Novel Mechanisms for Extracellular NAMPT Secretion

Abstract

This thesis dissertation is focused upon nicotinamide phosphoribosyltransferase (NAMPT), an ancient pluripotent enzyme present in multiple organisms composed of eukaryotic cells from simple genome-structured organisms, such as yeast, to complex biological systems such as humans. NAMPT is a foundational intracellular metabolic rate-limiting enzyme that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN) to supplement and maintain cellular NAD+ levels that are required for survival. An organism’s main source of NAD+ is gathered from NAM or nicotinic acid (NA) within dietary sources containing amino acid tryptophan with NAD+ critical for both eukaryotic and prokaryotic cellular energy metabolism. NAMPT utilizes NAM, NA, and nicotinamide riboside (NR) to synthesize NAD+ within the classical NAD+ salvage pathway to maintain cellular NAD+ levels. Beyond NAMPT’s well-defined intracellular function, when NAMPT is secreted/released extracellularly, eNAMPT serves as a novel damage-associated molecular pattern protein (DAMP) to regulate innate immunity, inflammatory, fibrotic, and neoplastic responses via ligation of the pathogen recognition receptor, TLR4. DAMPs are molecules that are released from damaged or dying cells due to trauma or pathogen triggering infections, to activate the innate immune response, and to potentially promote pathological inflammatory responses. Secreted levels of eNAMPT into the bloodstream directly influence the severity of the immune response and contribute to disease severity. Elevated plasma levels of eNAMPT serve as a biomarker of disease activity in patients (and pre-clinical models) with acute respiratory distress syndrome, sepsis, systemic lupus erythematosus, diabetes, sarcoidosis, lung, and hepatic fibrosis, chorioamnionitis, inflammatory bowel disease, and pulmonary hypertension. Based upon eNAMPT’s influence on innate immunity and disease severity, the graduate studies contained in this thesis sought to decipher the key mechanisms that regulate eNAMPT secretion/release as these events are fundamental to understanding eNAMPT’s involvement in human disease. These studies focused on unconventional protein secretion (UPS) mechanisms as NAMPT does not contain an endoplasmic reticulum (ER) mRNA signal sequence and conventional protein secretion mechanisms were ruled out. Specifically, Type 1 (self-sustained membrane translocation) and Type 3 (autophagy-based secretion) mechanisms were investigated. Utilizing human monocytic THP-1 cells as the platform for eNAMPT secretion, we demonstrated that eNAMPT release during pyroptosis evoked by Nigericin, and eNAMPT secretion by lipopolysaccharide (LPS, non-pyroptotic), requires NLRP3 inflammasome activation with substantial attenuation by NLRP3 inhibition (MCC-950). ENAMPT secretion/release was attenuated by targeted genetic deletion of key inflammasome components, including NLRP3, caspase-1, or gasdermin D (GSDMD). Pyroptosis-associated eNAMPT release involved cleavage of the pore-forming GSDMD protein resulting in plasma membrane rupture (PMR) whereas non-pyroptotic LPS-induced eNAMPT secretion involved neither GSDMD cleavage nor PMR (verified utilizing noncleavable GSDMD mutant constructs) but was highly dependent upon NAMPT ubiquitination catalyzed by a complex containing the NEDD4 E3 ligase, Hsp90 (a selective chaperone), and intact GSDMD verified by enzymatic inhibition or silencing of NEDD4, GSDMD, or Hsp90. NAMPT ubiquitination and secretion involves the activation of LC3 lipidation as super-resolution microscopy analyses demonstrate NAMPT co-localization with autophagosome marker LC3B. Additionally, eNAMPT secretion was significantly reduced by targeting inhibition of LC3 lipidation E1 and E3 enzymes, ATG5 and ATG7, respectively. In summary, this body of work represents major components of an eNAMPT secretion pathway via TLR4 ligation and NAMPT’s metabolic role in the inflammatory response as an immunomodulatory cytokine. These studies provide key insights into eNAMPT secretion that may accelerate the development of therapeutic strategies that address unmet therapeutic needs in inflammatory, fibrotic, and neoplastic disorders.