UC Davis

C-peptide exhibits beneficial effects, particularly in diabetic patients, but its clinical use has been hampered by a lack of mechanistic understanding. The work described in this thesis focuses on the advancement of C-peptide as a co-therapeutic with insulin for diabetic patients by elucidating the molecular mechanisms that drive the beneficial signaling effects of C-peptide. The work involved in understanding C-peptide’s mechanisms of action during circulation, internalization, and peptide signaling include 1) identifying which metal co-factors interact with C-peptide and alter its intracellular functions, 2) analyzing how selected metal co-factors mediate ternary interactions between C-peptide and serum albumin, another potential regulatory factor, and impacts the biochemical behavior of the peptide, 3) revealing how high glucose exposure alters structure of serum albumin and modifies ternary complexations with C-peptide, and 4) determining how metal-bound C-peptide internalizes in different endocrine organs and shifts intracellular Cu trafficking levels linking to copper-related diseases. Chapter 2 addresses the first goal in determining which essential first-row d-block transition metals play a role on the function of C-peptide using spectroscopic tools such as electronic absorption spectroscopy. Cr(III), Cu(II), and Zn(II) demonstrated binding to C-peptide with differing stoichiometries and biologically relevant binding affinities. Cr(III) and Cu(II) in particular modulated peptide internalization activity through subtle structural changes, pointing to the biological importance of metal ions as co-factors and its impact on the function of bioactive peptides. To this end, Chapter 3 expands on the regulatory factors associated with C-peptide by demonstrating the combined impact of Cu(II) and the serum protein albumin on the activity of C-peptide. By tackling the second goal, this chapter displays a combination of powerful tools such as FRET screening with Cu(II) and Zn(II) and spectroscopic tools. To complement the CD and electronic absorption spectroscopy studies, a collaborative effort with the Britt lab and Wang lab at UC Davis were extended to elucidate the coordination chemistry behind these ternary complexes using EPR and TDDFT studies. This work shows that Cu(II) distinctly mediates the formation of ternary complexes between albumin and C-peptide and that the resulting species depend on the order of addition. The resulting species revealed two schematics of binding, one of which is confirmed by both EPR and TDDFT simulations, whereas the other schematic points to a possible second binding site on C-peptide, as previously determined the Heffern lab. Due to differences in structure, both ternary complexes notably alter peptide activity, showing differences from the peptide or Cu(II)/peptide complexes alone in redox protection as well as in cellular internalization of the peptide. In standard clinical immunoassays for measuring C-peptide levels, the complexes inflate the quantitation of the peptide, suggesting that such adducts may affect biomarker quantitation. Altogether, our work points to the potential relevance of Cu(II)-linked C-peptide/albumin complexes in the peptide's mechanism of action and application as a clinical biomarker. Because the focus on C-peptide mechanisms is tailored towards diabetic patients, it is crucial to consider that the structure-function of the peptide alongside its regulatory factors may change in the presence of chronic glucose conditions. Serum albumin undergoes irreversible glycation after long-term exposure to high glucose levels, leading to modifications in its protein structure. The third goal addresses this challenge in Chapter 4, which reveals by spectroscopy (CD, UV-Vis) that glycated serum albumin alters ternary complexation with Cu(II) and C-peptide, as well as shifts cellular internalization and clinical measurements of C-peptide. To extend our understanding the molecular mechanisms of C-peptide with Cu(II) and albumin, this work looks into the impact of long-term incubation with high glucose on complexation with Cu(II), C-peptide and albumin. These studies begin to reveal formation of aggregation in collaboration with Dr. Duim at UC Davis, leading to the project development of determining what factors may drive aggregation. This warrants future studies by not only focusing on the mechanisms of C-peptide with its co-factors in normal conditions, but also considering the challenges that come with drug development of C-peptide in diabetic conditions. As the relationship between Cu(II) and C-peptide has been thoroughly characterized in the previous chapters, Chapter 5 expands on the fourth goal by exploring the potential impact of Cu(II)-bound C-peptide on peptide internalization in different endocrine targets and Cu intracellular pathways. This work displays trends in redistributing Cu trafficking levels in the presence and absence of serum albumin, pointing to the mechanism of C-peptide as a sequestration factor, as well as linking to the impact C-peptide may have on Cu-associated diseases.