Age-dependent upregulation of p53 and cytochrome c release and susceptibility to apoptosis in skeletal muscle fiber of aged rats: role of carnitine and lipoic acid

Mitochondrial dysfunction has been implicated in the regulation of myofiber loss during aging, possibly by apoptotic pathways. However, the mitochondrial-mediated pathway of apoptosis by cytochrome c in skeletal muscle remains ambiguous. To understand this, we have studied the upstream and downstream events of cytochrome c release, and assessed the efficacy of carnitine and lipoic acid cosupplementation. The results show that elevated levels of cytosolic cytochrome c activate apoptosis in aged rats, and was confirmed further by in vitro caspase-3 assay. Interestingly, the exogenous addition of cytochrome c results in a much higher increase of caspase-3 activity in aged treated rats than age-matched control rats, strongly suggesting that cytochrome c is a limiting factor for caspase-3 activation in the cytosol. Carnitine and lipoic acid supplement decreased apoptosis in aged rats by maintaining mitochondrial membrane integrity and thereby preventing further loss of cytochrome c in vivo. Furthermore, the upregulation of p53 observed in aged rats is attributed to the loss of outer mitochondrial membrane integrity and subsequent release of cytochrome c through BH3-only proteins. In conclusion, the p53-dependent activation of the mitochondrial-cytochrome c pathway of apoptosis in the present study suggests the existence of cross talk between mitochondria and nucleus. However, the exact molecular mechanism remains to be explored. Oral supplements of carnitine and lipoic acid play an antiapoptotic role in aged rat skeletal muscle by protecting mitochondrial membrane integrity.     

A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula

Many higher plants establish symbiotic relationships with arbuscular mycorrhizal (AM) fungi that improve their ability to acquire nutrients from the soil. In addition to establishing AM symbiosis, legumes also enter into a nitrogen-fixing symbiosis with bacteria known as rhizobia that results in the formation of root nodules. Several genes involved in the perception and transduction of bacterial symbiotic signals named "Nod factors" have been cloned recently in model legumes through forward genetic approaches. Among them, DMI3 (Doesn't Make Infections 3) is a calcium- and calmodulin-dependent kinase required for the establishment of both nodulation and AM symbiosis. We have identified, by a yeast two-hybrid system, a novel protein interacting with DMI3 named IPD3 (Interacting Protein of DMI3). IPD3 is predicted to interact with DMI3 through a C-terminal coiled-coil domain. Chimeric IPD3::GFP is localized to the nucleus of transformed Medicago truncatula root cells, in which split yellow fluorescent protein assays suggest that IPD3 and DMI3 physically interact in Nicotiana benthamiana. Like DMI3, IPD3 is extremely well conserved among the angiosperms and is absent from Arabidopsis. Despite this high level of conservation, none of the homologous proteins have a demonstrated biological or biochemical function. This work provides the first evidence of the involvement of IPD3 in a nuclear interaction with DMI3.     

Structural basis of kinetic variations in Sucrose Phosphate Phosphatase (SPP) of rice and Anabaena unveiled through computational analysis

Sucrose is an important storage form of assimilated carbon in many plant species. Unlike other sucrose biosynthetic enzymes, Sucrose Phosphate Phosphatase (SPP), the terminal enzyme in sucrose biosynthetic pathway, is the least understood. SPPs from different organisms have different kinetic properties. The current study focuses on the structural differences among SPP homologues and unveils the probable structural basis of kinetic variations. We have employed computational methods of molecular modeling and structure comparisons and identified structural variations in some of the substrate binding residues, amino acid substitutions in regions that are lining the active site and minute structural differences that can enhance the nucleophilicity of a catalytic nucleophile (Asp 9 ). We report a structurally and hence functionally important amino acid substitution (Asp 159 by Alanine) in one of the rice SPP isoforms, which can result in the disruption of a H-bond that helps in binding of sucrose at the active site of the enzyme. In this paper we discuss the structural basis of enhanced catalytic efficiency of rice SPP in comparison with a cyanobacterium (Anabaena variabilis). The natural mutations identified in our analysis of the SPP catalytic domain would be useful in re-designing the enzyme for enhanced catalytic efficiency and higher sucrose production.     

Uncommon presentation of a common tachycardia

We describe a patient with an implanted pacemaker for impaired AV conduction who presented with an incessant tachycardia. EP study showed that the tachycardia was atrioventricular nodal reentrant tachycardia (AVNRT) with repeated spontaneous initiation because of poor or absent antegrade fast pathway conduction. Slow pathway ablation was successful in terminating the tachycardia and making it non-inducible.     

A support vector machine-based method for predicting the propensity of a protein to be soluble or to form inclusion body on overexpression in Escherichia coli

MOTIVATION: Inclusion body formation has been a major deterrent for overexpression studies since a large number of proteins form insoluble inclusion bodies when overexpressed in Escherichia coli. The formation of inclusion bodies is known to be an outcome of improper protein folding; thus the composition and arrangement of amino acids in the proteins would be a major influencing factor in deciding its aggregation propensity. There is a significant need for a prediction algorithm that would enable the rational identification of both mutants and also the ideal protein candidates for mutations that would confer higher solubility-on-overexpression instead of the presently used trial-and-error procedures. RESULTS: Six physicochemical properties together with residue and dipeptide-compositions have been used to develop a support vector machine-based classifier to predict the overexpression status in E.coli. The prediction accuracy is approximately 72% suggesting that it performs reasonably well in predicting the propensity of a protein to be soluble or to form inclusion bodies. The algorithm could also correctly predict the change in solubility for most of the point mutations reported in literature. This algorithm can be a useful tool in screening protein libraries to identify soluble variants of proteins.     

Effects of salt on the thermal stability of human plasma high-density lipoprotein

High-density lipoproteins (HDL) mediate cholesterol removal and thereby protect against atherosclerosis. Mature spherical HDL contain the apolar lipid core and polar surface of proteins and phospholipids. Earlier, we showed that the structural integrity of HDL is modulated by kinetic barriers that prevent spontaneous protein dissociation and lipoprotein fusion and rupture. To determine the role of electrostatic interactions in the kinetic stability of mature HDL, here we analyze the effects of salt and pH on their thermal denaturation. In low-salt buffer at pH 5.7-7.7, HDL are highly thermostable. Increasing the salt concentration from 0 to 0.3 M NaCl causes low-temperature shifts in the calorimetric HDL transitions of up to -14 degrees C. This salt-induced destabilization leads to protein unfolding below 100 degrees C, facilitating the first Arrhenius analysis of HDL denaturation by circular dichroism spectroscopy. In 150 mM NaCl, two kinetic phases in HDL protein unfolding are observed: a faster phase with an activation energy E(a,fast) < or =15 kcal/mol and a slower phase with an E(a,slow) = 50 +/- 7 kcal/mol. Gel electrophoresis and electron microscopic data suggest that the faster phase involves partial protein unfolding but no significant protein dissociation or changes in HDL size, while the slower phase involves complete protein unfolding, partial protein dissociation, and HDL fusion. Hence, the slower phase may resemble HDL remodeling and fusion by plasma enzymes during metabolism. Analysis of the effects of various salts, sucrose, and pH suggests that HDL destabilization by salt results from ionic screening of favorable short-range electrostatic interactions such as salt bridges. Consequently, electrostatic interactions significantly contribute to the high thermostability of HDL in low-salt solutions.     

Uniform binding and negative catalysis at the origin of enzymes

Enzymes are well known for their catalytic abilities, some even reaching “catalytic perfection” in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate‐handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a “substrate‐handle‐binding‐first” mechanism (“binding‐first” for brevity) instead of “chemistry‐first” and we are, therefore, left to wonder what the role of non‐catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back‐to‐back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as “uniform binding” (parallel stabilization of all enzyme‐bound states to the same degree). Indeed, we show that a uniform‐binding proto‐catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate‐handle‐binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction''s course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of “binding‐first” enzyme families like the aldolase superfamily.