Browsing School, Graduate by Subject "S100A1"
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S100A1 modulation of skeletal muscle excitation-contraction couplingS100A1, a 21 kDa dimeric Ca2+ binding protein, enhances cardiac Ca2+ release and contractility, and is a potential therapeutic agent for the treatment of cardiomyopathy. A role of S100A1 in skeletal muscle is less well defined. Additionally, the molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release has not been fully elucidated. Here, utilizing a genetic approach to knock out (KO) S100A1, I demonstrate a physiologic role of S100A1 in skeletal muscle excitation-contraction (EC) coupling. Using high-speed confocal microscopy, I show that ablation of S100A1 leads to delayed myoplasmic Ca2+ transients with decreased amplitude following an action potential in isolated flexor digitorum brevis (FDB) muscle fibers. Through binding assays and competition experiments, I identify a novel S100A1 binding site on the cytoplasmic face of the ryanodine receptor (RyR1) that corresponds to a previously identified calmodulin (CaM) binding domain (CaMBD). I find that S100A1 competes with CaM for this site, which also interacts with the voltage sensor of EC coupling, the dihydropyridine receptor. To investigate effects of S100A1 on the voltage sensor, I utilized whole-cell patch clamp electrophysiology to record intra-membrane charge movement currents in WT and KO fibers. In contrast to recent reports, I find that FDB fibers exhibit two distinct components of charge movement, an initial rapid component (Qgamma) and a delayed, steeply voltage dependent "hump" component (Qbeta;) previously recorded primarily in amphibian but not mammalian fibers. Surprisingly, I find that Qgamma is selectively suppressed in S100A1 KO fibers. Finally, I explore the effects of S100A1 on whole muscle contractile force, to test if S100A1's modulation of single fiber Ca2+ release translates to altered contractile performance in vivo. I find that tibialis anterior muscles of S100A1-/- mice generate less contractile force and exhibit a greater rate of fatigue than WT counterparts. Taken together, these data suggest S100A1 binds to the CaMBD of RyR1 and enhances voltage-gated Ca2+ release, leading to elevated myoplasmic Ca2+ and increased contractile force following muscle fiber excitation. This thesis sheds light on voltage sensor activation of Ca2+ release in skeletal muscle, and supports S100A1 as a positive regulator of EC coupling.
Structural and functional studies on S100A1S100 proteins are small, dimeric calcium binding proteins that contain two EF-hands per subunit. While these proteins are expressed plentifully in a tissue-specific manner, the cellular functions of most S100 proteins remain understudied. This dissertation describes efforts to elucidate both the structure and the function of one member of this family, S100A1, in various biologically relevant states. Multidimensional NMR spectroscopy techniques were used to solve the solution structure of S100A1 in its Ca 2+-bound form. The protein folds as a symmetric homodimer, with an X-type helix bundle comprising the dimer interface. A large conformational change, involving the reorientation of helix 3, accompanies S100A1 calcium binding. This change exposes a previously hidden hydrophobic pocket which is the general target protein binding site and specifically the binding site for the calmodulin binding domain of the ryanodine receptor. The solution structure of S100A1 bound to a peptide from this region, along with whole-cell calcium transient measurements in S100A1 knockout mice, provide evidence that S100A1 directly increases RyR1-mediated calcium release by binding to a discrete area on the cytoplasmic face of RyR1. This binding event is driven by both hydrophobic and ionic interactions between S100A1 and the RyR peptide. A competition between S100A1 and calmodulin for the same region of RyR1 likely represents a cellular mechanism of modulating SR Ca2+ release in skeletal muscle fibers.;Studies detailing the internal backbone dynamics of the Ca2+-loaded form of another S100 protein, S100B, are also presented in this manuscript. These experiments were conducted to further our understanding of the molecular mechanisms driving S100 protein structure and function. Here, S100B is shown to be a stable protein in the Ca2+-bound form, with significant motions in the hydrophobic pocket. These motions may assist in peptide binding, and support a previously suggested coupling between Ca2+ and target peptide binding in S100 proteins.
Targeting Malignant Melanoma and Potential Off-target Effects in EC-couplingS100B belongs to the S100 family of Ca2+-binding proteins, a family known for calcium-dependent interactions that regulate biological processes. Upregulation of S100B in malignant melanoma (MM) downregulates p53 tumor suppressor function and is correlated with poor prognosis, making S100B a therapeutic target for MM. A fragment-based drug discovery program is underway to develop small-molecule S100B inhibitors. Compounds SC0025 and SC1990 occupy part of the S100B hydrophobic cleft, termed site 3, while compounds SBi5361 and 5363 occupy sites 1-3. Crystal structures show specific protein-inhibitor interactions to exploit in further studies for improving affinity and specificity. Heteronuclear RNA-binding protein (hnRNP) A18 is also involved in MM. A18 is upregulated in tumors and promotes tumor growth via coordination of pro-survival mRNA. The crystal structure for the RNA recognition motif (RRM) of A18 is reported here, with comparisons to the homologous RNA-binding protein, hnRNP A1. These comparisons show a conserved global fold and conservation of known RNA-binding residues. Given this, it would be impossible to design inhibitors specific for A18. Instead, it is the intrinsically disordered domain of A18 that must endow specificity, as this is not conserved. As such, this structure serves as a foundation for work with full-length A18 and drug-design efforts targeting A18 in MM. The sibling protein to S100B, S100A1 regulates several cellular processes, including Ca2+-signaling in striated muscle, through interaction with the ryanodine receptor. The crystal structure of S100A1, reported here, provides insights into S100A1-target binding specificity through key differences in the binding pockets of S100A1 and S100B. In cardiac cells, S100A1 increases Cav1 channel current amplitude, an effect blocked by inhibition of protein kinase A (PKA), implying a PKA-dependent process. As this did not require cAMP, its mechanism of activation remained unknown. Biochemical studies demonstrate that S100A1 directly activates PKA in a Ca2+-dependent manner. A functional role for this pathway is also established as PKA-dependent subcellular redistribution of HDAC4 was abolished in S100A1 knockout mice. Thus, the interaction between S100A1 and PKA provides a link between Ca2+- and PKA-signaling.