Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive ...target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.
The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids ...can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.
Melanolipofuscin (MLF) is a complex granule, exhibiting properties of both melanosomes and lipofuscin (LF) granules, which accumulates in retinal pigment epithelial (RPE) cells and may contribute to ...the etiology of age-related macular degeneration (AMD). MLF accumulation has been reported by Feeney-Burns to more closely reflect the onset of AMD than the accumulation of lipofuscin. In an effort to assess the possible contribution MLF may have to the onset of AMD, we analyzed the phototoxicity and protein composition of MLF and compared those results to that of LF.
Specifically, we observed the accumulation of MLF in human RPE from different decades of life, and assessed the phototoxicity of these granules. We also employed fluorescence spectroscopy, atomic force microscopy, transmission and scanning electron microscopy and proteomic analysis to examine the composition of MLF granules in an effort to ascertain their origin.
Our results show that MLF granules are phototoxic and their accumulation more closely reflects the onset of AMD than does LF accumulation. Our compositional analysis of MLF has shown that while these granules contain some similarities to LF granules, MLF is substantially different. Of significant interest is the finding that MLF, in contrast to LF, does not contain photoreceptor-specific proteins, suggesting that MLF may not originate from the phagocytosis of photoreceptor outer segments. Instead the presence of RPE- and melanosome-specific proteins would suggest that MLF accumulates as a result of the melanosomal autophagocytosis of RPE cells.
Our results provide significant insight into understanding the formation and toxicity of MLF and suggest a possible contribution to the etiology of retinal diseases.
Bmx nonreceptor tyrosine kinase has an established role in endothelial and lymphocyte signaling; however, its role in the heart is unknown. To determine whether Bmx participates in cardiac growth, we ...subjected mice deficient in the molecule (Bmx knockout mice) to transverse aortic constriction (TAC). In comparison with wild-type mice, which progressively developed massive hypertrophy following TAC, Bmx knockout mice were resistant to TAC-induced cardiac growth at the organ and cell level. Loss of Bmx preserved cardiac ejection fraction and decreased mortality following TAC. These findings are the first to demonstrate a necessary role for the Tec family of tyrosine kinases in the heart and reveal a novel regulator (Bmx) of pressure overload-induced hypertrophic growth.
Abstract only
Cardiovascular disease is the leading cause of death in the developed world and includes growth of the heart (hypertrophy) resulting from altered mechanical and neural/hormonal factors. ...As a major clinical problem resultant from atherosclerosis and heart attacks, novel insights into hypertrophy are needed, in particular regarding changes in the diseased nuclear and cytoskeletal proteomes. To this end, transverse aortic constriction (TAC) was performed to induce pressure overload hypertrophy in mice. Hearts were excised between 1‐8 weeks post‐TAC, nuclei isolated and proteins separated by 2D or 1D PAGE and subjected to LC/MS/MS. Of ~1000 nuclear proteins exhibiting reproducible changes in abundance (p>0.05) after pressure overload, myozenin‐a molecular link between the phosphatase calcineurin and the structural protein α‐actinin‐was found to be upregulated. Further analysis of myozenin via immunoblotting and MS/MS revealed a truncated form that appears during hypertrophy. In addition, novel phosphorylation of residues S106, S116, T107 and T111 on myozenin‐2 was observed. Comparison of apparent mass using electrophoresis, immunoblotting and MS peptide maps revealed regions of modification on myozenin following injury. These studies add a new dimension to the role of myozenin in the heart and provide insights into nuclear‐cytoskeletal communication during cardiac hypertrophy.
Support: NIH, AHA
Focal adhesions are anatomical structures in cardiac cells at the juxtaposition of the extracellular matrix and the sarcolemma and are involved in mechanotransduction, or the transmission of ...mechanical forces to enact intracellular responses. Focal adhesion kinase (FAK) is thought to mediate signaling between the membrane and intracellular organelles (e.g. nucleus), however, the proteins involved in FAK signaling at different subcellular locations remain unknown. Therefore, we examined specific subsets of FAK interacting proteins by optimized immunoprecipitation (IP). To identify robust interactions, we examined those that were retained when the IP procedure was conducted in 2% Triton X‐100, followed by SDS‐PAGE separation and identification by LC/MS/MS. This stringency of wash conditions retained interactions of FAK primarily with structural proteins including integrins, actin, myosin, vimentin and SERCA. We are repeating these analyses following isolation with less detergent (0.5% NP‐40) to identify proteins more transiently interacting with FAK and therefore potentially involved in intracellular signaling. We are also using proteomics and confocal microscopy to evaluate the proteins involved in FAK signaling specifically at the nucleus. Together these studies will provide important insights into the signaling microenvironment of FAK in cardiac cells.
Supported by AHA and NIH/NHLBI.
Ligand-gated ion channels underlie synaptic communication in the nervous system(1). In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X ...receptor channel family(2). In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions(3, 4), which have downstream signaling roles(5) (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes(6). In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain. P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release(7). P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function(8). P2X2 receptors are abundant in the nervous system(9) and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 & TM2) separated by an extracellular region(7) and intracellular N and C termini (Fig 1a)(7). P2X subunits(10) (P2X1-P2X7) show 30 50% sequence homology at the amino acid level(11). P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors(9) no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C terminus of P2X2 receptors.
To elucidate the origins of biologically active retinal lipofuscin (RLF) by examining its protein composition.
Total protein and total lipid were extracted and quantified. Proteins in this ...lipoprotein granule were identified by limited-scale proteomic analysis using both two-dimensional (2D) gel electrophoresis and SDS-PAGE coupled with MALDI-QqToF MSMS and automated LCMSMS, respectively.
RLF granules were 44% protein and 50% lipid. Proteomic analyses identified 41 constituent proteins. Hydrophobic proteins and several proteins specific to photoreceptors, including rhodopsin, that have not previously been reported, were identified. Extensive protein modification, especially oxidative damage, was observed.
Proteins identified support the model that RLF accumulates in RPE cells as a result of the buildup of undigested material from the phagocytosis of photoreceptor outer segments. Perhaps oxidative damage renders some of these proteins indigestible and thus leads to the accumulation of RLF granules.
The Tec family of non‐receptor tyrosine kinases (Tec, Btk, Bmx, Emt and Txk) have well established roles in cell growth and differentiation. These proteins are known to be activated by growth factors ...and cytokines and are critically involved in T‐cell activation. Recent studies have suggested a role for Bmx in ischemic injury to mouse heart and skeletal muscle, however the actions of the Tec family in cardiac signal transduction remain largely unknown. In the present study, we conducted comprehensive analyses of Tec family expression in whole heart and isolated cardiac myocytes under normal and ischemic conditions. Immunoblotting for Tec kinases revealed expression of Bmx and Tec in both whole heart and myocyte lysates. Interestingly, we reproducibly observe two distinct bands when blotting for Tec in myocyte lysates and only one band in whole heart lysate, suggesting the existence of two Tec isoforms in myocytes. We have optimized immunoprecipitations for isolation of Tec‐interacting proteins (including preclearing with IgG control to reduce nonspecific contamination) and are using this approach to evaluate the Tec signaling network in cardiac cells. These Tec‐associated proteins have been electrophoresed, trypsinized and are being identified by LC/MS/MS. These studies are an important first step to understanding the mechanistic role of Tec family kinases in myocardial ischemia.
Supported by AHA & NIH/NHLBI.
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