Dr. Aloysius Klingelhutz, Professor in the Department of Microbiology and Immunology and Director of Undergraduate Studies, has been awarded a National Institutes of Health/National Institute on Aging (NIH/NIA) R21 Research Grant entitled "The role of ISG15 and ISGylation in the senescence-associated secretory phenotype.”  The goals of this project are to explore how cellular senescence cause aging-associated conditions such as inflammation, tissue degradation, and cancer through a mechanism that involves secretion of detrimental factors by senescent cells to surrounding cells and tissue. ISG15 is a small protein activated by pathogen infection, DNA damage, and senescence that modifies other proteins to change their stability and function. The studies will determine how upregulation of ISG15, secretion of ISG15, and modification of specific cellular proteins by ISG15 play roles in causing and maintaining the secretory state of senescent cells, pointing to potential mechanisms to ameloriate aging associated disease. This project involves collaboration with Dr. Lilliana Radoshevich at National Jewish Health.

Abstract

Cellular senescence is a cellular state that affects age-related diseases such as inflammation, cancer development, and cancer progression. Cells that enter senescence have a senescence-associated secretory phenotype (SASP) that has major, and usually detrimental, effects on other cells in the surrounding microenvironment. ISG15 is a ubiquitin-like protein that can covalently modify host and pathogenic proteins, changing their stability and function. ISG15 can also be secreted and function as a cytokine. While ISG15 is classically regarded as an interferon-stimulated gene that is induced during infection by pathogens, it can also be upregulated by DNA damage and stress. Recent studies have demonstrated that ISG15 is among the most upregulated genes across tissues in aging rodents. It has also been shown by us and others that ISG15 is upregulated during replicative senescence of human fibroblasts. Our preliminary data further indicates that ISG15 is significantly upregulated in skin fibroblasts derived from patients with dyskeratosis congenita, a human model of dysfunctional telomerase and shortened telomeres. Immortalization of DC cells by telomerase activation restores ISG15 levels to normal. Little is known about how ISG15 upregulation contributes to senescence and what proteins are ISGylated in senescent cells. Further, it is unknown how ISGylation changes upon cell immortalization. We hypothesize that upregulation of ISG15 and concomitant ISGylation of proteins caused by DNA damage is important for the SASP. In this proposal, we will determine what the interplay is between secreted ISG15 and ISGylation in senescence and immortalization. Using our unique patient cells and cell lines, we will map the senescence-associated ISGylome using innovative proteomic strategies pioneered in our laboratories. We will compare the ISGylomes between cells that have been induced to senesce by different mechanisms and from human and mouse, and we will determine how secreted ISG15 affects surrounding cells. Further, we will assess how immortalization by telomerase or viral oncogenes changes the ISGylome. Finally, we will specifically determine how ISG15 and ISGylation affects the SASP through genetic manipulation of cells to have reduced and enhanced ISGylation. These studies will provide key insight into how upregulation of ISG15 and ISGylation of specific cellular proteins play a role in cellular senescence and, in particular, the SASP.