Friedreich cardiomyopathy is a desminopathy.

Heart disease is an integral part of Friedreich ataxia (FA) and the most common cause of death in this autosomal recessive disease. The result of the mutation is lack of frataxin, a small mitochondrial protein. The clinical and pathological phenotypes of FA are complex, involving brain, spinal cord, dorsal root ganglia, sensory nerves, heart, and endocrine pancreas. The hypothesis is that frataxin deficiency causes downstream changes in the proteome of the affected tissues, including the heart. A proteomic analysis of heart proteins in FA cardiomyopathy by antibody microarray, Western blots, immunohistochemistry, and double-label laser scanning confocal immunofluorescence microscopy revealed upregulation of desmin and its chaperone protein, αB-crystallin. In normal hearts, these two proteins are co-localized at intercalated discs and Z discs. In FA, desmin and αB-crystallin aggregate, causing chaotic modification of intercalated discs, clustering of mitochondria, and destruction of the contractile apparatus of cardiomyocytes. Western blots of tissue lysates in FA cardiomyopathy reveal a truncated desmin isoprotein that migrates at a lower molecular weight range than wild type desmin. While desmin and αB-crystallin are not mutated in FA, the accumulation of these proteins in FA hearts allows the conclusion that FA cardiomyopathy is a desminopathy akin to desmin myopathy of skeletal muscle.

Profiling of p5, a 24 Amino Acid Inhibitory Peptide Derived from the CDK5 Activator, p35 CDKR1 Against 70 Protein Kinases.

Cyclin-dependent kinase 5 (CDK5) is a multifunctional serine/threonine kinase that regulates a large number of neuronal processes essential for nervous system development and function with its activator p35 CDK5R1. Upon neuronal insults, p35 is proteolyzed and cleaved to p25 producing deregulation and hyperactivation of CDK5 (CDK5/p25), implicated in tau hyperphosphorylation, a pathology in some neurodegenerative diseases. A truncated, 24 amino acid peptide, p5, derived from p35 inhibits the deregulated CDK5 phosphotransferase activity and ameliorates Alzheimer's disease (AD) phenotypes in AD model mice. In the present study, we have screened a diverse panel of 70 human protein kinases for their sensitivities to p5, and a subset of these to p35. At least 16 of the tested protein kinases exhibited IC50 values that were 250 µM or less, with CAMK4, ZAP70, SGK1, and PIM1 showing greater sensitivity to inhibition by p5 than CDK5/p35 and CDK5/p25. In contrast, the p5 peptide modestly activated LKB1 and GSK3ß. A sub set of kinases screened against p35 showed that activity of CAMK4 in the absence of calcium and calmodulin was also markedly inhibited by p35. The Cyclin Y-dependent kinases PFTK1 (CDK14) and PCTK1 (CDK16) were activated by p35 at least 10-fold in the absence of Cyclin Y and by approximately 50% in its presence. These findings provide additional insights into the mechanisms of action for p5 and p35 in the regulation of protein phosphorylation in the nervous system.

Kinetworks protein kinase multiblot analysis.

The proteomics analysis of protein kinases and other cell-signaling proteins in tumor samples by traditional two-dimensional (2-D) gel electrophoresis is complicated by the low abundance of these regulatory proteins relative to metabolic enzymes and structural proteins. We present an antibody-based method called Kinetworks that relies on sodium dodecyl sulfate (SDS)-poly-acrylamide minigel electrophoresis and multilane immunoblotters to permit the specific and quantitative detection of 45 or more protein kinases or other signal transduction proteins at once. The technique can also permit the resolution of these proteins based on differences in their phosphorylation state and other forms of covalent modification. Kinetworks profiling of protein kinases in solid human tumors and cell lines can reveal profound differences in their expression and phosphorylation states, which can serve for the identification of cancer diagnostic markers and therapeutic targets for drug discovery.