Department of Pathololgy
Dr. Shenker’s laboratory is focused on mechanisms of immunoregulation, microbial pathogenesis and immunotoxicity. Over the past several years, significant progress has been made in our understanding of the etiology and pathogenesis of oral infectious diseases. However, the nature and contribution of the immune system to these disorders remain unclear. It is our hypothesis that the immune system plays a primary role to minimize and/or prevent infection. Furthermore, we propose that immunoregulatory abnormalities contribute to the pathogenesis of and susceptibility to oral infectious disorders such as periodontal disease. In this regard, our investigations have demonstrated that several periodontal pathogens produce factors capable of impairing human T- and B-cell function; these include Aggregatibactor (formerly Actinobacillus) actinomycetemcomitans, Fusobacterium nucleatum and Treponema denticola. While each of these immunoinhibitoiry proteins are distinct gene products, they share a common mode of action: cell cycle arrest.
We have focused much of our recent effort on the immunotoxin produced by A.actinomycetemcomitans which is a member of the family of the cytolethal distending toxins; this toxin induces G2 arrest in lymphocytes and eventually leads to activation of the apoptotic cascade. The objectives of our study have been to define the role of membrane lipid microdomains in relation to the cascade of events responsible for toxin-induced G2 arrest and cell death and to determine the relationship between Cdt subunit structure and function. To date, we have demonstrated that the heterotrimeric holotoxin functions as an AB2 toxin where CdtB is the active (A) unit and the complex of CdtA and CdtC comprise the binding (B) unit. Moreover, we have determined that the CdtC subunit binds to membrane cholesterol via a cholesterol recognition/interaction amino acid consensus (CRAC) region which results in the association of the Cdt holotoxin with membrane lipid rafts.
More recently, we have determined that the CdtB subunit exhibits phosphophatidylinositol-3,4,5-phosphate (PIP3) phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PIP3 to PI-3,4-P2 and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Mutation of conserved amino acids critical to catalysis in this family of enzymes reduced phosphatase activity along with decreased ability to induce G2 arrest. Consistent with this activity, CdtB induces time-dependent reduction of PIP3 in cells. Collectively, our studies demonstrate that CdtB not only exhibits PIP3 phosphatase activity, but also that toxicity in lymphocytes is dependent upon this activity. A major focus of our research continues to focus on the molecular mode of action of and the relationship between PIP3 depletion and cell cycle arrest
Another area of research is focused on harnessing the pharmacologic potential of CdtB as an agent for treating both cancer and inflammatory disorders. In this regard, phosphoinositides (PIs) are derivatives of phosphatidylinositol and while they represent minor components of membrane lipids, PIs regulate a wide range of cellular functions by serving as site-specific membrane signals that mediate membrane recruitment and regulation of effector/signaling proteins. The best characterized of the PIs, PIP3, is synthesized from PI(4,5)P2 following the activation of PI-3K and has received much attention for its critical role as a second messenger in the PI-3K/PIP3/Akt signaling pathway. PIP3 is negatively regulated via dephosphorylation by the tumor suppressor phosphatase, PTEN (ptase and tensin homolog deleted on chromosome ten) or SHIP (src homology 2-containing inositol phosphatase; CdtB mimics these enzymatic activities. There is a growing body of evidence that mutations in the PI-3K/PIP3/Akt pathway are highly oncogenic leading to increased cell proliferation and survival; indeed, the PI-3K/PIP3/Akt signaling pathway is unique in that every major component of this signaling system is frequently altered in cancer. It is clear that the PI-3K/PIP3/Akt pathway represents a potent target for pharmacologic intervention for a wide range of cancers. We are currently developing immunotoxins and chimeric fusion toxins that specifically target CdtB to tumor cells and inflammatory cells.