Ranjana Mehta

Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans

Anti-Aging Several human neurodegenerative diseases, such as Alzheimers and Huntingtons, are caused by aberrant protein aggregation. These disorders typically develop after the fifth decade of life, suggesting a connection with the aging process. In a number of different species, life span can be extended by dietary restriction and reduced insulin and insulin-like growth factor–1 (IGF-1) signaling. These pathways can also decrease toxic protein aggregation, mechanistically linking aging with proteotoxic diseases. While searching for regulators of proteotoxicity in Caenorhabditis elegans, Mehta et al. (p. 1196, published online 16 April) found that reduction of the von Hippel–Lindau tumor suppressor homolog VHL-1 significantly increased life span and enhanced resistance to proteotoxicity. VHL-1 is an E3 ubiquitin ligase that negatively regulates the hypoxic response, and animals grown under hypoxic conditions lived longer. This alternative longevity pathway was distinct from both dietary restriction and insulin/IGF-1–like signaling. Induction of the hypoxic response in a worm slows aging and enhances resistance to proteotoxicity. The Caenorhabditis elegans von Hippel–Lindau tumor suppressor homolog VHL-1 is a cullin E3 ubiquitin ligase that negatively regulates the hypoxic response by promoting ubiquitination and degradation of the hypoxic response transcription factor HIF-1. Here, we report that loss of VHL-1 significantly increased life span and enhanced resistance to polyglutamine and β-amyloid toxicity. Deletion of HIF-1 was epistatic to VHL-1, indicating that HIF-1 acts downstream of VHL-1 to modulate aging and proteotoxicity. VHL-1 and HIF-1 control longevity by a mechanism distinct from both dietary restriction and insulin-like signaling. These findings define VHL-1 and the hypoxic response as an alternative longevity and protein homeostasis pathway.

Regulation of mRNA Translation as a Conserved Mechanism of Longevity Control

Appropriate regulation of mRNA translation is essential for growth and survival and the pathways that regulate mRNA translation have been highly conserved throughout eukaryotic evolution. Translation is controlled by a complex set of mechanisms acting at multiple levels, ranging from global protein synthesis to individual mRNAs. Recently, several mutations that perturb regulation of mRNA translation have also been found to increase longevity in three model organisms: the buddingyeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Many of these translation control factors can be mapped to a single pathway downstream of the nutrient responsive target of rapamycin (TOR) kinase. In this chapter, we will review the data suggesting that mRNA translation is an evolutionarily conserved modifier of longevity and discuss potential mechanisms by which mRNA translation could influence aging and age-associated disease in different species.

Adenylylation and Catalytic Properties of Mycobacterium tuberculosis Glutamine Synthetase Expressed in Escherichia coli versus Mycobacteria

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456–464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially (∼25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated (∼30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.

Adenylylation and catalytic properties of M. tuberculosis glutamine synthetase expressed in E. coli vs. mycobacteria

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456–464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially (∼25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated (∼30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.

Characterization of a Recombinant Form of Annexin VI for Detection of Apoptosis

We developed a recombinant form of human annexin VI called annexin VI-601 (M(r) 76,224) with the N-terminal extension of Ala-Gly-Gly-Cys-Gly-His to allow ready attachment of fluorescent or radioactive labels. The protein was produced by expression in E. coli and was purified by calcium-dependent membrane binding, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. The protein could be readily labeled with iodoacetamidofluorescein and with (99m)Tc. The protein bound with high affinity to PS-containing phospholipid vesicles and to erythrocytes with exposed phosphatidylserine. Fluorescent annexin VI-601 readily detected apoptosis of Jurkat cells by flow cytometry at much lower calcium concentrations than those required for equivalent detection by annexin V. In vivo administration of radiolabeled protein showed that blood clearance was much slower than annexin V. In conclusion, annexin VI may have advantages over annexin V in certain situations for both in vitro and in vivo detection of apoptosis and therapeutic targeting of PS due to its lower calcium requirement for membrane binding and its higher molecular weight.

Rapid Extension of Single and Double Stranded DNA on Atomically Flat Conductive Surfaces

We report a simple method for extending DNA on atomically flat conductive graphite and gold surfaces employing molecular combing and using either coordinating ions on graphite or self-assembled molecular monolayers on gold. Extended DNA molecules on conductive surfaces can be used as platforms for building nanoscale electronic devices or used for direct electronic sequencing.

Nanoscale Sensors for Health Monitoring in Prolonged Space Missions.

We present a nanoscale biomolecule sensor for use in immune system monitoring in astronauts on long space flights in conditions of microgravity. Silicon nanowire based sensors were fabricated in single crystal Silicon-on-Insulator (SOI) wafers and have a footprint of approximately 1 μm. The versatile sensing transduction mechanism, through functionalization of the nanowire surface, allows for sensor adaptation to many relevant biomolecular targets. The ability to directly read out sensor data electronically eliminates the need for large optical analysis equipment necessary in traditional biomolecule sensing devices. Despite the fact that the sensors were found to have excellent transistor characteristics and promising results have been previously reported, much work remains in making the sensors behave in a more reliable and reproducible fashion necessary for critical tasks such as health monitoring.