Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2014)

Activation of cell surface receptors by ligands leads to the recruitment of a number of signaling molecules that mediate cellular responses. RAS-GTPases are signaling proteins that bring about major changes in cell growth, differentiation, gene expression and cytoskeletal organization upon receptor stimulation. The RAS effector RIN1 is involved in receptor trafficking and signaling. RIN1 is recruited to the receptor following ligand stimulation, but the function of its individual effector domains in this process remained uncharacterized. RIN1 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase RAB5, which is involved in the formation of early endosomes. RIN1 can also directly bind and activate ABL tyrosine kinases, which have been implicated in cellular actin remodeling and receptor trafficking. RIN1 also forms a complex with STAM, an ESCRT complex protein, which favors maturation of receptor-containing endosomes. As all of RIN1's known effectors are involved in modulating receptor trafficking, we resorted to examine the role of individual domains in receptor internalization and fate. Chapters two and three debrief the mechanisms by which RIN1 effectors function in an integrated manner to regulate receptor trafficking and corresponding cellular responses.

Using domain-specific mutants of RIN1, we show that the RAB5 and ABL signaling pathways mediate opposing functions, maintaining a balance in the route and rate of receptor internalization. RAB5 promotes actin remodeling, EGF induced macropinocytosis and migration, facilitating receptor degradation. ABL kinases, on the other hand, prevent receptor degradation and migration towards EGF. Our study also shows that regulation of RIN1 localization by the 14-3-3 proteins is a major factor in determining signaling intensity. We characterized a novel binding partner of RIN1, namely, BIN1, a BAR domain protein involved in membrane bending.

Intracellular pathogens invade host cells by exploiting host cell surface receptors and signaling pathways. Listeria monocytogenes in a food-borne pathogen that binds host MET to invade epithelial cells and modulates RAB5 GTPases at several stages of the infection. The effect of host RAB5 regulators on pathogenesis remained uncharacterized. We analyzed the role of RIN1, a major RAB5-GEF in epithelial cells, in the process of bacterial invasion and spread (chapter four). This study leads the way to determining the mechanism of pathogenesis of several pathogens that depend on host RAB5-GTPases for effective infection progress. Finally, chapter five discusses the significance of our studies from a therapeutic viewpoint in the context of cancers and infectious diseases.

    Cover page: The RAS Effector RIN1 Modulates Endocytosis via Activation of RAB5 GTPases and ABL Tyrosine Kinases
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2014)
    Listeria monocytogenes is a food-borne pathogenic bacterium that invades intestinal epithelial cells through a phagocytic pathway that relies on the activation of host cell RAB5 GTPases. Listeria monocytogenes must subsequently inhibit RAB5, however, in order to escape lysosome-mediated destruction. Relatively little is known about upstream RAB5 regulators during L. monocytogenes entry and phagosome escape processes in epithelial cells. Here we identify RIN1, a RAS effector and RAB5-directed guanine nucleotide exchange factor (GEF), as a host cell factor in L. monocytogenes infection. RIN1 is rapidly engaged following L. monocytogenes infection and is required for efficient invasion of intestinal epithelial cells. RIN1-mediated RAB5 activation later facilitates the fusion of phagosomes with lysosomes, promoting clearance of bacteria from the host cell. These results suggest that RIN1 is a host cell regulator that performs counterbalancing functions during early and late stages of L. monocytogenes infection, ultimately favoring pathogen clearance.
      Cover page: The RAB5‐GEF Function of RIN1 Regulates Multiple Steps During Listeria monocytogenes Infection
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2014)
      Listeria monocytogenes is a food-borne pathogenic bacterium that invades intestinal epithelial cells through a phagocytic pathway that relies on the activation of host cell RAB5 GTPases. Listeria monocytogenes must subsequently inhibit RAB5, however, in order to escape lysosome-mediated destruction. Relatively little is known about upstream RAB5 regulators during L. monocytogenes entry and phagosome escape processes in epithelial cells. Here we identify RIN1, a RAS effector and RAB5-directed guanine nucleotide exchange factor (GEF), as a host cell factor in L. monocytogenes infection. RIN1 is rapidly engaged following L. monocytogenes infection and is required for efficient invasion of intestinal epithelial cells. RIN1-mediated RAB5 activation later facilitates the fusion of phagosomes with lysosomes, promoting clearance of bacteria from the host cell. These results suggest that RIN1 is a host cell regulator that performs counterbalancing functions during early and late stages of L. monocytogenes infection, ultimately favoring pathogen clearance.
        Cover page: The RAB5-GEF function of RIN1 regulates multiple steps during Listeria monocytogenes infection.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2013)
        Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.
          Cover page: Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses
          Top