Targeting protein–protein interactions within the cyclic AMP signaling system as a therapeutic strategy for cardiovascular disease

The cAMP signaling system can trigger precise physiological cellular responses that depend on the fidelity of many protein–protein interactions, which act to bring together signaling intermediates at defined locations within cells. In the heart, cAMP participates in the fine control of excitation–contraction coupling, hence, any disregulation of this signaling cascade can lead to cardiac disease. Due to the ubiquitous nature of the cAMP pathway, general inhibitors of cAMP signaling proteins such as PKA, EPAC and PDEs would act non-specifically and universally, increasing the likelihood of serious ‘off target’ effects. Recent advances in the discovery of peptides and small molecules that disrupt the protein–protein interactions that underpin cellular targeting of cAMP signaling proteins are described and discussed

Spinal Cord Injury Markedly Altered Protein Expression Patterns in the Affected Rat Urinary Bladder during Healing Stages

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27 (Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

<p>Abstract</p> <p>Background</p> <p>A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.</p> <p>Methods</p> <p>Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.</p> <p>Results</p> <p>Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell <it>in vitro </it>and attenuated active force development of intact tissue <it>ex vivo</it>.</p> <p>Conclusions</p> <p>This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.</p

HDAC8 substrates: Histones and beyond

The lysine deacetylase family of enzymes (HDACs) was first demonstrated to catalyze deacetylation of acetyllysine residues on histones. In subsequent years, HDACs have been shown to recognize a large pool of acetylated nonhistone proteins as substrates. Recently, thousands of acetylated proteins have been discovered, yet in most cases, the HDAC that catalyzes deacetylation in vivo has not been identified. This gap has created the need for better in vivo, in vitro, and in silico approaches for determining HDAC substrates. While HDAC8 is the best kinetically and structurally characterized HDAC, few efficient substrates have yet been substantiated in vivo. In this review, we delineate factors that may be important for determining HDAC8 substrate recognition and catalytic activity, including structure, complex formation, and post‐translational modifications. This summary provides insight into the challenges of identifying in vivo substrates for HDAC8, and provides a good vantage point for understanding the variables important for predicting HDAC substrate recognition. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 112–126, 2013.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94512/1/22135_ftp.pd

BAG3: a multifaceted protein that regulates major cell pathways

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression

Muscle wasting and the temporal gene expression pattern in a novel rat intensive care unit model

<p>Abstract</p> <p>Background</p> <p>Acute quadriplegic myopathy (AQM) or critical illness myopathy (CIM) is frequently observed in intensive care unit (ICU) patients. To elucidate duration-dependent effects of the ICU intervention on molecular and functional networks that control the muscle wasting and weakness associated with AQM, a gene expression profile was analyzed at time points varying from 6 hours to 14 days in a unique experimental rat model mimicking ICU conditions, i.e., post-synaptically paralyzed, mechanically ventilated and extensively monitored animals.</p> <p>Results</p> <p>During the observation period, 1583 genes were significantly up- or down-regulated by factors of two or greater. A significant temporal gene expression pattern was constructed at short (6 h-4 days), intermediate (5-8 days) and long (9-14 days) durations. A striking early and maintained up-regulation (6 h-14d) of muscle atrogenes (muscle ring-finger 1/tripartite motif-containing 63 and F-box protein 32/atrogin-1) was observed, followed by an up-regulation of the proteolytic systems at intermediate and long durations (5-14d). Oxidative stress response genes and genes that take part in amino acid catabolism, cell cycle arrest, apoptosis, muscle development, and protein synthesis together with myogenic factors were significantly up-regulated from 5 to 14 days. At 9-14 d, genes involved in immune response and the caspase cascade were up-regulated. At 5-14d, genes related to contractile (myosin heavy chain and myosin binding protein C), regulatory (troponin, tropomyosin), developmental, caveolin-3, extracellular matrix, glycolysis/gluconeogenesis, cytoskeleton/sarcomere regulation and mitochondrial proteins were down-regulated. An activation of genes related to muscle growth and new muscle fiber formation (increase of myogenic factors and JunB and down-regulation of myostatin) and up-regulation of genes that code protein synthesis and translation factors were found from 5 to 14 days.</p> <p>Conclusions</p> <p>Novel temporal patterns of gene expression have been uncovered, suggesting a unique, coordinated and highly complex mechanism underlying the muscle wasting associated with AQM in ICU patients and providing new target genes and avenues for intervention studies.</p

PKA phosphorylation of the small heat-shock protein Hsp20 enhances its cardioprotective effects

The small heat-shock protein Hsp20 (heat-shock protein 20), also known as HspB6, has been shown to protect against a number of pathophysiological cardiac processes, including hypertrophy and apoptosis. Following β-adrenergic stimulation and local increases in cAMP, Hsp20 is phosphorylated on Ser&lt;sup&gt;16&lt;/sup&gt; by PKA (protein kinase A). This covalent modification is required for many of its cardioprotective effects. Both Hsp20 expression levels and its phosphorylation on Ser&lt;sup&gt;16&lt;/sup&gt; are increased in ischaemic myocardium. Transgenic mouse models with cardiac-specific overexpression of Hsp20 that are subject to ischaemia/reperfusion show smaller myocardial infarcts, and improved recovery of contractile performance during the reperfusion phase, compared with wild-type mice. This has been attributed to Hsp20's ability to protect against cardiomyocyte necrosis and apoptosis. Phosphomimics of Hsp20 (S16D mutants) confer improved protection from β-agonist-induced apoptosis in the heart, whereas phospho-null mutants (S16A) provide no protection. Naturally occurring mutants of Hsp20 at position 20 (P20L substitution) are associated with markedly reduced Hsp20 phosphorylation at Ser&lt;sup&gt;16&lt;/sup&gt;, and this lack of phosphorylation correlates with abrogation of Hsp20's cardioprotective effects. Therefore phosphorylation of Hsp20 at Ser&lt;sup&gt;16&lt;/sup&gt; by PKA is vital for the cardioprotective actions of this small heat-shock protein. Selective targeting of signalling elements that can enhance this modification represents an exciting new therapeutic avenue for the prevention and treatment of myocardial remodelling and ischaemic injury