Life Sciences Commons^™

Proteomic Approaches To Identify Unique And Shared Substrates Among Kinase Family Members, Charles Lincoln Howarth

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a reversible post-translational modification that is a critical component of almost all signaling pathways. Kinases regulate substrate proteins through phosphorylation, and nearly all proteins are phosphorylated to some extent. Crucially, breakdown in phosphorylation signaling is an underlying factor in many diseases, including cancer. Understanding how phosphorylation signaling mediates cellular pathways is crucial for understanding cell biology and human disease.

Targeted protein degradation (TPD) is a strategy to rapidly deplete a protein of interest (POI) and is applicable to any gene that is amenable to CRISPR-Cas9 editing. One TPD approach is the auxin-inducible degron (AID) system, which relies …

Novel Mechanistic Insight Into Ciliary Regulation: Old Pathways Yield New Mechanisms, Larissa L. Dougherty

Dartmouth College Ph.D Dissertations

Cilia are structures present on most eukaryotic cells which provide important signaling and motile components to cells from early development to fully differentiated and matured cells. Regulation of these structures is critical to proper functioning of the cell and is known to be tied to the cell cycle. Preparation for ciliary assembly following cell cycle exit and ciliary disassembly following cell cycle reentry requires components throughout the cell body and within the cilium to facilitate this process. Here I identify how the cell adapts to ensure modifications to cilia occur for assembly or disassembly using the model organism Chlamydomonas reinhardtii. …

Mitochondrial Division: Synergizing In Mitochondrial Divisome, Ao Liu

Dartmouth College Ph.D Dissertations

Mitochondria are the energy factories of the cell. The dynamic nature of cells demands routine changes in mitochondrial morphology by fusion and division. The dynamin GTPase Drp1 is a central mitochondrial division protein, driving constriction of the outer mitochondrial membrane via oligomerization. At least four regulatory factors control Drp1 activity on the outer mitochondrial membrane (OMM): 1) receptor proteins (Mff, MiD49, MiD51, and Fis1); 2) actin filaments; 3) the mitochondrial phospholipid cardiolipin (CL); and 4) Drp1 post-translational modifications, of which two phosphorylation sites (S579 and S600) are the most well studied. However, the molecular mechanism of how these factors work …

Deciphering Phosphoprotein Phosphatase Signaling Networks Using Proteomics Approaches, Brooke Brauer

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a highly regulated mechanism of cell signaling control and its deregulation is implicated in disease. The kinases that catalyze the addition of phosphate groups onto their substrate proteins have been well studied, their signaling pathways mapped, and their effects on cell and organismal health observed. Knowledge of the phosphatases that reverse the reaction only recently began to come into focus. Phosphoprotein phosphatases (PPPs), long thought to be housekeeping enzymes, are now known to be exquisitely specific towards their substrates, but the exact nature of phosphatase regulation—both upstream and downstream of the phosphatase—is unclear.

PPPs recognize substrates through …

Cysteine Metallochemistry And Metal Binding: Quantification Of The Thermodynamic Foundations Of Cellular Homeostasis, Matthew R. Mehlenbacher

Dartmouth College Ph.D Dissertations

Metals are required for life. Many metalloproteins contain cysteine in their metal-binding site (MBS) and cysteines are unique in that they are reactive, and strongly bind certain metals, which aid in metal selectivity and specificity. Using isothermal titration calorimetry (ITC), the thermodynamic foundation for metal binding, cellular protection, and transcriptional regulation, which all utilize cysteines in their MBS, are quantified.

In bacteria there are metalloprotein pathways that actively uptake mercury, which are regulated by the metalloregulatory protein MerR. MerR de-represses the transcription of these mer proteins in a metal-dependent manner. Using ITC, the thermodynamic foundation of the negative allosteric coupling …