+-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 Ca 2+-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 Ca 2+-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 Ca 2+-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 Ca 2+-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 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.

S100A1 binds to the calmodulin-binding site of ryanodine receptor and modulates skeletal muscle excitation-contraction coupling.

S100A1, a 21-kDa dimeric Ca2+-binding protein, is an enhancer of cardiac Ca2+ release and contractility and a potential therapeutic agent for the treatment of cardiomyopathy. The role of S100A1 in skeletal muscle has been less well defined. Additionally, the precise molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release in striated muscle has not been fully elucidated. Here, utilizing a genetic approach to knock out S100A1, we demonstrate a direct physiological role of S100A1 in excitation-contraction coupling in skeletal muscle. We show that the absence of S100A1 leads to decreased global myoplasmic Ca2+ transients following electrical excitation. Using high speed confocal microscopy, we demonstrate with high temporal resolution depressed activation of sarcoplasmic reticulum Ca2+ release in S100A1-/- muscle fibers. Through competition assays with sarcoplasmic reticulum vesicles and through tryptophan fluorescence experiments, we also identify a novel S100A1-binding site on the cytoplasmic face of the intact ryanodine receptor that is conserved throughout striated muscle and corresponds to a previously identified calmodulin-binding site. Using a 12-mer peptide of this putative binding domain, we demonstrate low micromolar binding affinity to S100A1. NMR spectroscopy reveals this peptide binds within the Ca2+-dependent hydrophobic pocket of S100A1. Taken together, these data suggest that S100A1 plays a significant role in skeletal muscle excitation-contraction coupling, primarily through specific interactions with a conserved binding domain of the ryanodine receptor. This warrants further investigation into the use of S100A1 as a therapeutic target for the treatment of both cardiac and skeletal myopathies.

The three-dimensional solution structure of Ca(2+)-bound S100A1 as determined by NMR spectroscopy.

S100A1 is an EF-hand-containing Ca(2+)-binding protein that undergoes a conformational change upon binding calcium as is necessary to interact with protein targets and initiate a biological response. To better understand how calcium influences the structure and function of S100A1, the three-dimensional structure of calcium-bound S100A1 was determined by multidimensional NMR spectroscopy and compared to the previously determined structure of apo. In total, 3354 nuclear Overhauser effect-derived distance constraints, 240 dihedral constraints, 160 hydrogen bond constraints, and 362 residual dipolar coupling restraints derived from a series of two-dimensional, three-dimensional, and four-dimensional NMR experiments were used in its structure determination (>21 constraints per residue). As with other dimeric S100 proteins, S100A1 is a symmetric homodimer with helices 1, 1', 4, and 4' associating into an X-type four-helix bundle at the dimer interface. Within each subunit there are four alpha-helices and a short antiparallel beta-sheet typical of two helix-loop-helix EF-hand calcium-binding domains. The addition of calcium did not change the interhelical angle of helices 1 and 2 in the pseudo EF-hand significantly; however, there was a large reorientation of helix 3 in the typical EF-hand. The large conformational change exposes a hydrophobic cleft, defined by residues in the hinge region, the C terminus, and regions of helix 3, which are important for the interaction between S100A1 and a peptide (TRTK-12) derived from the actin-capping protein CapZ.

Structure of human brain fructose 1,6-(bis)phosphate aldolase: linking isozyme structure with function.

Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 A resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.

Molecular mechanisms of S100-target protein interactions.

S100 proteins have no known enzymatic activity and exert their intracellular effects via interaction with and regulation of the activity of other proteins, termed target proteins, in both a Ca(2+)-dependent and Ca(2+)-independent manner. Structural studies have identified the linker region between the two EF-hand Ca(2+) binding domains and the C-terminus as Ca(2+)-dependent target protein binding sites in several S100 family members. In fact, C-terminal aromatic residues are obligatory for interaction of S100A1 with several of its Ca(2+)-dependent target proteins. Pharmacological studies suggest the presence of additional Ca(2+)-dependent binding motifs on some family members. A minimum of seven family members interact with and regulate the activity of aldolase A in a Ca(2+)-independent manner. In the case of S100A1, Ca(2+)-independent target protein interactions utilize a binding motif distinct from the C-terminal Ca(2+)-dependent target protein binding site. Several studies suggest that ionic interactions participate in the interaction of S100 family members with Ca(2+)-independent target proteins. While some target proteins are activated by multiple family members, other target proteins exhibit family member-specific activation, i.e., they are activated by a single family member. As predicted, family member specific interactions appear to be mediated by regions that exhibit the most divergence in amino acid sequence among family members, the linker or "hinge" region and the C terminus. Further specificity in S100-target protein interactions may arise from the different biochemical/biophysical properties of the individual family members, including affinity for metal ions (Ca(2+), Zn(2+), and Cu(2+)), oligomerization properties, heterodimerization, post-translational modifications, and lipid-binding. Delineation of the structural motifs that mediate S100-target protein interactions and determination of the in vivo relevance of these interactions are needed to fully understand the role of S100 proteins in normal and diseased cells.

The myocardial protein S100A1 plays a role in the maintenance of normal gene expression in the adult heart.

S100A1 and S100B are members of a family of 20 kDa Ca2+-binding homodimers that play a role in signal transduction in mammalian cells. S100A1 is the major isoform in normal heart and S100B, normally a brain protein, is induced in hypertrophic myocardium and functions as an intrinsic negative modulator of the hypertrophic response. In order to examine the function of S100A1, we first showed that, in contrast to S100B, S100A1 was downregulated in rat experimental models of myocardial hypertrophy following myocardial infarction or pressure overload. Second, in co-transfection experiments in cultured neonatal rat cardiac myocytes, S100A1 inhibited the alpha1-adrenergic activation of promoters of genes induced during the hypertrophic response including the fetal genes skeletal alpha actin (skACT), and beta-myosin heavy chain (MHC) and S100B, but not the triiodothyronine (T3) activation of the promoter of the alpha-MHC gene, that is normally expressed in adult myocardium. These results suggest that S100A1 is involved in the maintenance of the genetic program that defines normal myocardial function and that its downregulation is permissive for the induction of genes that underlie myocardial hypertrophy.