Cellular Adaptation Relies on Regulatory Proteins Having Episodic Memory

The ability to memorize changes in the environment is present at all biological levels, from social groups and individuals, down to single cells. Trans-generational memory is embedded subcellularly through genetic and epigenetic mechanisms. Evidence that cells process and remember features of the immediate environment using protein sensors is reviewed. It is argued that this mnemonic ability is encapsulated within the protein conformational space and lasts throughout its lifetime, which can overlap with the lifespan of the organism. Means to determine diachronic changes in protein activity are presented.     

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca²⁺ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His₁₀) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.     

Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.     

RESOURCERER: a database for annotating and linking microarray resources within and across species

Microarray expression analysis is providing unprecedented data on gene expression in humans and mammalian model systems. Although such studies provide a tremendous resource for understanding human disease states, one of the significant challenges is cross-referencing the data derived from different species, across diverse expression analysis platforms, in order to properly derive inferences regarding gene expression and disease state. To address this problem, we have developed RESOURCERER, a microarray-resource annotation and cross-reference database built using the analysis of expressed sequence tags (ESTs) and gene sequences provided by the TIGR Gene Index (TGI) and TIGR Orthologous Gene Alignment (TOGA) databases [now called Eukaryotic Gene Orthologs (EGO)].     

Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.     

Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae

The product of the ARO10 gene from Saccharomyces cerevisiae was initially identified as a thiamine diphosphate-dependent phenylpyruvate decarboxylase with a broad substrate specificity. It was suggested that the enzyme could be responsible for the catabolism of aromatic and branched-chain amino acids, as well as methionine. In the present study, we report the overexpression of the ARO10 gene product in Escherichia coli and the first detailed in vitro characterization of this enzyme. The enzyme is shown to be an efficient aromatic 2-keto acid decarboxylase, consistent with it playing a major in vivo role in phenylalanine, tryptophan and possibly also tyrosine catabolism. However, its substrate spectrum suggests that it is unlikely to play any significant role in the catabolism of the branched-chain amino acids or of methionine. A homology model was used to identify residues likely to be involved in substrate specificity. Site-directed mutagenesis on those residues confirmed previous studies indicating that mutation of single residues is unlikely to produce the immediate conversion of an aromatic into an aliphatic 2-keto acid decarboxylase. In addition, the enzyme was compared with the phenylpyruvate decarboxylase from Azospirillum brasilense and the indolepyruvate decarboxylase from Enterobacter cloacae. We show that the properties of the two phenylpyruvate decarboxylases are similar in some respects yet quite different in others, and that the properties of both are distinct from those of the indolepyruvate decarboxylase. Finally, we demonstrate that it is unlikely that replacement of a glutamic acid by leucine leads to discrimination between phenylpyruvate and indolepyruvate, although, in this case, it did lead to unexpected allosteric activation.     

Chronic exposure to beta-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents

To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.