Multiomics of -Dependent Replicative Lifespan Extension Models Reveals Gcn4 as a Regulator of Protein Turnover in Yeast.

We have shown that multiple tRNA synthetase inhibitors can increase lifespan in both the nematode and the budding yeast by acting through the conserved transcription factor Gcn4 (yeast)/ATF-4 (worms). To further understand the biology downstream from this conserved transcription factor in the yeast model system, we looked at two different yeast models known to have upregulated Gcn4 and -dependent increased replicative lifespan. These two models were yeast and yeast treated with the tRNA synthetase inhibitor borrelidin.

Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved.

Mapping protein-protein interactions in homodimeric CYP102A1 by crosslinking and mass spectrometry.

Covalent crosslinking and mass spectrometry techniques hold great potential in the study of multiprotein complexes, but a major challenge is the inability to differentiate intra- and inter- protein crosslinks in homomeric complexes. In the current study we use CYP102A1, a well-characterized homodimeric P450, to examine a subtractive method that utilizes limited crosslinking with disuccinimidyl dibutyric urea (DSBU) and isolation of the monomer, in addition to the crosslinked dimer, to identify inter-monomer crosslinks. The utility of this approach was examined with the use of MS-cleavable crosslinker DSBU and recently published cryo-EM based structures of the CYP102A1 homodimer.

SPOCK, an R based package for high-throughput analysis of growth rate, survival, and chronological lifespan in yeast.

Plate reader-based methods for high-throughput measurement of growth rate, cellular survival, and chronological lifespan are a compelling addition to the already powerful toolbox of budding yeast genetics. These methods have overcome many of the limits of traditional yeast biology techniques, but also present a new bottleneck at the point of data-analysis. Herein, we describe SPOCK (Survival Percentage and Outgrowth Collection Kit), an R-based package for the analysis of data created by high-throughput plate reader based methods.

lifespan measurement.

Aging is a fundamental biological process that is still not fully understood. As many of the most significant human diseases have aging as their greatest risk factor, a better understanding of aging potentially has enormous practical implications in treating these diseases. The nematode is an exceptionally useful genetic model organism that had been used with great success to shed light on many genes and pathways that are involved in aging.