High Mobility Group Box-1 (HMGB1) is a transcription factor-like protein that has recently been characterized as a prototypical Damage -Associated Molecular Pattern molecule (DAMP). HMGB1 is a crucial late-acting mediator of sepsis in patients with sepsis, severe sepsis and septic shock. While much attention has been focused on the function of extracellular HMGB1, the mechanisms of HMGB1 release in sepsis have received little consideration. HMGB1 lacks a secretory signal peptide;therefore, it cannot be secreted via the endoplasmic reticulum-Golgi system. The newly synthesized HMGB1 undergoes extensive post-translational modifications, e.g., acetylation of lysine residues that promote active transport of HMGB1 from the nucleus to the endosomal compartment and prevent its re-entry into the nucleus. Another nuclear protein, which is activated in similar inflammatory conditions, is Poly (ADP-ribose) Polymerase-1(PARP-1). PARP-1 is the most abundant isoform of the PARPenzyme family and its continued activation leads to depletion of its substrate, nicotinamide adenine dinucleotide (NAD+), with consequent depletion of adenosine-5'-triphosphate (ATP), energy failure and cell death. The proposed research plan will define the role of PARP-1 in the modulation of lipopolysaccharide (LPS)-mediated HMGB1 transcription, post-translational modification and secretion. The central hypothesis for this grant application is that PARP-1 is essential for LPS-mediated HMGB1 secretion. In this R01 grant application, we propose a comprehensive approach including in vitro and in vivo studies that will define the role of PARP-1 in modulating LPS-mediated HMGB1 release.
In Aim 1, we determine the molecular mechanisms whereby PARP-1 regulates LPS-induced HMGB1 secretion in monocytes. We hypothesize that 1) chemical and genetic PARP-1 inhibition modulates mitogen-activated protein kinase (MAPK) pathway activity;2) PARP-1 inhibition modulates extracellular signal-regulated kinase (ERK) 1/2-mediated histone acetyl-transferase (HAT) activity of p300 and a closely related protein, cAMP response element-binding protein (CREB)-binding protein (CBP);3) PARP-1 inhibits LPS-mediated HMGB1 gene transcription.
In Specific Aim 2, we will determine if PARP-1 inhibition modulates the nuclear export and delivery of HMGB1 to the endosomal compartment for LPS-mediated secretion in monocytes. Under this aim, we will determine: 1) if LPS-mediated HMGB1 trafficking to the lysosomes requires PARP-1 activity;2) HMGB1 concentration in the lysosomes with or without PARP-1 inhibition 3) HMGB1 acetylation and its correlation with ADP-ribosylation. To verify the results of our in vitro studies we will tet PARP-1 inhibitors in relevant clinical models of sepsis;therefore, in Aim 3 we will assess the potential role of PARP activation in sepsis using cecal ligation and puncture (CLP) procedure in wild-type and PARP-/- mice. Additional studies will be performed to determine the therapeutic window for use of PARP-1 inhibitors in murine sepsis.
Sepsis continues to be a global public health problem and results in significant mortality and morbidity. Despite advances in the understanding of the mechanisms that govern sepsis, the discovery of new and efficacious therapeutic agents has lagged behind. In this proposal, we will explore the mechanisms involved in the release of High Mobility Group Box-1, a crucial late - mediator of sepsis.
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