Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies.

Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on delivering external electron donors or acceptors to initiate electron transfer reactions. This is a challenge. Small-molecule redox reagents can act indiscriminately, poisoning the cell. Natural redox proteins are more selective, but finding the right partner can be difficult due to the limited number of redox potentials and difficulty tuning them. De novo proteins offer an alternative path. They are robust and can withstand mutations that allow for tailorable changes. They are also devoid of evolutionary artifacts and readily bind redox cofactors. However, no reliable set of engineering principles have been developed that allow for these proteins to be fine-tuned so their redox midpoint potential (Em) can form donor/acceptor pairs with any natural oxidoreductase. This work dissects protein-cofactor interactions that can be tuned to modulate redox potentials of acceptors and donors using a mutable de novo designed tetrahelical protein platform with iron tetrapyrrole cofactors as a test case. We show a series of engineered heme b-binding de novo proteins and quantify their resulting effect on Em. By focusing on the surface charge and buried charges, as well as cofactor placement, chemical modification, and ligation of cofactors, we are able to achieve a broad range of Em values spanning a range of 330 mV. We anticipate this work will guide the design of proteinaceous tools that can interface with natural oxidoreductases inside and outside the cell while shedding light on how natural proteins modulate Em values of bound cofactors.

Analysis of alternative splicing associated with aging and neurodegeneration in the human brain.

Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of individuals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)-dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)-dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration.

Understanding splicing regulation through RNA splicing maps.

Alternative splicing is a highly regulated process that greatly increases the proteome diversity and plays an important role in cellular differentiation and disease. Interactions between RNA-binding proteins (RBPs) and pre-mRNA are the principle regulator of splicing decisions. Findings from recent genome-wide studies of protein-RNA interactions have been combined with assays of the global effects of RBPs on splicing to create RNA splicing maps. These maps integrate information from all pre-mRNAs regulated by single RBPs to identify the global positioning principles guiding splicing regulation. Recent studies using this approach have identified a set of positional principles that are shared between diverse RBPs. Here, we discuss how insights from RNA splicing maps of different RBPs inform the mechanistic models of splicing regulation.

Complex genetic changes in strains of Saccharomyces cerevisiae derived by selection in the laboratory.

Selection of model organisms in the laboratory has the potential to generate useful substrates for testing evolutionary theories. These studies generally employ relatively long-term selections with weak selective pressures to allow the accumulation of multiple adaptations. In contrast to this approach, we analyzed two strains of Saccharomyces cerevisiae that were selected for resistance to multiple stress challenges by a rapid selection scheme to test whether the variation between rapidly selected strains might also be useful in evolutionary studies. We found that resistance to oxidative stress is a multigene trait in these strains. Both derived strains possess the same major-effect adaptations to oxidative stress, but have distinct modifiers of the phenotype. Similarly, both derived strains have altered their global transcriptional responses to oxidative stress in similar ways, but do have at least some distinct differences in transcriptional regulation. We conclude that short-term laboratory selections can generate complex genetic variation that may be a useful substrate for testing evolutionary theories.

C/EBP and Cdx family factors regulate liver fatty acid binding protein transgene expression in the small intestinal epithelium.

A transgene constructed from the rat liver fatty acid binding protein gene (Fabp1) promoter is active in all murine small intestinal crypt and villus epithelial cells. Coincident Cdx and C/EBP transcription factor binding sites were identified spanning Fabp1 nucleotides -90 to -78. CDX-1, CDX-2, C/EBPalpha, and C/EBPbeta activated the Fabp1 transgene in CaCo-2 cells, and mutagenizing the -78 site prevented activation by these factors. CDX but not C/EBP factors bound to the site in vitro, although C/EBP factors competed with CDX factors for transgene activation. The -78 site adjoins an HNF-1 site, and CDX and C/EBP family factors cooperated with HNF-1alpha but not HNF-1beta to activate the transgene. Furthermore, CDX-1, CDX-2, C/EBPalpha, and C/EBPbeta bound to HNF-1alpha and HNF-1beta. The transgene with a mutagenized -78 site was silenced in vivo specifically in small intestinal crypt epithelial cells but remained active in villus cells. These results demonstrate functional interactions between HNF-1, C/EBP, and CDX family factors and suggest that these interactions may contribute to differential transcriptional regulation in the small intestinal crypt and villus compartments.