Resolución 66 del COMEX
4.7 Elaboración de la matriz DOFA sectorizando por cuadrante los factores
Mammalian immunity is composed of two systematic layers: innate and adaptive immunity. They are responsible for recognition of pathogens and elimination of them, respectively. As the first line of host defense, pathogen recognition in innate immunity is achieved through activation of pattern recognition receptors (PRRs). PRRs are activated through recognition of the specific pathogen-‐associated molecular patterns
(RLR), cytosolic DNA sensors (CDS), and the C-‐type lectin receptors (CLR) (Kawai & Akira, 2011). The first PRR family to be identified and most widely studied was TLR that recognizes a wide range of PAMPs and activates downstream pathways.
As type I transmembrane proteins, TLRs are composed of an extracellular ectodomain containing Leucine-‐rich-‐repeat (LRR) motif and a cytoplasmic Toll-‐
interleukin-‐1 Receptor (Toll-‐IL-‐1R or TIR) domain responsible for relaying the signal to the downstream adapters (Akira et al, 2006) (Figure1A). LRR domain consists of 19-‐25 tandem copies of the leucine-‐rich motif of 24-‐29 amino acids such as XLXXLXLXX and XΦXXΦXXXXFXXLX where X represents any amino acid and Φ represents a hydrophobic amino acid (Figure 1B) (Akira & Takeda, 2004). These repeats are composed of a β-‐ strand and an α-‐helix connected with loops creating a concave surface responsible for ligand recognition (Figure 1C-‐D). On the other hand, TIR domains have only 20-‐30% sequence homology with three conserved regions required for signaling.
TLRs are divided into subfamilies based on the ligand they recognize: TLR1, TLR2, and TLR6 recognize lipids, TLR7, TLR8, and TLR9 recognize nucleic acids, TLR3 recognizes double stranded DNA (dsDNA) from viruses, TLR4 recognizes
lipopolysaccharide (LPS) from Gram-‐negative bacteria, and TLR5 recognizes flagellin from Flagellated bacteria (Figure 2, Table 1) (Akira et al, 2006).
Upon ligand recognition, TLRs induce host defense genes through activation of two major downstream pathways based on the adapter protein involved: Myeloid
differentiation primary response gene 88 (MyD88)-‐dependent and MyD88-‐independent, also known as TIR-‐domain containing adapter-‐inducing interferon-‐β (TRIF)-‐dependent, pathways (Janeway & Medzhitov, 2002). All TLRs except TLR3 are MyD88-‐dependent pathways while TLR4 stimulation activates both MyD88-‐dependent and –independent pathways (Figure 2). Throughout this dissertation, I focused mainly on studying the events downstream MyD88-‐dependent TLR4 signaling.
MyD88-‐Dependent Signaling
a short intermediate domain (ID) (Lin et al, 2010). TIR domain is responsible for the recruitment of MyD88 as a scaffold to the activated TLR to relay the signal to
downstream elements (Ohnishi et al, 2009). On the other hand, DD is responsible for homophilic interaction with the downstream elements such as IL-‐1R-‐associated kinases (IRAKs) IRAK-‐1 and IRAK-‐4 (Akira et al, 2006). Activated IRAKs associate with Tumor necrosis factor (TNF) receptor (TNFR)-‐associated factor 6 (TRAF6), inducing
ubiquitination of the Inhibitor of kappaB (IκB) kinase (IKK)/Nuclear factor kappaB (NFκB) essential modulator (NEMO) complex and itself. In addition, another complex composed of Transforming growth factor-‐β (TGF-‐β)-‐activated kinase 1 (TAK1) and TAK1 binding proteins is recruited to TRAF6. TAK1 recruitment is crucial for
phosphorylation and subsequent activation of IKK-‐β for the degradation of IκB, which in return exposes the nuclear-‐localization signal, thus enables the early-‐phase
activation of NFκB transcription factor for the pro-‐inflammatory cytokine expression (Figure 2).
NFκB IN INFLAMMATION RESPONSE
Downstream TLR pathways, transcription factor NFκB regulates the transcription of many immune response-‐related genes such as cytokines, growth factors, effector enzymes in response to activation of immunity-‐related receptors (Hayden & Ghosh, 2004). Proteins in the NFκB family are divided into two subfamilies called the ‘NFκB’ and the ‘Rel’ proteins, all of which include an amino-‐terminal
conserved DNA-‐binding/dimerization domain called the Rel-‐homology domain (RHD) (Figure 3) (Gilmore, 2006). The NFκB subfamily proteins p100 and p105 are
activated through removal of these inhibitory domains by proteolysis leading to the exposure of the active forms p52 and p50, respectively (Hoffmann & Baltimore, 2006). These activated forms, as they don’t have trans-‐activation domains (TADs), require heterodimerization with the Rel proteins to initiate transcription (Hayden & Ghosh, 2012). The Rel proteins p65/RelA, RelB, and RelC have a C-‐terminal TADs, which can activate transcription across many species although their sequences are not conserved the same way (Gilmore, 2006). All five members of NFκB family can form homodimers or heterodimers in vivo, except RelB, which exists only in heterodimers in vivo (Figure 4). The variety of different homodimers and heterodimers create a multi-‐layered regulation system as different dimers have different DNA-‐binding site specificities and protein-‐protein interactions at the promoters.
In this thesis, I mainly focus on the transcriptional activity of the p65 subunit downstream MyD88-‐dependent TLR4 pathways, because its nuclear translocation and DNA-‐binding are indicators of transcriptional activation of NFκB target genes.
IκB Family Proteins Regulate p65 Activity
NFκB activity is regulated by interaction with seven IκB family proteins IκBα, IκBβ, IκBγ, IκBε, B-‐cell lymphoma-‐3 (BCL-‐3), IκBζ, and IκBζ as well as the precursor proteins p100 and p105 (Hayden & Ghosh, 2012). IκB family proteins contain five to seven ankyrin repeats that modulate the interaction NFκB proteins (Hayden & Ghosh, 2004). This interaction covers the Nuclear localization signal (NLS) of p65 to
and cytoplasmic localization of NFκB, respectively (Sun & Andersson, 2002).
Regulation of Transcriptional Activity of p65 via Phosphorylation
NFκB activity is also regulated by post-‐translational modifications (PTMs), such as phosphorylation, acetylation, and methylation. Currently, phosphorylation of p65 is the most common and most widely studied NFκB PTM because it is associated with rapid transactivation and transcriptional activity of p65 (Figure 6, Figure 7) (Vermeulen et al, 2006); thus, in this overview I will focus on the p65 regulation through phosphorylation.
Currently, five phosphorylation sites have been discovered to regulate p65 activity: S276, S311, S468, S529, and S536 (Figure 6). S276 resides in the RHD and gets phosphorylated by cyclic AMP-‐dependent kinase (PKAc) (Zhong et al, 1997) and by Mitogen-‐ and stress-‐activated protein kinase 1 (MSK1) upon TNF treatment
(Vermeulen et al, 2003). Phosphorylation of this site is essential for gene activation (Zhong et al, 1998). Another important phosphorylation is the phosphorylation of S311 by Protein kinase C-‐ζ (PKCζ) that is responsible for recruitment of cAMP response element binding protein (CREB)-‐binding Protein (CBP) and RNA polymerase II (RNA-‐ PII) to the IL-‐6 promoter (Duran et al, 2003). Moreover, S529 is phosphorylated by casein kinase II (CKII) (Wang & Baldwin, 1998) and IKK2 enhancing transactivity of p65 (Sakurai et al, 1999). In addition to S529, IKK2 also phosphorylates S536 for
transactivation upon IκB phosphorylation leading to enhanced binding to promoters with LPS stimulation. Ribosomal S6-‐kinase 1 (RSK1) also phosphorylates S536 through DNA damage response via p53 signaling (Bohuslav et al, 2004). In contrary to the other
transcription factor activity of p65 upon TNF or IL-‐1 (Buss et al, 2004; Mattioli et al, 2004).
It is important to note that the dynamic regulation of p65 phosphorylation status is sustained by the interplay between kinases and phosphatases. Protein phosphatase-‐ 2A (PP2A), protein phosphatase 4 (PP4), protein phosphatase 1γ (PP1γ), wild-‐type p53-‐induced phosphatase 1 (WIP1), and phosphoserine phosphatase (SerB) are some of the known negative regulators of p65 keeping it in the inactive state (Peng et al, 2015; Takeuchi et al, 2013; Yang et al, 2001; Yeh et al, 2004).