Centromere binding factor 1 (Cbf1) is a transcription factor that controls the transcription of many genes involved in cellular respiration and lipid biogenesis and, as such, has been associated with hypolipidemia in humans. It is a known substrate for PAS kinase, which phosphorylates Cbf1 and alters its activity. Our hypothesis is that this phosphorylation affects the genes it regulates and the DNA motifs it binds to, perhaps due to different transcription complexes being formed. In this study, we conduct a chromatin immunoprecipitation in Saccharomyces cerevisiae to determine what genes and DNA motifs Cbf1 binds to in its wild type versus phosphosite mutant forms. We discovered several motifs that may be specific to each Cbf1 form, however further evidence is necessary. We were able to identify five motifs in reads associated with phosphosite Cbf1, while reads associated with wild type Cbf1 had 16 motifs, with no overlap between the motifs found from the two forms. This may be due to phosphorylated Cbf1 having more binding partners. Cbf1 regulated genes and possible transcription complex binding partners are proposed.

Glucose allocation is an important cellular process that is misregulated in the interrelated diseases obesity, diabetes and cancer. Cells have evolved critical mechanisms for regulating glucose allocation, one of which is sensory protein kinases. PAS kinase is a key sensory protein kinase that regulates glucose allocation in yeast, mice and man; and is a novel therapeutic target for the treatment of metabolic diseases such as obesity, diabetes and cancer. Despite its importance, the molecular mechanisms of PAS kinase function are largely unknown. Through large-scale protein-interaction studies, we have identified 93 novel binding partners for PAS kinase which help to expand its role in glucose allocation as well as suggest novel roles for PAS kinase including mitochondrial metabolism, cell growth/division, protein modification, stress tolerance, and gene/protein expression. From a subset of these binding partners, we identified 5 in vitro substrates of PAS kinase namely Mot3, Utr1, Zds1, Cbf1 and Pbp1. Additionally, we have further characterized Pbp1 and Cbf1 as PAS kinase substrates through both in vitro and in vivo evidence as well as phenotypic analysis. Evidence is provided for the PAS kinase-dependent phosphorylation and activation of Pbp1, which in turn inhibits cell proliferation through the sequestration of TORC1. In contract, PAS kinase-dependent phosphorylation of Cbf1 inhibits its activity, decreasing cellular respiration. This work elucidates novel molecular mechanisms behind PAS kinase function in both mitochondrial and cell growth pathways in eukaryotic cells, increasing our understanding of the regulation of central metabolism.

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The evolution and adaptation of microorganisms is so rapid it can be seen in the time frame of days. The root cause for their evolution comes from selective environmental pressures that see organisms with beneficial mutations survive otherwise deadly encounters or outperform members of its population who fail to adapt. This does not always result in strict improvement of the individual as in the case of antibiotic resistant bacteria who often display fitness tradeoffs to avoid death (see Reviews [1-3]). For example, when an ampicillin resistance gene (ampC) containing plasmid that is occasionally found in the wild was transformed into S. typhimurium the bacteria had slower growth and impaired invasiveness [4]. In another example, capreomycin use with mycobacteria resulted in lower binding of the drug to the ribosome through mutations in rRNA methylase TlyA 16S rRNA, which decreases the overall fitness of the mycobacteria [5]. The evolutionary interactomes between bacteria and antibiotics do not end there, as antibiotic resistant bacteria often accumulate compensatory mechanisms to regain fitness. These range in effect with some altering individual cellular pathways and others having systemic affects [1]. My work has focused on the intersection of diabetes and related antibiotic resistant bacterial infections. Diabetes is one of the leading health issues in the United States, with over 10% of the adult population and over 26% of the elderly diagnosed (American Diabetes Association) [6]. Herein I further characterize the molecular pathways involved in diabetes, through the study of PAS kinase (PASK) function. PAS kinase is a serine-threonine protein kinase which regulates the pathways disrupted in diabetes, namely triglyceride accumulation, metabolic rate (respiration), adiposity and insulin production and sensitivity [7-9]. In this study I specifically focus on the effects of PAS kinase and its substrate, USF1/Cbf1p, and how their altered metabolic deficiencies can be suppressed using yeast cells. Through this study I further characterized the molecular function of USF1/Cbf1p through the identification of putative co-transcriptional regulators, identify novel genes involved in the regulation of respiration, and uncover a function or a previous uncharacterized protein, Pal1p. Part of the diabetes healthcare challenge results from the wide range of diseases that are associated with diabetes, including obesity [10, 11], renal failure [12, 13], neuropathies and neurodegeneration [14, 15], endocrine dysfunctions [16, 17], and cancers [18]. In addition, diabetes is a leading cause of lower limb amputations, due to poor circulation and the prevalence of ulcers [19-21], many of which are antibiotic resistant [22-25]. Phage therapy, based on the administration of bacterial viruses, is a viable option for the treatment of these diseases, with our lab recently isolating bacteriophages for several clinical cases. In the second half of my thesis, I present the study of the adaptation of bacteriophages to their hosts as well as report contributions of local ecology to their evolution.

The second identified substrate of PAS kinase discussed is Pbp1. The human homolog of Pbp1 is ataxin-2, mutations in which are a known risk factor for amyotrophic lateral sclerosis (ALS). As diet and sex have been shown to be important factors regarding PAS kinase function, they also are strong contributing factors to ALS and are extensively reviewed herein. Pbp1 is known to be sequestered by PAS kinase under glucose depravation, and it can sequester additional proteins along with it to regulate different cellular pathways. To shed light on the pathways affected by Pbp1, we performed a yeast two-hybrid assay and mass spectrometry, identifying 32 novel interacting partners of Pbp1 (ataxin-2). We provide further analysis of the direct binding partner Ptc6, measuring mitophagy, mitochondrial content, colocalization, and respiration. This work elucidates novel molecular mechanisms behind the function of PAS kinase and yields valuable insights into the role of PAS kinase in disease.

Nutrient sensing kinases lie at the heart of cellular health and homeostasis, allowing cells to quickly adapt to changing environments. Target of Rapamycin (TOR) and PAS kinase (PASK, or PASKIN) are two such nutrient kinases, conserved from yeast to man. In yeast, these kinases each have paralogs. The two TOR paralogs in yeast mimic the mammalian TORC1 and TORC2 complexes, except both Tor1 and Tor2 may contribute to TORC1 or TORC2 function. The two PAS kinase paralogs are paired with the TOR paralogs, meaning that both Psk1 and Psk2 regulate TORC1, while Psk2 suppresses a temperature-sensitive allele of Tor2. Herein we review the evolutionary models for these paralogs, their function in yeast and mammalian cells, as well as the overlapping function of PAS kinase and TOR. We also use Rice University’s Direct Coupling Analysis algorithms to analyze co-evolutionary relationships and identify potential interaction sites between PAS kinase and several of its substrates.