UCSF

Dynamic changes in intracellular pH (pHi) are confirmed to regulate myriad cell behaviors, including proliferation, cell-substrate adhesion, cell migration, dysplasia and tumorigenesis, and stem cell differentiation for lineage specification. Previous work revealed how the reversible protonation of endogenous pH sensitive proteins, collectively termed “pH sensors,” is a mechanism whereby pHi dynamics regulates these cell behaviors. Often, these proteins contain a structurally or functionally critical histidine which can titrate between protonated and neutral within the narrow physiological range and affect the electrostatics both in cis and with binding partners. My thesis research focused on three predicted pH sensors, transcription factors regulating gene expression, the muscle isoform of the enzyme phosphofructokinase (PFKM) regulating glycolysis, and a charge changing mutant p53-R273H promoting cancer progression. For transcription factors, we identified at least 65 in distinct families, including FOX, SOX and MITF/Myc, that contain a conserved histidine in their DNA binding domain (DBD) that in available structures forms a direct hydrogen bond with nucleotides. Focusing on FOX family transcription factors, we identified pH-regulated binding affinities for a canonical FkhP sequence with higher affinity pH 7.0 compared with pH 7.5 for FOXC2, FOXM1, and FOXN1. For FOXC2, we determined greater activity at lower pHi in cells and confirmed that pH-dependent binding and activity is mediated a conserved histidine (His122) in the DBD. Additionally, using an unbiased in vitro screen (SELEX) we identified differences in binding DNA motif preferences between pH 7 and 7.8. For PFKM, we determined that His242 is required but not sufficient for a pH-sensing mechanism of allosteric relief of ATP inhibition of enzyme activity. For p53-R273H we screened bioactive compounds from three commercially available libraries to identify two compounds that restore DNA binding, which we previously showed is decreased at the higher pHi of cancer cells. Collectively, these data reveal how pH broadly regulates protein electrostatics specifically at titratable histidine residues to regulate diverse behaviors including gene expression, metabolism, and cancer. Moreover, our current findings establish a previously unreported mode of regulation for transcription factors across diverse families, and confirm the feasibility of targeting charge changing mutations in disease by using small molecules to restore protein function.