Molecular Dynamics Simulation of Dioxygen Pathways through Mini Singlet Oxygen Generator (miniSOG), a Genetically Encoded Marker and Killer Protein

In this work, molecular dynamics (MD) simulations of the permeation of proteins by small gases of biological significance have been extended from gas carrier, sensor, and enzymatic proteins to genetically encoded tags and killer proteins. To this end, miniSOG was taken as an example of current high interest, using a biased form of MD, called random‐acceleration MD. Various egress gates and binding pockets for dioxygen, as an indistinguishable mimic of singlet dioxygen, were found on both above and below the isoalloxazine plane of the flavin mononucleotide cofactor in miniSOG. Of such gates and binding pockets, those lying within two opposite cones, coaxial with a line normal to the isoalloxazine plane, and with the vertex at the center of such a plane are those most visited by the escaping gas molecule. Out of residues most capable of quenching ¹O₂, Y30, lying near the base of one such a cone, and H85, near the base of the opposite cone, are held to be most responsible for the reduced quantum yield of ¹O₂ with folded miniSOG with respect to free flavin mononucleotide in solution.     

Mpl Traffics to the Cell Surface Through Conventional and Unconventional Routes

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA‐mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER‐tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER‐Golgi and autolysosome secretory pathways, as well as recycling.      

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio‐temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore‐assisted light inactivation (CALI) of proteins, as well as for photo‐induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini‐antibodies or selective scaffold proteins and photo‐induced cytotoxicity of miniSOG, which is particularly promising for selective non‐invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

     

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 µm, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.