We show that repeating units from all reported disease genes are capable of forming hairpins of common structure and threshold stability. The threshold stability is roughly -50 kcal per hairpin and is influenced by the flanking sequence of the gene. Hairpin stability has two components, sequence and length; only DNA of select sequences and the correct length can form hairpins of threshold energy. There is a correlation among the ability to form hairpins of threshold stability, the sequence selectivity of expansion, and the length dependence of expansion. Additionally, hairpin formation provides a potential structural basis for the constancy of the CCG region of the Huntington's disease gene in individuals and explains the stabilizing effects of AGG interruptions in FMR1 alleles.

BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding.

Rapid exchange of high energy carrying molecules between intracellular compartments is essential in sustaining cellular energetic homeostasis. Adenylate kinase (AK)-catalyzed transfer of adenine nucleotide ␤-and ␥-phosphoryls has been implicated in intracellular energy communication and nucleotide metabolism. To demonstrate the significance of this reaction in cardiac energetics, phosphotransfer dynamics were determined by [18 O]phosphoryl oxygen analysis using 31 P NMR and mass spectrometry. In hearts with a null mutation of the AK1 gene, which encodes the major AK isoform, total AK activity and ␤-phosphoryl transfer was reduced by 94% and 36%, respectively. This was associated with up-regulation of phosphoryl flux through remaining minor AK isoforms and the glycolytic phosphotransfer enzyme, 3-phosphoglycerate kinase. In the absence of metabolic stress, deletion of AK1 did not translate into gross abnormalities in nucleotide levels, ␥-ATP turnover rate or creatine kinase-catalyzed phosphotransfer. However, under hypoxia AK1-deficient hearts, compared with the wild type, had a blunted AK-catalyzed phosphotransfer response, lowered intracellular ATP levels, increased P i /ATP ratio, and suppressed generation of adenosine. Thus, although lack of AK1 phosphotransfer can be compensated in the absence of metabolic challenge, under hypoxia AK1-knockout hearts display compromised energetics and impaired cardioprotective signaling. This study, therefore, provides first direct evidence that AK1 is essential in maintaining myocardial energetic homeostasis, in particular under metabolic stress. Adenylate kinase (AK)1 catalyzes reversible phosphotransfer, 2 ADP 7 AMP ϩ ATP, and participates in de novo synthesis, regeneration and salvage of adenine nucleotides (1-5). AK is particularly abundant in tissues with high energy turnover, where it facilitates transfer of energy-rich ␤-and ␥-phosphoryls and regulates vital ATP-dependent cellular processes (6 -10).In fact, AK may serve as an integral component of phosphotransfer networks, along with creatine kinase (CK) and glycolysis, effectively coupling ATP-generating with ATP-consuming or ATP-sensing intracellular sites (11-15).In the heart, CK-catalyzed phosphotransfer is the major pathway that can transfer high energy phosphoryls derived from the ␥-phosphoryl of ATP (10, 16 -18). Although less active than CK, AK catalysis provides a unique mechanism for transfer and utilization of both ␥-and ␤-phosphoryls in the ATP molecule (10, 15). In this way, AK-catalyzed phosphotransfer doubles the energetic potential of ATP and could provide an additional energetic source under conditions of increased energy demand (10, 19). However, due to lack of membrane permeant and selective AK inhibitors, the biological importance of AK in heart muscle and its role in sustaining myocardial energetics under conditions of metabolic stress have not been established.We have recently demonstrated that deletion of the AK1 gene, which encodes the major AK isoform, produces a phenotype with reduced skelet...

Cell survival is critically dependent on the preservation of cellular bioenergetics. However, the metabolic mechanisms that confer resistance to injury are poorly understood. Phosphotransfer reactions integrate ATPconsuming with ATP-producing processes and could thereby contribute to the generation of a protective phenotype. Here, we used ischemic preconditioning to induce a stress-tolerant state and 18 O-assisted 31 P nuclear magnetic resonance spectroscopy to capture intracellular phosphotransfer dynamics. Preconditioning of isolated perfused hearts triggered a redistribution in phosphotransfer flux with significant increase in creatine kinase and glycolytic rates. High energy phosphoryl fluxes through creatine kinase, adenylate kinase, and glycolysis in preconditioned hearts correlated tightly with post-ischemic functional recovery. This was associated with enhanced metabolite exchange between subcellular compartments, manifested by augmented transfer of inorganic phosphate from cellular ATPases to mitochondrial ATP synthase. Preconditioning-induced energetic remodeling protected cellular ATP synthesis and ATP consumption, improving contractile performance following ischemia-reperfusion insult. Thus, the plasticity of phosphotransfer networks contributes to the effective functioning of the cellular energetic system, providing a mechanism for increased tolerance toward injury.Cells with high energy turnover are particularly vulnerable to insults induced by deprivation of oxygen and metabolic substrates (1-3). Recently, endogenous defense mechanisms have been discovered that can "precondition" cells to withstand metabolic stress (3, 4). Preconditioning underlying cytoprotection has implicated multiple metabolic, signal transduction, and electrical events (3-10). However, the actual mechanisms responsible for preservation of cellular energetic systems and, ultimately, functional recovery remain poorly understood.In the heart, coupling of energetics with contractile function is facilitated through phosphotransfer relays, catalyzed by creatine kinase, adenylate kinase, and glycolysis (11-15). Poor contractile performance in the failing myocardium is associated with deficits in phosphotransfer-dependent metabolic signaling (16 -19). Furthermore, disruption in phosphotransfer enzymes compromises the ability of heart muscle to respond to metabolic stress (20 -23). Alterations in cellular energy metabolism triggered by ischemic preconditioning, including a characteristic creatine phosphate overshoot, indicates that this protective process targets phosphotransfer reactions (24 -27). However, direct evidence demonstrating the protective role of phosphotransfer networks in the preconditioned state is still lacking.Here, we demonstrate that ischemic preconditioning of heart muscle induces remodeling in cellular energy transduction, transfer, and utilization processes, thereby promoting preservation of energy metabolism. Post-ischemic contractile recovery was tightly associated with preconditioning-induced adjustment in me...

No abstract