Copper(II) Affects the Biochemical Behavior of Proinsulin C-peptide by Forming Ternary Complexes with Serum Albumin.

Peptide hormones are essential signaling molecules with therapeutic importance. Identifying regulatory factors that drive their activity gives important insight into their mode of action and clinical development. In this work, we demonstrate the combined impact of Cu(II) and the serum protein albumin on the activity of C-peptide, a 31-mer peptide derived from the same prohormone as insulin. C-peptide exhibits beneficial effects, particularly in diabetic patients, but its clinical use has been hampered by a lack of mechanistic understanding. We show that Cu(II) mediates the formation of ternary complexes between albumin and C-peptide and that the resulting species depend on the order of addition. These 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 biomarker.

Analysis of Metal Effects on C-Peptide Structure and Internalization.

The connecting peptide (C-peptide) has received increased attention for its potential therapeutic effects in ameliorating illnesses such as kidney disease and diabetes. Although the mechanism of C-peptide signaling remains elusive, evidence supports its internalization and intracellular function. Emerging research is uncovering the diverse biological roles metals play in controlling and affecting the function of bioactive peptides. The work presented herein investigates interactions between C-peptide and first-row d-block transition metals, as well as their effects on C-peptide internalization into cells. Through spectroscopic techniques, it is demonstrated that CrIII , CuII , and ZnII bind to C-peptide with differing stoichiometries and biologically relevant affinities. In addition, metal binding elicits both subtle changes in secondary structure and inhibits adoption of an α-helical character in environments where the dielectric constants are reduced. This study shows how metal ions can modulate peptide hormone activity through subtle structural changes to disrupt cellular uptake.

Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores.

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been reported to directly bind to a variety of peptides and play both necessary and pathological roles ranging from mediating structure to electron transfer properties to imparting catalytic function. Quantifying the binding affinity and thermodynamics of these Cu(II)-peptide complexes in vitro provides insight into the thermodynamic driving force of binding, potential competitions between different metal ions for the peptide or between different peptides for Cu(II), and the prevalence of the Cu(II)-peptide complex in vivo. However, quantifying the binding thermodynamics can be challenging due to a myriad of factors, including accounting for all competing equilibria within a titration experiment, especially in cases where there are a lack of discrete spectroscopic handles representing the peptide, the d-block metal ion, and their interactions. Here, a robust set of experiments is provided for the accurate quantification of Cu(II)-peptide thermodynamics. This article focuses on the use of electronic absorption spectroscopy in the presence and absence of chromophoric ligands to provide the needed spectroscopic handle on Cu(II) and the use of label-free isothermal titration calorimetry. In both experimental techniques, a process is described to account for all competing equilibria. While the focus of this article is on Cu(II), the described set of experiments can apply beyond Cu(II)-peptide interactions, and provide a framework for accurate quantification of other metal-peptide systems under physiologically relevant conditions.

Fatty Acid Uptake in Liver Hepatocytes Induces Relocalization and Sequestration of Intracellular Copper.

Copper is an essential metal micronutrient with biological roles ranging from energy metabolism to cell signaling. Recent studies have shown that copper regulation is altered by fat accumulation in both rodent and cell models with phenotypes consistent with copper deficiency, including the elevated expression of the copper transporter, ATP7B. This study examines the changes in the copper trafficking mechanisms of liver cells exposed to excess fatty acids. Fatty acid uptake was induced in liver hepatocarcinoma cells, HepG2, by treatment with the saturated fatty acid, palmitic acid. Changes in chaperones, transporters, and chelators demonstrate an initial state of copper overload in the cell that over time shifts to a state of copper deficiency. This deficiency is due to sequestration of copper both into the membrane-bound copper protein, hephaestin, and lysosomal units. These changes are independent of changes in copper concentration, supporting perturbations in copper localization at the subcellular level. We hypothesize that fat accumulation triggers an initial copper miscompartmentalization within the cell, due to disruptions in mitochondrial copper balance, which induces a homeostatic response to cytosolic copper overload. This leads the cell to activate copper export and sequestering mechanisms that in turn induces a condition of cytosolic copper deficiency. Taken together, this work provides molecular insights into the previously observed phenotypes in clinical and rodent models linking copper-deficient states to obesity-associated disorders.