p66Shc in Cardiovascular Pathology.

p66Shc is a widely expressed protein that governs a variety of cardiovascular pathologies by generating, and exacerbating, pro-apoptotic ROS signals. Here, we review p66Shc's connections to reactive oxygen species, expression, localization, and discuss p66Shc signaling and mitochondrial functions. Emphasis is placed on recent p66Shc mitochondrial function discoveries including structure/function relationships, ROS identity and regulation, mechanistic insights, and how p66Shc-cyt c interactions can influence p66Shc mitochondrial function. Based on recent findings, a new p66Shc mitochondrial function model is also put forth wherein p66Shc acts as a rheostat that can promote or antagonize apoptosis. A discussion of how the revised p66Shc model fits previous findings in p66Shc-mediated cardiovascular pathology follows.

P66Shc (Shc1) Zebrafish Mutant Line as a Platform for Testing Decreased Reactive Oxygen Species in Pathology.

Reactive oxygen species (ROS) dysregulation exacerbates many pathologies but must remain within normal ranges to maintain cell function. Since ROS-mediated pathology and routine cell function are coupled, in vivo models evaluating low-ROS background effects on pathology are limited. Some models alter enzymatic antioxidant expression/activity, while others involve small molecule antioxidant administration. These models cause non-specific ROS neutralization, decreasing both beneficial and detrimental ROS. This is detrimental in cardiovascular pathology, despite the negative effects excessive ROS has on these pathologies. Thus, current trends in ROS-mediated pathology have shifted toward selective inhibition of ROS producers that are dysregulated during pathological insults, such as p66Shc. In this study, we evaluated a zebrafish heterozygote p66Shc hypomorphic mutant line as a low-ROS myocardial infarction (MI) pathology model that mimics mammalian MI. Our findings suggest this zebrafish line does not have an associated negative phenotype, but has decreased body mass and tissue ROS levels that confer protection against ROS-mediated pathology. Therefore, this line may provide a low-ROS background leading to new insights into disease.

Overproduction of Membrane-Associated, and Integrated, Proteins Using Saccharomyces cerevisiae.

Structural and functional eukaryotic membrane protein research continues to grow at an increasing rate, placing greater significance on leveraging productive protein expression pipelines to feed downstream studies. Bacterial expression systems (e.g., E. coli) are often the preferred system due to their simple growth conditions, relative simplicity in experimental workflow, low overall cost per liter of cell growth, and ease of genetic manipulation. However, overproduction success of eukaryotic membrane proteins in bacterial systems is hindered by the limited native processing ability of bacterial systems for important protein folding interactions (e.g., disulfide bonds), post-translational modifications (e.g., glycosylation), and inherent disadvantages in protein trafficking and folding machinery compared to other expression systems.In contrast, Saccharomyces cerevisiae expression systems combine positive benefits of simpler bacterial systems with those of more complex eukaryotic systems (e.g., mammalian cells). Benefits include inexpensive growth, robust DNA repair and recombination machinery, amenability to high density growths in bioreactors, efficient transformation, and robust post-translational modification machinery. These characteristics make S. cerevisiae a viable first-alternative when bacterial overproduction is insufficient. Thus, this chapter provides a framework, using methods that have proven successful in prior efforts, for overproducing membrane anchored or membrane integrated proteins in S. cerevisiae. The framework is designed to improve yields for all levels of overexpression expertise, providing optimization insights for the variety of processes involved in heterologous protein expression.

Purification of Membrane Proteins Overexpressed in Saccharomyces cerevisiae.

Membrane protein (MP) functional and structural characterization requires large quantities of high-purity protein for downstream studies. Barriers to MP characterization include ample overexpression, solubilization, and purification of target proteins while maintaining native activity and structure. These barriers can be overcome by utilizing an efficient purification protocol in a high-yield eukaryotic expression system such as Saccharomyces cerevisiae. S. cerevisiae offers improved protein folding and posttranslational modifications compared to prokaryotic expression systems. This chapter contains practices used to overcome barriers of solubilization and purification using S. cerevisiae that are broadly applicable to diverse membrane associated, and membrane integrated, protein targets.