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Characterization of Signaling Pathways Involved in Cell Death and Survival

Investigators
Srinivasula, Srinivasa M
Institutions
DHHS/NIH - National Cancer Institute
Start date
2012
End date
2012
Objective

AutophagyHost defenses against microorganisms include targeting the pathogens for degradation in acidic lysosomal organelles, generating adaptive immune responses by orchestrating successful antigen presentation and maintenance of cellular homeostasis. Infected cells employ evolutionarily-conserved cellular autophagic machinery that is normally used in the degradation of intracellular protein aggregates and damaged organelles to accomplish these responses. This catabolic pathway, known as autophagy, helps cells survive under starvation conditions by restoring nutrient balance.

During autophagy, cytoplasmic material is engulfed in de novo double-membrane vesicles (referred to as autophagosomes) and is delivered to lysosomes where the cargo is degraded into its constituent parts for reuse by the cell. This process is controlled by ATG5 and ATG7, which by mechanisms similar to ubiquitin-conjugation are involved in the conjugation of lipid to microtuble-associated protein 1 light chain 3 (LC3, a homolog of yeast ATG8). Conjugation with phosphatidylethnolamine (PE) converts the soluble form of LC3 (LC3-I) to another (LC3-II) that specifically associates with autophagic membrane vesicles. Unlike classical autophagy that involves nonselective bulk degradation of cytosolic material, infected cells target intracellular bacteria (or bacteria-containing phagosomes) for sequestration into LC3-positive vacuoles for eventual destruction.

This process of selective removal of invading microbes using autophagic machinery, termed xenophagy, plays a key role in the restriction by destruction of several kinds of bacteria, including Escherchia coli, Salmonella enterica, Mycobacterium tuberculosis, Listeria monocytogenes, and Group A Streptococcus as well as parasites such as Toxoplasma gondii. Genetic and pharmacological interference with the autophagic machinery has been shown to increase the number of intracellular bacteria. Xenophagy can also protect against infection by the Sindbis virus in mice and the singlestranded tobacco mosaic virus in plants. In addition to eliminating intracellular microbes, xenophagy elicits adaptive immune responses by contributing to the cross-presentation of microbial peptide antigens on both MHC class I and II molecules. Autophagic signaling pathways involved in host defense are just beginning to be understood. Toll-like receptors (TLRs) on macrophages recognize pathogen-associated molecular patterns (PAMPs) and engage autophagic processes to clear pathogens. Lysosomal maturation of bacteria-containing phagosomes appears to be enhanced by TLR-mediated recruitment of the autophagosomal marker LC3 to phagosomes and their fusion with lysosomes. Moreover, engagement of TLR4 is also known to induce do novo formation of LC3-positive autophagosomes. Whereas these findings have linked TLRs and autophagy in host defense processes, many questions, such as how TLR-initiated signaling leads to the assembly of LC3-positive vesicles, remain unanswered. Given the importance of autophagy for immunity, cancer and neurodegenerative diseases we have focused our efforts on identifying molecules that regulate these processes. We found that TLR4-mediated autophagy is selective autophagy of aggresome-like induced structures (ALIS).

We found that in response to infection with Gram-negative bacteria, macrophages upregulate the expression of p62 (also known as SQSTM1), a ubiquitin-and LC3- binding molecule and ubiquitinated proteins, which together form ALIS. ALIS formation but not their clearance occurs independently of the classic autophagic machinery. Reactive oxygen species (ROS)- and p38-medited activation of NRF2 transcription factor contributes to p62 upregulation and ALIS formation, indicating a link between oxidative-stress and immune responses. Knockdown of NRF2 or p62 with siRNA completely abrogates ALIS formation and TLR4-mediated autophagy.

Additionally, we found that p62 like molecule known as NBR1 is recruited toALIS. Unlike p62, NBR1 is not essential for TLR4-mediated ALIS formation but is required its autophagic clearance suggesting that distinct roles for p62 and NBR1 in TLR4-mediated autophagy.Investigations also revealed that FYVE motif-containing molecules, CARP1 and CARP2 regulate the kinetics of the formation and degradation of ALIS. Macrophages isolated from bone marrow of mice deficient in either CARP1 or CARP2 showed defects in ALIS accumulation. Experiments are underway in the laboratory to understand the physiological role of ALIS in antigen presentation and regulation of autophagic and oxidative-stress signaling pathways. To understand the role of ALIS in host defense we are using Mycobacterium tuberculosis and pathogenic E. coli as infectious models.

sApoptosis is a conserved programmed cell death process triggered in many cell types by exposure to a variety of stimuli including soluble or membrane-bound death ligands such as Fas-L. Apoptosis ccurs in all cell types in the vessel wall and is implicated in the normal vasculature and disease conditions including arteriosclerosis, aortic aneurysms, arterial remodeling and medial degeneration. Fas expressed on the cell surface predominantly determine the sensitivity of cells to its ligand, and specific stimuli such as p53 activation and interferon-c (IFN-c) treatment potentiate apoptosis by increasing cell surface Fas levels.

Many cell types including vascular cells retain the receptor in the Golgi compartment, thus rendering the cells more resistant to Fas-mediated apoptosis. Very little is known of the mechanisms by which, Fas is retained in the Golgi ompartments of vascular cells. PP1201/RECS1 has been identified as a gene responsive to centrifugal force and shear stress (RECS). PP1201-deficient mice show no phenotypic abnormalities, but aged knockout mice are prone to cystic medial degeneration (CMD), a pathological condition encountered in aortic aneurysms. These animals display abnormal aortic media with large CMD foci manifested by absence of smooth muscle cells and by the accumulation of basophilic ground substance referred as mucoid degeneration. The aneurysms observed in PP1201-deficient mice are noninflammatory as the loss of cells in CMD lesions is not associated with infiltration of T lymphocytes or macrophages. Whereas these studies propose a protective role for PP1201 in vascular homeostasis, they do not address signaling pathways modulated by PP1201 and whether increased levels of PP1201 are obligatory for such a function. PP1201 encodes for a protein with seven transmembrane domains.

Our studies demonstrated that PP1201 localizes to Golgi compartments and can control trafficking of receptors to the plasma membrane. We found that high levels of PP1201 can attenuate Fas surface expression and protects cells against Fas-L-induced apoptosis. Our results suggest that increased expression of PP1201 in response to shear stress could help to maintain vascular homeostasis by dampening the effects of Fas signaling.

Funding Source
Nat'l. Cancer Inst.
Project source
View Project >>
Project number
1ZIABC010629-09
Categories
Parasites
Salmonella
Listeria
Escherichia coli