Structural and Functional Versatility of Interferon-Inducible Gtpases
Sayantan Roy, Bing Wang, and Qian Yin
Florida State University
GTP triphosphatases (GTPases) have been meticulously documented for their roles to regulate multiple cellular processes spanning from cell mobility, membrane fusion and fission, to cytokinesis and vesicle transport. Interferon (IFN)-inducible GTPases, consisting of more than forty members in human and mice, are among the most highly expressed interferon stimulated genes (ISGs), sometimes accounting for twenty percent of all proteins induced by IFN-γ. Recent genetic and cell studies start to reveal the important roles of IFN-inducible GTPases in restriction and elimination of pathogens, yet the molecular mechanisms are largely unclear. Using a combination of biochemical, biophysical, and structural tools, we start to understand the functional forms of two IFN-inducible GTPases, GBP2 and IRGM, and the mechanisms governing their activation. We characterized their oligomeric status, enzymatic activity, and their interactions with potential binding partners.
Understanding How CRISPR-CAS9 Controls DNA Specificity
Travis Hand1, Anuska Das1, and Hong Li1,2
1Institute of Molecular Biophysics, 2Department of Chemistry and Biochemistry
Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the Cas9-bound guide RNA and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition in time and space of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from thermophile Acidothermus cellulolyticus (AceCas9). Acidothermus cellulolyticus has been used for producing the enzyme endo-1, 4-β-glucanase (E1) which is used for the commercial hydrolysis of cellulose into glucose. Genome editing in A. cellulylyticus could impact its utilization in bioenergy development. We found that AceCas9 depends strictly on a 5’-NNNCC-3’ PAM and is more efficient in cleaving negative supercoils than relaxed DNA. We further characterized the dependence of AceCas9 on temperature, guide length, divalent metal ions, and mismatches to the guide RNA. The thermostability, cytosine-specific and DNA topology-sensitive properties of the AceCas9 maybe explored for specific genome editing applications.
The Effect of Li+ Binding on Secondary and Tertiary Structure, Hydrophobicity, Thermodynamics, and Interactions with Interacting Partners of DREAM.
Samiol Azam* and Jaroslava Miksovska.
Florida International University, Miami, FL, United States.
Li+ is widely used for the treatment of manic-depressive illness. Studies on neuronal calcium sensor protein 1 (NCS-1) have found that Li+ can inhibit the interaction between NCS-1 and inositol 1,4,5-triphosphate receptor protein, alleviating hyperarousal of insomnia patient. Li+ can have neuroprotective or neurotoxic effects depending on the concentration. In a rat model, chronic exposure of Li+ has shown a significant decrease in membrane-associated protein kinase C (PKC) in the hippocampus, suggesting potential clinical relevance. Here, we show that Li+ binds to a neuronal calcium sensor protein named Downstream Element Antagonist Modulator (DREAM), a protein expressed in the hippocampus region of the brain. Li+ association results in a decrease in the emission intensity of tryptophan, suggesting rearrangements of the tertiary structure of the protein. Li+ binding also exposes hydrophobic cavity of the protein as evidenced by binding of the hydrophobic molecule 1,8-ANS. CD data reveals that Li+ binding increases the rigidity of the protein. Tryptophan emission and CD data are further supported by lifetime data. Thermodynamics parameters of Li+ association to DREAM have been obtained through isothermal titration calorimetry (ITC) measurements. Li+-bound DREAM interactions with binding partners have been obtained by titrating FITC-tagged presenilin-1 and potassium channel against Li+-bound DREAM. Findings of this project suggest potentials for the Li+ compound in the animal model to investigate if acute and chronic exposure of Li+ has any effect on the expression level of DREAM and if Li+-based compounds influence interactions between DREAM and intracellular partners.
The Novel Disaggregase Activity of Protein Disulfide Isomerase
Albert Serrano,1 Michael Taylor,1 Helen Burress,1 Xin Qiao,2 Lucia Cilenti,1 Lauren Farley,1 Jason O. Matos,1 Bo Chen,2 Suren A. Tatulian,2 and Ken Teter1
1 Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32826, USA
2 Department of Physics, University of Central Florida, Orlando, FL 32816, USA
Protein disulfide isomerase (PDI) exhibits linked but independent functions as an oxidoreductase and chaperone. We have identified a unique property of PDI that is related to its chaperone activity. Using isotope-edited Fourier transform infrared spectroscopy, we have shown that PDI partially unfolds upon contact with the catalytic A1 subunit of cholera toxin, aggregated alpha-synuclein, or aggregated amyloid beta peptide. The substrate-induced unfolding of PDI allows it to act as a disaggregase to break apart the multimeric cholera toxin and to both prevent and reverse amyloid formation at a 1:10 molar ratio of PDI:substrate (alpha-synuclein or amyloid beta peptide). Chaperones that function through conditional disorder and chaperones that function as a disaggregase have been described, but neither property has been attributed to PDI. Furthermore, no disaggregase is known to act by conditional disorder. Our observations thus provide a conceptual advance to understand the chaperone activity of PDI and its neuroprotective function.