Selenium and its Unique Biochemistry in Cellular and Human Health
The element selenium plays an important role in cellular and human health. In the chemistry of life, selenium expands the chemical mechanisms for proteins to execute enzymatic reactions. In contrast to metals or organic co-factors, however, selenium is covalently incorporated into these proteins in the form of the rare amino acid selenocysteine (Sec, U). The resulting selenoproteins play important roles in diverse pathways such as reactive oxygen species (ROS) mediated signaling and detoxification, regulation of redox pathways, hormone synthesis, calcium signaling and the cellular response to dysfunctional proteins.
A part of our team studies selenoproteins particularly in the context of oxidative stress –an imbalance between cellular oxidants such as free radicals and antioxidants. Prolonged oxidative stress caused by environmental factors or improper cellular response, not only leads to impaired signaling but to the actual damage of proteins, lipids and DNA. Accumulation of such damages over time can severely compromise human health and lead, for example, to diabetes, cardiovascular diseases, cancer and aging.
We work on understanding the function of individual selenoproteins in the management and clearance of oxidative stress in cells. For this important biological function, the noncanonical amino acid selenocysteine plays a central role and its differences and similarities to the “normal” cysteine in the context of protein functions is of great interest to us. Our desire to thoroughly study selenoproteins and the unique biochemistry of selenium has naturally led us to the development of novel biochemical and biophysical tools, such as the efficient preparation of selenoproteins by expressed chemical ligation or the advancement of 77Se-NMR spectroscopy methods to study selenium containing proteins. An overview of these related research topics is given below.
Selenoproteins: Exploiting Selenium’s Properties for Advanced Functionalities
While selenoproteins are low in number in any given genome, their contribution is often disproportionally important if not critical for survival when an organism is under oxidative stress. However, we currently know the precise physiological function for only a small fraction of the selenoproteome and still have yet to fully capture and describe the chemical diversity of reactivities within the selenoprotein family. To that end, we pursuing the following fundamental questions regarding selenoproteins:
• What physiological advantages does a protein gain by using selenocysteine instead of cysteine?
• Do selenium-containing proteins resist damage by oxidative stress better than their sulfur-containing counterparts?
• Are selenoproteins more efficient at detecting and signaling the presence of reactive oxygen species?
• How does selenium’s low redox potential translate into selenoproteins’ unique chemistry?
• Do selenoproteins’ ability of rapid redox (reduction-oxidation) reactions turn them into signal transduction sensors?
Selenium NMR Spectroscopy:
Probing Structure, Dynamics, and Function of Macromolecules
We are engaged in the development of biological 77Se-NMR for two reasons: Firstly, this technique enables us to directly study the selenium nuclei and their chemical environment in selenoproteins. Secondly, selenium is an exquisite surrogate for sulfur, whose only NMR-sensitive isotope, 33S, is a low-sensitivity quadrupolar nucleus that cannot be utilized to study biological systems. Yet, the sulfur-containing amino acids cysteine and methionine are critical parts of many important biological functions. By substituting sulfur with selenium, a nucleus with very similar physicochemical properties, but minimally impact on protein structure, 77Se-NMR offers great potential to study the sulfur-based mechanisms in protein’s enzymatic functions, their interactions with other proteins and ligands as well as the dynamics and structure of biological systems.
To further advance studies of macromolecules by 77Se spectroscopy, we have by now developed a variety of facile and cost-effective methods to perform the sulfur to selenium substitution and isotopically enrich the resulting selenoproteins with 77Se. Because the NMR response of this selenium nucleus is very sensitive to its chemical environment, we observe a wide range of chemical shifts for 77Se in these biological systems which permits us to follow chemical reactions in selenium-rich proteins. At this point, we have built a biologically relevant library of solution and solid-state NMR parameters of selenium-containing proteins, that not only enables us and others to extract more information from 77Se-NMR spectra, but also aids the development of computational approaches for data analysis.
Some of the questions we are now trying to address are:
• Can selenium NMR be used to measure weak protein-protein or protein-ligand interactions?
• Can selenium NMR be used to map protein interfaces?
• How to best extract information about protein motion?
Preparation of Selenoproteins
The wide gap in our knowledge regarding selenoproteins is primarily caused by the difficulty of obtaining selenoproteins in sufficient quantities for meaningful biochemical and biophysical characterization. This supply challenge has a biomolecular cause: Unlike the 20 canonical amino acids with their individual codons, selenocysteine (Sec, U) is genetically encoded by an UGA codon, which is unfortunately also used as signal to stop translation. Nature provided a workaround this ambiguity by employing dedicated ancillary proteins to aid the incorporation of Sec into the polypeptide chain during translational. To overcome the limitation this process puts on selenoprotein production, we have developed new and innovative ways to increase the amount of selenoproteins we can produce to levels that enable us to conduct biochemical and biophysical characterizations in the laboratory. In a particularly efficient approach, we were able to exploit the high chemical reactivity of selenocysteine to join two separately expressed protein fragments to form the final protein. This technique enables us also to introduce selenocysteine in a protein at any position. Because of selenium’s chemical properties, this selenocysteine represents a unique, bioorthogonal, chemical handle that expands the utility of this method beyond protein production.
There are several avenues that we are interested in exploring in the future:
• Can we extend this method to multistep ligations to form final proteins from more than two fragments?
• Can methods for selenopeptide synthesis be adapted turned into biochemistry-friendly preparations?
• Is it possible to adopt cell free expression systems for the production of selenoproteins?