Fluorescent Sensors for Subcellular Metal Ion Imaging in Living Systems

Metal ions, such as iron and copper, play diverse roles in physiological and pathological processes.  They are essential to life and play crucial roles in biological metabolism but are also detrimental as their free forms (labile ions) may promote the generation of highly deleterious reactive oxygen species under cellular conditions.  Studying the cellular labile forms of these metal ions are very important however has been hindered by the lack of easily traceable markers for the metal ions. The goal of this research is to develop fluorescent imaging probes to monitor and quantification these metal ions at subcellular resolution in living organisms.  These rationally designed fluorescent sensors are created by chemical synthesis and evaluated in live-cell or small animal settings under a laser confocal microscope. These novel probes not only enable the direct study of the cell biology of these metal ions but also provide new tools for the study of the pathogenesis of diseases related to dyshomeostasis of metal ions.

Figure 1. A turn-on fluorescent sensor for imaging labile Fe³⁺in live neuronal cells at subcellular resolution

Molecular Imaging Probes for Reactive Oxygen/Nitrogen Species (ROS/RNS)

Reactive Oxygen/Nitrogen Species (ROS/RNS) are reactive molecules or free radicals derived from molecular oxygen or nitrogen. They are produced by cellular aerobic respiration, redox enzymes, or promoted by environmental factors such as exposing to radiation or toxins. ROS/RNS play essential roles in immune response, cell signaling and apopotosis but high levels of ROS/RNS cause cellular deleterious events which cause oxidative stress and related diseases.  Studying the cellular ROS/RNS has been difficult due to the lack of sensitive traceable markers for ROS/RNS. The goal of this research is to develop fluorescent imaging probes to monitor and quantification ROS/RNS at subcellular resolution in living organisms.  These rationally designed ROS/RNS probes are chemically synthesized and evaluated in live-cell or small animal settings under a confocal microscope. These novel ROS/RNS probes not only enable the direct imaging of these reactive species but also provide new tools for the study of the pathogenesis of diseases related to ROS/RNS.

Figure 2. Direct observation of internalization and ROS generation of amyloidb-peptide in neuronal cells at subcellular resolution

Genetically-Codedincorporation of Fluorescent Amino Acids into Proteins for Protein Imaging in Live-Cells

Harnessing a chemical biology approach, this project develops a novel platform formolecular imaging of proteins in real-time in living cells. First, we create small unnatural fluorescent amino acids via chemical synthesis. Second, we engineer a cognate amber suppressor tRNA,which will be charged with the fluorescent amino acid via a rationally engineered tRNA synthetases which will recognize the fluorescent amino acid and the tRNA. Third, we introduce the amber codon to a desired position on a gene coding a target protein. Upon introducing into a host cell, these engineered biomolecules can insert the small fluorescent amino acid into a desired position within thepolypeptide chain of the target protein without perturbing its native structure or function. This incorporated fluorescent amino acid probe will allow protein imaging in live cells and their locations will be observed in real time at a microscopic level. This novel platform also has a broad capacity for studying protein trafficking, protein-protein/DNA/RNA/membrane interactions, signal transduction, and monitoring mislocated proteins in the cells. In addition, this novel platform can reveal the pathogenesis of mistrafficking pathways, validate suspected mistrafficking mutations, and enable a greater understanding of disease-related phenotypes at the subcellular level.

Figure 3.General schematic for the amber suppression method of unnatural amino acid incorporation.

Targeted Anticancer Drugs

Anticancer drugs normally target cells that are rapidly dividing, such as cancer cells. However, side effects are common because some of our normal cells such as those in our gastrointestinal tract, hair, and bone marrow are also rapidly dividing. Specific delivery of drugs to cancer cells is of great interest. Targeted anticancer drugs (smart drugs) are a novel strategy to improvetherapeutic efficiency and reduce toxic side effects. We have been developing a novel class of potent and selective targeted drug conjugates by conjugating the high targeting capability of a small peptide with potent cytotoxic agents. Our targeted anticancer drugs specifically deliver the anticancer drugs to cancer cells viareceptor-mediated endocytosis by targeting Her2 receptor or transferrin receptor which is over-expressed on the surface of cancer cells.

Health Benefits of Cranberry and Other Polyphenols

Health benefits of the North American native fruit cranberry (Vaccinium macrocarpon, Ait. Ericaceae) have been recognized since the early 1900s. The effectiveness of cranberry in preventing and treatment of urinary tract infections (UTIs) has been confirmed by randomized, double-blind placebo-controlled clinical trials. Recent research shows that cranberries and cranberry products may be beneficial in the treatment of stomach ulcers, gum diseases and dental infections. More interestingly, anti-oxidative effects have been demonstrated recently which are linked to potential protection against aging, stroke, cardiovascular diseases neurological disorders and cancer, etc. However, little is known about the biochemical mechanism of action of the health benefits of cranberry. We have proposed that regulating of iron homeostasis and ROS pathways may play a pivotal role in the bio-effects of cranberry. In collaboration with other researchers in our UMass Cranberry Health Research Center (https://www.umassd.edu/chrc/), we investigate the biochemical mechanisms of action of cranberry’s health benefits and develop research tools and assays to test our hypothesis.