Nucleophosmin (NPM1) is a mainly nucleolar phosphoprotein that shuttles between the nucleolus and cytoplasm. NPM1 is involved in a variety of biological processes such as centrosome duplication, ribosome biogenesis, intracellular transport, apoptosis and mRNA splicing (Lindström MS 2011). NPM1 is also implicated in various viral infections including human immunodeficiency virus type 1 (HIV-1) (Gadad SS et al. 2011), adenovirus (Samad MA et al. 2007), herpes simplex virus 1 (HSV-1) (Lymberopoulos MH et al. 2011) and severe acute respiratory syndrome coronavirus type 1 (SARS-CoV-1) (Zeng Y et al. 2008).

The glutathione S-transferase (GST)-tagged N protein of SARS-CoV-1 binds to endogenous NPM1 in HeLa cell lysates (Zeng Y et al. 2008). Co-immunoprecipitation assay further confirmed the interaction of NPM1 and the viral N protein in N-expressing HeLa cells. An in vitro phosphorylation assay using HeLa cell lysates showed that the binding of N protein inhibited the phosphorylation of NPM1 at Thr199 by CDK2 which can lead to cell cycle arrest (Zeng Y et al. 2008). SARS-CoV -1 N protein co-localized with the NPM1 protein in the perinuclear region of HeLa cells. NPM1 usually plays a role in nuclear import of the viral proteins to which it binds. It is unclear if the binding of SARS-CoV-1 N with NPM1 is involved with sub-cellular localization of N (Zeng Y et al, 2008). Similar findings were reported for the N protein of porcine epidemic diarrhea virus (PEDV), which belongs to the Alphacoronavirus genus in the Coronaviridae family (Shi D et al. 2017). Binding of the PEDV N protein to NPM1 prevented proteolytic cleavage of NPM1 by caspase-3 leading to increased cell survival (Shi D et al. 2017).

This Reactome event shows the interaction between SARS-CoV-1 N and host NPM1.

Thiopurines were originally developed for cancer treatment in the early 1950s, with 6-mercaptopurine (6MP) being the first thiopurine approved by the FDA for the treatment of leukaemia, just two years after its discovery. Azathioprine (AZA), a prodrug of 6MP, was developed by the addition of a nitroimidazol group a few years later to bypass the high first-pass metabolism of 6MP due to oxidation in intestinal cells by xanthine oxidase (XDH). AZA is a thiopurine prodrug, and its pharmacological action is based on the release of the active metabolite 6-mercaptopurine (6MP) which is further metabolised to pharmacoligically active 6-thioguanine nucleotides (6-TGNs). These 6-TGNs achieve their cytotoxic effects in one of four ways

1. Incorporation of 6-thioguanosine triphosphate (6TGTP) into RNA
2. Incorporation of 6-thiodeoxyguanosine triphosphate (6TdGTP) into DNA
3. Inhibition of de novo purine synthesis by methylmercaptopurine nucleotides such as methylthioinosine monophosphate (meTIMP)
4. Inhibition of RAC1 by 6TGTP which induces apoptosis in activated T-cells.

While AZA has been supplanted as an antitumour drug, it remains useful as an immunosuppressant antimetabolite drug indicated to treat rheumatoid arthritis, Crohn's disease, ulcerative colitis, cancer and to prevent rejection in kidney transplant patients (Axelrad et al. 2016, Tominaga et al. 2021).

The molecular steps of AZA metabolism are described in this pathway (Cuffari et al. 1996, Dubinsky 2004). Briefly, oral AZA is rapidly converted to 6MP. Initial 6MP metabolism occurs along competing catabolic (XDH, TPMT) and anabolic (HPRT) enzymatic pathways. Once formed, 6-thiosine 5′-monophosphate (6TIMP) is further metabolized by inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate synthetase (GMPS) to 6-thioguanosine 5′monophosphate (6TGMP). 6TGMP is then converted to the pharmacologically-active di- and tri- derivatives by their respective kinases.

The mitogen activated protein kinases (MAPKs) are a family of conserved protein serine threonine kinases that respond to varied extracellular stimuli to activate intracellular processes including gene expression, metabolism, proliferation, differentiation and apoptosis, among others.
The classic MAPK cascades, including the ERK1/2 pathway, the p38 MAPK pathway, the JNK pathway and the ERK5 pathway are characterized by three tiers of sequentially acting, activating kinases (reviewed in Kryiakis and Avruch, 2012; Cargnello and Roux, 2011). The MAPK kinase kinase kinase (MAPKKK), at the top of the cascade, is phosphorylated on serine and threonine residues in response to external stimuli; this phosphorylation often occurs in the context of an interaction between the MAPKKK protein and a member of the RAS/RHO family of small GTP-binding proteins. Activated MAPKKK proteins in turn phosphorylate the dual-specificity MAPK kinase proteins (MAPKK), which ultimately phosphorylate the MAPK proteins in a conserved Thr-X-Tyr motif in the activation loop.
Less is known about the activation of the atypical families of MAPKs, which include the ERK3/4 signaling cascade, the ERK7 cascade and the NLK cascade. Although the details are not fully worked out, these MAPK proteins don't appear to be phosphorylated downstream of a 3-tiered kinase system as described above (reviewed in Coulombe and Meloche, 2007; Cargnello and Roux, 2011) .
Both conventional and atypical MAPKs are proline-directed serine threonine kinases and, once activated, phosphorylate substrates in the consensus P-X-S/T-P site. Both cytosolic and nuclear targets of MAPK proteins have been identified and upon stimulation, a proportion of the phosphorylated MAPKs relocalize from the cytoplasm to the nucleus. In some cases, nuclear translocation may be accompanied by dimerization, although the relationship between these two events is not fully elaborated (reviewed in Kryiakis and Avruch, 2012; Cargnello and Roux, 2011; Plotnikov et al, 2010).

RHOBTB2 is an atypical member of the RHO GTPase family that is predicted not to cycle between a GTP-bound form and a GDP-bound form (Berthold et al. 2008). RHOBTB family proteins, in contrast to other RHO GTPases, possess other conserved domains in addition to the GTPase domain. The GTPase domain at the N terminus is followed by a proline rich region, a tandem of two BTB (broad complex, tramtrack, bric à brac) domains, and a conserved C terminal BACK (BTB and C terminal Kelch) domain (Berthold et al. 2008, Ji and Rivero 2016). RHOBTB proteins can form homo- and heterodimers, but the role of dimerization in RHOBTB function is not known (Berthold et al. 2008, Ji and Rivero 2016). RHOBTB2 is usually expressed weakly (Berthold et al. 2008), at a lower level than RHOBTB1 (Ji and Rivero 2016). Relatively high levels of RHOBTB2 can be detected in neural and cardiac tissues (Berthold et al. 2016). RHOBTB2 is involved in COP9 signalosome-regulated and CUL3-dependent protein ubiquitination (Berthold et al. 2008; Ji and Rivero 2016). RHOBTB2 suppresses cellular proliferation and promotes apoptosis (Ji and Rivero 2016). RHOBTB2 takes part in vesicle transport (Ji and Rivero 2016). RHOBTB2 was initially discovered as the gene homozygously deleted in breast cancer and was named DBC2 (deleted in breast cancer 2) (Berthold et al. 2008). RHOBTB2 level is decreased in many tumor types and it is proposed to act as a tumor suppressor. Genomic deletions and a small number of pathogenic mutations in RHOBTB2 have been reported in cancer (Berthold et al. 2008; Ji and Rivero 2016). Mutations of RHOBTB2 that result in impaired interaction with CUL3 have been found to cause epileptic encephalopathy (Belal et al. 2018).

Many GSK-3β inhibitors (GSKi) have been identified. They are known to induce apoptosis in leukemia and pancreatic cancer cells, and can destabilize p53, which may promote cellular death in response to DNA damaging agents (Wang et al, 2008; Beurel et al, 2009). Administration of GSKi inhibited cochlear destruction in cisplatin-injected mice (Park et al, 2009).

Lithium is a selective ATP competitive inhibitor of GSK-3 (Ryves and Harwood, 2001). Lithium carbonate is used with bipolar disorder patients (Moore et al, 2009). In a retrospective study of 162,118 COVID-19 patients from several U.S. health systems 7% of patients taking lithium developed COVID-19 compared with 15% among the general population (Liu et al, 2021). LY2090314 has been in clinical trials for metastatic pancreatic cancer and acute leukemia ([NCT01632306], [NCT01287520], [NCT01214603]). Clinical trials of GSKi for Alzheimer's disease were unsuccessful.

The use of GSKi remains controversial because of their possibly oncogenic properties. Evaluation of GSKi in clinical trials has been hampered by the fear that inhibition of GSK-3 may stimulate or aid in malignant transformation as GSK-3 can phosphorylate pro-oncogenic factors such as beta-catenin, c-Jun and c-Myc which targets them for degradation (Patel & Woodgett, 2008). However, no studies have been reported suggesting that treatment of mice with GSKi resulted in an increase in cancer incidence. In fact, many patients with bipolar disorder have been treated with lithium for prolonged periods of time, with no evidence that these patients have increased incidences of cancer (McCubrey et al, 2014).

The GSKi kenpaullone and lithium chloride were found to reduce viral Nucleoprotein phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic symptoms. Effects of GSK-3 inhibition were reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus (Wu et al, 2009).

This Reactome event shows the SARS‑CoV‑1 viroporin 3a‑triggered potassium (K+) efflux across the plasma membrane.

SARS-CoV-1 viroporin 3a is a membrane protein that can translocate to the plasma membrane and, via shedding, to other cells. The cytoplasmic domain of 3a contains the tyrosine based sorting motif (YXXΦ, where X can be any residue and Φ is a residue with a bulky hydrophobic side chain) which is responsible for Golgi to plasma membrane transport (Minakshi R & Padhan K 2014). In the membranes, 3a forms a homotetrameric inward-rectifying potassium ion channel which is inhibited by barium ions and 4-aminopyridine (Chan CM et al. 2009; Chien TH et al. 2013). The ion channel activity of 3a is linked to SARS-CoV-1-induced cell death in various mammalian cells (Law PTW et al. 2005; Freundt EC et al. 2010; Chan CM et al. 2009; Chien TH et al. 2013; Chen IY et al. 2019; Yue Y et al. 2018). Blocking potassium efflux from the cell impaired the ability of viral 3a protein to induce the caspase-dependent extrinsic apoptotic signalling pathway (Chan CM et al, 2009; Chien TH et al, 2013). In addition, membrane-bound 3a oligomers were found to activate the NLRP3 inflammasome and interleukin 1 beta (IL-1β) secretion via potassium efflux causing necrotic cell death (Chen IY et al. 2019; Yue Y et al. 2018). These data suggest that the ion channel activity of SARS-CoV-1 3a oligomers regulates both non-lytic apoptosis and highly inflammatory pyroptosis.Further, mutagenesis studies revealed that membrane association (Ren Y et al. 2020) and ion channel activities (Yue Y et al. 2018) of SARS-CoV-1 3a are involved but not critical for triggering cell death suggesting that 3a can also induce apoptotic or pyroptotic cell death in a membrane-independent manner (Ren Y et al. 2020).

ATR kinase activity is stimulated upon binding of the ATR-ATRIP complex to an RPA-ssDNA complex. ATR can subsequently phosphorylate and activate the checkpoint kinase Chk1, allowing further amplification of the checkpoint signal. The ATR and Chk1 kinases then modify a variety of factors that can lead to stabilization of stalled DNA replication forks, inhibition of origin firing, inhibition of cell cycle progression, mobilization of DNA repair factors, and induction of apoptosis. This checkpoint signaling mechanism is highly conserved in eukaryotes, and homologues of ATR and ATRIP are found in such organisms as S. cerevisiae (Mec1 and Ddc2, respectively), S. pombe (rad3 and rad26, respectively), and X. laevis (Xatr and Xatrip, respectively).

The ATR (ATM- and rad3-related) kinase is an essential checkpoint factor in human cells. In response to replication stress (i.e., stresses that cause replication fork stalling) or ultraviolet radiation, ATR becomes active and phosphorylates numerous factors involved in the checkpoint response including the checkpoint kinase Chk1. ATR is invariably associated with ATRIP (ATR-interacting protein) in human cells. Depletion of ATRIP by siRNA causes a loss of ATR protein without affecting ATR mRNA levels indicating that complex formation stabilizes the ATR protein. ATRIP is also a substrate for the ATR kinase, but modification of ATRIP does not significantly regulate the recruitment of ATR-ATRIP to sites of damage, the activation of Chk1, or the modification of p53.

While the ATR-ATRIP complex binds only poorly to RPA complexed with ssDNA lengths of 30 or 50 nt, binding is significantly enhanced in the presence of a 75 nt ssDNA molecule. Complex formation is primarily mediated by physical interaction between ATRIP and RPA. Multiple elements within the ATRIP molecule can bind to the RPA-ssDNA complex, including residues 1-107 (highest affinity), 218-390, and 390-791 (lowest affinity). Although the full-length ATRIP is unable to bind ssDNA, an internal region (108-390) can weakly bind ssDNA when present in rabbit reticulocyte lysates. ATR can bind to the ssDNA directly independent of RPA, but this binding is inhibited by ATRIP. Upon binding, the ATR kinase becomes activated and can directly phosphorylate substrates such as Rad17.

NPAS4 (Neuronal PAS domain containing protein 4) is a calcium dependent transcription factor predominantly expressed in neurons that regulates activation of genes involved in neuronal circuit formation, function, and plasticity (Ooe et al. 2004; Lin et al. 2008; Ramamoorthi et al. 2011; Maya-Vetencourt 2013; Sun and Lin 2016; Weng et al. 2018). NPAS4 possesses a conserved basic helix loop helix (bHLH) motif and a PAS domain (Fahim et al. 2018). NPAS4 is among the most rapidly induced immediate early genes (IEGs), which are activated after sensory and behavioral experience and thought to be crucial for formation of long term memory (Ramamoorthi et al. 2011; Sun et al. 2016; Heslin and Coutellier 2018; Weng et al. 2018). NPAS4 is activated within minutes of neuronal stimulation to regulate the formation of inhibitory synapses (Lin et al. 2008). NPAS4 enables gene regulation to be tailored to the type of depolarizing activity along the somato dendritic axis of a neuron (Brigidi et al. 2019). Transcriptional targets of NPAS4 include transcription factors and proteins involved in signal transduction and protein trafficking (Lin et al. 2008, Brigidi et al. 2019). NPAS4 regulates development of glutamatergic and GABAergic synapses essential for information processing and memory formation (Lin et al. 2008, Weng et al. 2018). NPAS4 induced gene expression programs differ between excitatory and inhibitory neurons (Spiegel et al. 2014), leading to a circuit wide homeostatic response. Besides directly regulating function of neurons, NPAS4 may be involved in the regulation of neuroinflammation and neuronal apoptosis (Zhang et al. 2009; Choy et al. 2015; Fan et al. 2016; Zhang et al. 2021). NPAS4 is expressed in the pancreatic beta cells and regulates their function under stress conditions (Sabatini et al. 2018). For review, please refer to Sun and Lin 2016, and Fu et al. 2020.

Calpains (EC 3.4.22.17; CAPN, Clan CA, family C02) constitute a distinct group of intracellular cysteine proteases found in almost all eukaryotes and a few bacteria. Calpains can be described as cytosolic proteases exhibiting Ca2+-dependent limited proteolytic activity which function to transform or modulate their substrate proteins' structures and activities; they are therefore called modulator proteases. As calpains selectively cleave proteins in response to calcium signals, they have the potential to influence cellular functions such as signal transduction, cytoskeletal remodelling, cell motility, membrane repair, cell cycle progression, gene expression and apoptosis (Sorimachi et al. 2010). Calpain deficiencies are linked to a variety of defects in many different organisms, including lethality, muscular dystrophies, gastropathy and diabetes (Sorimachi et al. 2011).

The human genome has 15 genes (named using formal nomenclature as CAPN1, CAPN2, etc.) that encode a calpain-like protease domain. The two best-characterised members of the calpain family, CAPN1 and 2, are ubiquitously expressed and locate to the cytosol of the cell (Goll et al. 2003). All other calpains annotated here are assumed to be functionally similar to these two based on their structural similarites. They are heterodimers, consisting of a common small regulatory subunit (CAPNS1 or CAPNS2; ca. 30kDa) and a large, isoform-specific catalytic subunit. Three-dimensional structural analyses reveal the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to ca. four Ca2+ ions via well-conserved amino acids. So, despite the fact that they have divergent domain structures, calpains share this mechanistic functional character (Croall & Ersfeld 2007, Sorimachi et al. 2011, Ono & Sorimachi 2012).

Calpain activity is tightly regulated by the endogenous inhibitor calpastatin (CAST), which is capable of reversibly binding and inhibiting four molecules of calpain in the presence of calcium. This suggests calpains are transiently activated by high Ca2+ concentrations such as a Ca2+ influx, and then return to an inactive state ready for reactivation (Campbell & Davies 2012).

The stearoyl CoA desaturase (SCD) gene is transcribed to yield mRNA and the mRNA is translated to yield protein. SCD is an endoplasmic reticulum (ER) enzyme that catalyzes the biosynthesis of monounsaturated fatty acids (MUFAs) from saturated fatty acids that are either synthesized de novo or derived from the diet (Bene H et al. 2001; Paton CM & Ntambi JM 2009). Liver X receptors (LXRα/NR1H3 or LXRβ/NR1H2) are oxysterol receptors that regulate the gene expression of SCD. The evidence for the Reactome event of the induction of SCD gene expression by NR1H3 or NR1H2 comes from the studies with T0901317, the synthetic NR1H2,3 agonist, that has been shown to increase SCD gene expression in mouse liver and human arterial endothelial cells (HAEC) by 10- and 3-fold, respectively (Chu K et al. 2006; Peter A et al. 2008). Treatment with 22(R)-hydroxycholesterol, a natural ligand of NR1H2,3, increased the SCD activity in mouse macrophages J774 and thus supported the ability of NR1H2,3 to regulate SCD (Wang Y et al. 2004). NR1H2,3 has been presumed to regulate SCD protein level through the activation of sterol regulatory element-binding protein (SREBP1) and its consequent binding to SREBP1 binding site (SRE) (Schultz JR et al. 2000; Zhang X et al. 2014). However, studies with SREBP1c -/- mice have suggested that NR1H2,3 upregulate SCD in an SREBP1c-independent manner (Chu K et al. 2006). In HAEC cells, the NR1H2,3 activation increased SCD mRNA and protein expression, which served to protect the cells from saturated fatty acid-induced lipotoxicity, apoptosis and IL-6 and IL-8 expression (Peter A et al. 2008). Analysis of hepatic lipogenic gene expression indicated that nuclear receptor-interacting protein 140 (RIP140 or NRIP1) was required for the ability of NR1H3 to stimulate the expression of the SCD gene in WT and NRIP1 null mice after administration of T0901317 (Herzog B et al. 2007). These findings are supported by the failure of T0901317 to stimulate the expression of SCD gene in cultured human hepatoma HuH7 cells depleted of NRIP1 by siNRIP1 (Herzog B et al. 2007). 2007). Studies performed with T0901317 in wildtype vs. NR1H3-/- (LXRα-/-) and NR1H2 (LXRβ -/-) mice suggest that SCD1 is primarily regulated by NR1H3 (Zhang X et al. 2014).

Receptor-interacting serine/threonine-kinase protein 1 (RIPK1) and RIPK3-dependent necrosis is called necroptosis or programmed necrosis. The kinase activities of RIPK1 and RIPK3 are essential for the necroptotic cell death in human, mouse cell lines and genetic mice models (Cho YS et al. 2009; He S et al. 2009, 2011; Zhang DW et al. 2009; McQuade T et al. 2013; Newton et al. 2014). The initiation of necroptosis can be stimulated by the same death ligands that activate extrinsic apoptotic signaling pathway, such as tumor necrosis factor (TNF) alpha, Fas ligand (FasL), and TRAIL (TNF-related apoptosis-inducing ligand) or toll like receptors 3 and 4 ligands (Holler N et al. 2000; He S et al. 2009; Feoktistova M et al. 2011; Voigt S et al. 2014). In contrast to apoptosis, necroptosis represents a form of cell death that is optimally induced when caspases are inhibited (Holler N et al. 2000; Hopkins-Donaldson S et al. 2000; Sawai H 2014). Specific inhibitors of caspase-independent necrosis, necrostatins, have recently been identified (Degterev A et al. 2005, 2008). Necrostatins have been shown to inhibit the kinase activity of RIPK1 (Degterev A et al. 2008). Importantly, cell death of apoptotic morphology can be shifted to a necrotic phenotype when caspase 8 activity is compromised, otherwise active caspase 8 blocks necroptosis by the proteolytic cleavage of RIPK1 and RIPK3 (Kalai M et al. 2002; Degterev A et al. 2008; Lin Y et al. 1999; Feng S et al. 2007). When caspase activity is inhibited under certain pathophysiological conditions or by pharmacological agents, deubiquitinated RIPK1 is engaged in physical and functional interactions with the cognate kinase RIPK3 leading to formation of necrosome, a necroptosis-inducing complex consisting of RIPK1 and RIPK3 (Sawai H 2013; Moquin DM et al. 2013; Kalai M et al. 2002; Cho YS et al. 2009, He S et al. 2009, Zhang DW et al. 2009). Within the necrosome RIPK1 and RIPK3 bind to each other through their RIP homotypic interaction motif (RHIM) domains. The RHIMs can facilitate RIPK1:RIPK3 oligomerization, allowing them to form amyloid-like fibrillar structures (Li J et al. 2012; Mompean M et al. 2018). RIPK3 in turn interacts with mixed lineage kinase domain-like protein (MLKL) (Sun L et al. 2012; Zhao J et al. 2012; Murphy JM et al. 2013; Chen W et al. 2013). The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). Mouse MLKL activation relies on transient engagement of RIPK3 to facilitate phosphorylation of the pseudokinase domain (Murphy JM et al. 2013; Petrie EJ et al. 2019a), while it appears that stable recruitment of human MLKL by necrosomal RIPK3 is an additional crucial step in human MLKL activation (Davies KA et al. 2018; Petrie EJ et al. 2018, 2019b). RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2020; Samson AL et al. 2020). Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014), but it is trafficking via a Golgi-microtubule-actin-dependent mechanism that facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive. The precise oligomeric form of MLKL that mediates plasma membrane disruption has been highly debated (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Petrie EJ et al. 2017, 2018; Samson AL et al. 2020 ). However, microscopy data revealed that MLKL assembles into higher molecular weight species upon cytoplasmic necrosomes within human cells, and upon phosphorylation by RIPK3, MLKL is trafficked to the plasma membrane (Samson AL et al. 2020). At the plasma membrane, phospho-MLKL forms heterogeneous higher order assemblies, which are thought to permeabilize cells, leading to release of DAMPs to invoke inflammatory responses. MLKL also exerts non-necroptotic functions such as regulation of endosomal trafficking or MLKL-induced activation of the NLRP3 inflammasome (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). While RIPK1, RIPK3 and MLKL are the core signaling components in the necroptosis pathway, many additional molecules have been proposed to positively and negatively tune the signaling pathway. Currently, this picture is evolving rapidly as new modulators continue to be discovered.

The Reactome module describes MLKL-mediated necroptotic events on the plasma membrane.

In contrast to NOD1/2 some NLRPs function as large macromolecular complexes called 'Inflammasomes'. These multiprotein platforms control activation of the cysteinyl aspartate protease caspase-1 and thereby the subsequent cleavage of pro-interleukin 1B (pro-IL1B) into the active proinflammatory cytokine IL1B. Activation of caspase-1 is essential for production of IL1B and IL18, which respectively bind and activate the IL1 receptor (IL1R) and IL18 receptor (IL18R) complexes. IL1R and IL18R activate NFkappaB and other signaling cascades.

As the activation of inflammasomes leads to caspase-1 activation, inflammasomes can be considered an upstream step of the IL1R and IL18R signaling cascades, linking intracellular pathogen sensing to immune response pathways mediated by Toll-Like Receptors (TLRs). Monocytes and macrophages do not express pro-IL1B until stimulated, typically by TLRs (Franchi et al. 2009). The resulting pro-IL1B is not converted to IL1B unless a second stimulus activates an inflammasome. This requirement for two distinct stimuli allows tight regulation of IL1B/IL18 production, necessary because excessive IL-1B production is associated with numerous inflammatory diseases such as gout and rheumatoid arthritis (Masters et al. 2009).

There are at least four subtypes of the inflammasome, characterized by the NLRP. In addition the protein AIM2 can form an inflammasome. All activate caspase-1. NLRP1 (NALP1), NLRP3 (Cryopyrin, NALP3), IPAF (CARD12, NLRC4) and AIM2 inflammasomes all have clear physiological roles in vivo. NLRP2, NLRP6, NLRP7, NLRP10 and NLRP12 have been demonstrated to modulate caspase-1 activity in vitro but the significance of this is unclear (Mariathasan and Monack, 2007).

NLRP3 and AIM2 bind the protein 'apoptosis-associated speck-like protein containing a CARD' (ASC, also called PYCARD), via a PYD-PYD domain interaction. This in turn recruits procaspase-1 through a CARD-CARD interaction. NLRP1 and IPAF contain CARD domains and can bind procaspase-1 directly, though both are stimulated by ASC. Oligomerization of NLRPs is believed to bring procaspases into close proximity, leading to 'induced proximity' auto-activation (Boatright et al. 2003). This leads to formation of the active caspase tetramer. NLRPs are generally considered to be cytoplasmic proteins, but there is evidence for cytoplasmic-nuclear shuttling of the family member CIITA (LeibundGut-Landmann et al. 2004) and tissue/cell dependent NALP1 expression in the nucleus of neurons and lymphocytes (Kummer et al. 2007); the significance of this remains unclear.

The protease caspase‑9 (CASP9) is normally present as an inactive monomeric propeptide (procaspase‑9 or zymogen). Upon apoptosis procaspase‑9 (CASP9(1‑416) is recruited to APAF1:cytochrome C (CYCS):ATP complex to form the caspase‑activating apoptosome (Hu Q et al. 2014; Cheng TC et al. 2016). The cryo-EM structures have established that the nucleotide-binding oligomerization domain (NOD) of APAF1 mediates the heptameric oligomerization of APAF1, while its tryptophan-aspartic acid (WD40) domain interacts with CYCS (Yuan S & Akey CW 2013). The caspase recruitment domain (CARD) of APAF1 recruits the N‑terminal CARD of CASP9(1‑416) through homotypic CARD:CARD interactions (Li P et al. 1997; Qin H et al. 1999; Yuan S et al. 2010; Yuan S & Akey CW 2013). These homotypic interaction motifs are thought to interact with each other through three types of interfaces, type I, II, and III, which cooperate to generate homo- and hetero-oligomers from relatively small assemblies to open-ended filaments (Ferrao R & Wu H 2012). Structural and mutagenesis studies showed that all type I, II, and III interfaces are involved in the caspase-9 activation by APAF1-mediated helical oligomerization of CARDs (Hu Q et al. 2014; Cheng TC et al. 2016; Su TW et al. 2017; Li Y et al. 2017). Cryo-EM structure of the holo-apoptosome revealed an oligomeric CARD disk above the heptameric apoptosome ring with estimated molecular ratios between 2-5 zymogens per 7 APAF1 molecules (Hu Q et al. 2014; Cheng TC et al. 2016). The structural and biochemical studies showed that APAF1-CARD and CASP9-CARD initially formed a 1:1 complex in solution, which at higher concentrations is further oligomerized into a 3:3 complex. The 3:3 complex was reported as a core arrangement of the 4:3 or 4:4 APAF1-CARD:CASP9-CARD complex in the helical assembly of the CARD disk (Cheng TC et al. 2016; Su TW et al. 2017; Li Y et al. 2017; Dorstyn L et al. 2018). Thus, APAF1:CASP9 (1-416) heterodimers may be recruted to the assembling apoptosome as part of its activation.

The Reactome event describes the apoptosome assembly with the stoichiometry of 4 procaspase-9 zymogens per 7 APAF1 molecules. The formation of 1:1 and other combinations of APAF1:CASP9(1-416) complexes is not shown.

Receptor-interacting protein 1 (RIPK1) polyubiquitination is required for the recruitment of the downstream signaling complexes such as the IkB kinase (IKK) complex in the response to TNFR1 stimulation (Ea CK et al. 2006; Blackwell K et al. 2013). RIPK1 is polyubiquitinated at Lys377 (K377, equivalent to K376 of mouse RIPK1) (Ea CK et al. 2006). A point mutation of RIPK1 at Lys377 (K377R) was found to abolish its polyubiquitination and prevent the recruitment and activation of IKK and the TGF-β activated kinase 1 (TAK1) complex (Ea CK et al. 2006). TNFα-induced recruitment of the IKK complex to TNFR1 is completely impaired, recruitment of TAK1 is severely reduced, and recruitment of the LUBAC E3 ligase complex, is also reduced in human RIPK1-deficient Jukart T-cells (Blackwell K et al. 2013). In vivo studies showed that RIPK1 K376R mutation activated cell death resulting in embryonic lethality in mice (Tang Y et al. 2019; Zhang X et al. 2019; Kist M et al. 2021). These data suggest that K63-linked ubiquitylation of RIPK1 at K377 prevents TNF-induced cell death.

Several E3 ligases are involved in TNF-α signaling to initiate an immediate and effective host response to infection or injury. Among them are anti-apoptotic regulators BIRC2 and BIRC3, also known as inhibitor of apoptosis proteins (cIAP1/2). BIRC2/3 were found to constitutively associate with TRAF2 and via TRAF2 they were recruited to the TNFR1 signaling complex (Samuel T et al. 2006; Bertrand et al, 2008; Varfolomeev E et al, 2008). BIRC2/3 can directly ubiquitinate RIPK1 within the TNFR1 receptor complex allowing it to bind to the TAB2:TAK1 complex, a process reversed by the deubiquitinase CYLD and A20 (Bertrand et al, 2008; Varfolomeev et al, 2008; Moquin DM et al. 2013; Shembade N et al. 2010; Wertz IE et al. 2004). In conjunction with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, BIRC2/3 was shown to mediate polymerization of both K63-linked and linear Met1-linked chains on RIPK1 (Varfolomeev E et al, 2008; Bertrand et al, 2008; Blackwell K et al. 2013). TRAF2 promotes BIRC-mediated linear and K63-linked ubiquitination of RIP1 (Blackwell K et al. 2013). K11-linked polyubiquitination of RIPK1 may also depend on BIRC2 and BIRC3 (Dynek JN et al. 2010).

Germline and somatic defects in genes that encode proteins that participate in DNA repair give rise to genetic instability that can lead to malignant transformation or trigger cellular senescence or apoptosis. Germline defects in DNA repair genes are an underlying cause of familial cancer syndromes and premature ageing syndromes. Somatic defects in DNA repair genes are frequently found in tumors. For review, please refer to Tiwari and Wilson 2019.

We have so far annotated diseases of mismatch repair, diseases of base excision repair and diseases of DNA double-strand break repair.

Defects in mammalian DNA mismatch repair (MMR) genes (MLH1, PMS2, MSH2, and MSH6) result in microsatellite instability (MSI) and reduced fidelity during replication and repair steps. Defective variants of MMR genes are associated with sporadic cancers with hypermutation phenotypes as well as hereditary cancer syndromes such as Lynch syndrome (hereditary non-polyposis colorectal cancer) and constitutional mismatch repair deficiency syndrome (CMMRD). MSI is an important predictor of sensitivity to cancer immunotherapy as the high mutational burden renders MSI tumors immunogenic and sensitive to programmed cell death-1 (PD-1) immune checkpoint inhibitors (Mandal et al. 2019). For review, please refer to Pena-Diaz and Rasmussen 2016, Sijmons and Hofstra 2016, Tabori et al. 2017, Baretti and Le 2018.

Germline mutations, single nucleotide polymorphisms (SNPs) and somatic mutations in several genes involved in base excision repair (BER), a DNA repair pathway where a damaged DNA base is excised and replaced with a correct base, are involved in the development of cancer and several oxidative stress-related diseases. For review, please refer to Fu et al. 2012, Fletcher and Houlston 2010, Brenerman et al. 2014, Patrono et al. 2014, and D'Errico et al. 2017.

Germline mutations in genes involved in repair of DNA double-strand breaks (DSBs) are the underlying cause of several cancer predisposition syndromes, some of which also encompass developmental disorders associated with immune dysfunction, radiosensitivity and neurodegeneration. Somatic mutations in genes involved in DSB repair also occur in sporadic cancers. For review, please refer to McKinnon and Caldecott 2007, Keijzers et al. 2017, and Jachimowicz et al. 2019.

During viral infection, cytosolic viral RNA triggers activation of mitochondrial antiviral-signaling protein (MAVS) and the formation of MAVS signalosome (Kawai T et al. 2005; Seth RB et al. 2005; Xu LG et al. 2005). Activated MAVS recruits TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3) and IRF7 to the mitochondria leading to the activation of IRF3/IRF7 and subsequent production of type I interferons (Kawai T et al. 2005; Seth RB et al. 2005; Xu LG et al. 2005).

Co-immunoprecipitation assay showed that both TBK1 and IRF3 associated with heat shock protein 90kDa (HSP90), which facilitated signal transduction from TBK1 to IRF3 in Sendai virus (SeV)-infected human embryonic kidney (HEK293) cells (Yang K et l. 2006). MAVS, TBK1 and IRF3 were found to associate with mitochondrial import receptor subunit TOM70 (TOMM70) in HEK293 cells (Liu XY et al. 2010). TOMM70 localizes on the outer membrane of the mitochondria to mediate the translocation of mitochondrial protein precursors from the cytosol into the mitochondria (reviewed in Fan AC & Young JC 2011; Sokol AM et al. 2014; Kreimendahl S & Rassow J 2020). The molecular chaperone complexes of HSP90 and HSP70 were shown to deliver precursor proteins to TOMM70 for subsequent import (Young JC et al. 2003; Zanphorlin LM et al. 2016). The C-terminal motif (EEVD) of HSP90 was found to bind the N-terminal TPR clamp-type domain of TOMM70 (Liu XY et al. 2010; Gava LM et al. 2011). Knockdown of HSP90 by small interfering RNA (siRNA) decreased the association of TOMM70 with TBK1 and IRF3 in HEK293T cells (Liu XY et al. 2010). Further, in SeV-stimulated HEK293 cells, cytosolic BAX translocated to the mitochondrial outer membrane and induced apoptosis in the IRF3-dependent manner via the formation of the TOMM70:HSP90:IRF3:BAX protein complex (Wei B et al. 2015). The data suggest that HSP90 forms a complex with TBK1 and IRF3 in the cytosol and deliver them to the MAVS signalosome on the mitochondria.

Interaction between HSP90 and US11, a viral protein derived from human herpesvirus 1 (HHV-1, also known as herpes simplex virus 1, HSV-1) disrupted the formation of the HSP90:TBK1:IRF3 complex and induced degradation of TBK1 through a proteasome-dependent pathway in mouse embryonic fibroblasts (MEFs) (Liu X et al. 2018).

The apoptosome is a cytoplasmic protein complex of two major components ‑ the adapter protein apoptotic protease activating factor 1 (APAF1) and the protease caspase‑9 (CASP9) which interact with each other through their caspase recruitment domains (CARD) (Qin et al. 1999; Yuan S et al. 2010; Yuan S & Akey CW 2013). The function of the apoptosome is to assemble a multimeric complex between APAF1 and procaspase-9 CARDs to facilitate CASP9 activation (Jiang X and Wang X 2000; Srinivasrula SM et al. 2001; Shiozaki EN et al. 2002). The apoptosome is assembled upon APAF1 interaction with cytochrome c (CYCS), which is released from the mitochondrial intermembrane space during apoptosis (Zou H et al. 1997; Yuan S et al. 2013; Shakeri R et al. 2017). CYCS‑bound APAF1 undergoes ATP-mediated conformational changes and in the presence of CARD of CASP9 oligomerizes into a heptameric complex, which activates procaspase 9 (Zou H et al. 1997; Bratton SB et al. 2010; Acehan D et al. 2002; Yu X et al. 2005; Yuan S et al. 2010; Su TW et al. 2017). In the apoptosome, recruitment of caspase-9 may occur before oligomerization in the CARD disk, which presumably brings the caspase domain into proximity for their dimerization and activation (Su TW et al. 2017; Hu Q et al. 2014; Cheng TC et al. 2016). Once activated, CASP9 activates downstream effector caspases‑3 and ‑7. The activated effector caspases then cleave various cellular proteins.

Different models have been proposed to explain CASP9 activation: the “proximity‑driven dimerization model” and the “induced conformation model”. The first models states that upon binding to heptameric APAF1, monomers of procaspase‑9 are brought into close proximity at a high concentration (Acehan et al. 2002; Renatus et al. 2001). This induces dimerization which is sufficient for CASP9 activation whereas autoprocessing within the apoptosome complex merely stabilizes CASP9 dimer (Boatright KM et al. 2003; Pop C et al. 2006). The “induced conformation model” is based on the observation that CASP9 has a much higher level of catalytic activity when it's bound to the apoptosome. The model suggests that a conformational change occurs at the active site of CASP9 upon binding to APAF1 thus inducing CASP9 homodimerization and stabilizing it in the catalytically active conformation (Shiozaki EN et al. 2002). CASP9 activation may also involve formation of a multimeric CARD:CARD assembly between APAF1 and procaspase‑9 (Hu Q et al. 2014).

Ubiquitinated TRAF2 and BIRC2/3 were reported to recruit an additional E3 ligase complex, the linear ubiquitin (Ub) chain assembly complex (LUBAC). LUBAC is thought to bind K63 chains on BIRC2/3 but produce Met1 (M1)-linked (also known as linear) Ub chains to facilitate recruitment and Met1-linked ubiquitination of NEMO (IKBKG), the regulatory subunit of the IKK complex (Haas TL et al. 2009). LUBAC enhances NEMO interaction with the TNFR1 receptor signaling complex and thus IKK complex, stabilizes this protein complex, and promotes efficient TNF-induced activation of NFkappaB resulting in apoptosis inhibition (Haas TL et al. 2009)

LUBAC consists of the chain-assembling E3 ligase HOIP as well as HOIL-1 and SHARPIN (Kirisako T et al. 2006; Walczak H et al. 2012). Importantly, deletion of the LUBAC component SHARPIN in mice or mutation of HOIL-1 in humans, lead to hyperinflammatory phenotypes, indicating key roles of LUBAC and linear Ub chains in the response to infection and inflammation (Gerlach B et al. 2011; Ikeda F et al. 2011; Tokunaga F et al. 2011; Boisson B et al. 2012).

HOIP belongs to the RING-between-RING (RBR) family of E3 ligases and is the catalytic component of LUBAC (Spratt DE et al. 2014). RBR E3 ligase domain and a conserved C-terminal extension of HOIP are responsible for assembling Met1-linked chains (Smit JJ et al., 2012; Stieglitz B et al. 2012 and 2013). HOIP was reported to act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 domain subsequently forms a thioester intermediate with the transferred or “donor” ubiquitin (Stieglitz B et al. 2013). A Ub-associated (UBA) domain mediates interactions with HOIL-1L (Yagi H et al. 2012), while N-terminal PUB domain may be involved in interaction with regulatory proteins such as OTULIN to control NFkappaB signaling (Elliott PR et al. 2014). HOIP also comprises several NPL4 zinc finger (NZF) Ub binding domains (UBDs) that target it to ubiquitinated proteins (Haas et al. 2009; Fujita H et al. 2014).

Structural analysis revealed that NZF1 of HOIP can simultaneously bind both leucine zipper (CoZi) domains of NEMO (IKBKG) and ubiquitin and that both interactions are involved in TNF α-mediated NFkappaB activation (Fujita H et al. 2014). In addition, NEMO (IKBKG) ubiquitination required RBR domain of HOIL-1L (Smit JJ et al. 2013).

PDE3A inhibitors are drugs which inhibits the action of the phosphodiesterase enzyme isoform 3A (PDE3A). They are used for the therapy of acute heart failure and cardiogenic shock. The cardiovascular bipyridines amrinone and milrinone are positive inotropic agents with vasodilator properties. PDE3A inhibitors, as their name suggests, blocks the breakdown of cAMP, increasing cytosolic calcium levels and leading to a positive inotropic effect: they increase the force of cardiac contraction. Amrinone, milrinone and enoximone are used clinically for short-term treatment of cardiac failure (el Allaf et al. 1984, Honerjäger 1989).

Milrinone (brand name Primacor) is a PDE3A inhibitor drug used in patients with heart failure (Hamada et al. 1999, DiBianco et al. 1989, Benotti et al. 1985). Studies in dogs indicate that by inhibiting PDE3A, milrinone and amrinone prevent cAMP degradation and thereby increase cAMP levels. This increases the activation of protein kinase A (PKA), an enzyme that phosphorylates components of the contractile machinery in muscle cells, including the heart (Alousi & Johnson 1986). Amrinone (INN, inamrinone), is a pyridine phosphodiesterase 3A (PDE3A) inhibitor drug that may improve the prognosis in patients with congestive heart failure (Hamada et al. 1999, Wilmshurst et al. 1984). It may also possess a vasodilatory effect (Ono et al. 1996, Wilmshurst et al. 1984). Enoximone is an imidazole PDE3A inhibitor (Dage et al. 1982) used in the treatment of congestive heart failure (Weber et al. 1986). Anagrelide is an oral imidazoquinazoline agent possessing anti-PDE3A activity and inhibits platelet aggregation in both humans and animals (Tefferi et al. 1997). Cilostazol is a quinolinone derivative acting as an antiplatelet agent with vasodilating properties (Schror 2002). It is used in the symptomatic treatment of intermittent claudication (pain in legs when walking but paid disappears on rest) in patients with peripheral ischaemia (Farkas et al. 2020). Ibudilast is an anti-inflammatory and neuroprotective oral agent used for the treatment of multiple sclerosis, asthma, and cerebrovascular disease (Yamazaki et al. 2011).

PDE activity may increase in a variety of tumours therfore PDE inhibitors may be potential targets for tumour cell growth inhibition and induction of apoptosis (Davari et al. 2014, Nazir et al. 2017, An et al. 2019).

Given that PDE inhibitors are used in the treatment of pathologies (eg thrombosis, inflammation, fibrosis) that can significantly overlap Covid-19 clinical symptoms, these drugs may be beneficial for the treatment of Covid-19 infection (Giorgi et al. 2020).

In addition to its well-characterized role in estrogen-dependent transcription, estrogen (beta-estradiol, also known as E2) also plays a rapid, non-genomic role through interaction with receptors localized at the plasma membrane by virtue of dynamic palmitoylation. Estrogen receptor palmitoylation is a prerequisite for the E2-dependent activation of extra-nuclear signaling both in vitro and in animal models (Acconcia et al, 2004; Acconcia et al, 2005; Marino et al, 2006; Marino and Ascenzi, 2006). Non-genomic signaling through the estrogen receptor ESR1 also depends on receptor arginine methylation by PMRT1 (Pedram et al, 2007; Pedram et al, 2012; Le Romancer et al, 2008; reviewed in Arnal, 2017; Le Romancer et al, 2011 ).
E2-evoked extra-nuclear signaling is independent of the transcriptional activity of estrogen receptors and occurs within seconds to minutes following E2 administration to target cells. Extra-nuclear signaling consists of the activation of a plethora of signaling pathways including the RAF/MAP kinase cascade and the PI3K/AKT signaling cascade and governs processes such as apoptosis, cellular proliferation and metastasis (reviewed in Hammes et al, 2007; Handa et al, 2012; Lange et al, 2007; Losel et al, 2003; Arnal et al, 2017; Le Romancer et al, 2011). ESR-mediated signaling also cross-talks with receptor tyrosine kinase, NF- kappa beta and GPCR signaling pathways by modulating the post-translational modification of enzymes and other proteins and regulating second messengers (reviewed in Arnal et al, 2017; Schwartz et al, 2016; Boonyaratanakornkit, 2011; Biswas et al, 2005). In the nervous system, E2 affects neural functions such as cognition, behaviour, stress responses and reproduction in part by inducing such rapid extra-nuclear responses (Farach-Carson and Davis, 2003; Losel et al, 2003), while in endothelial cells, non-genomic ESR-dependent signaling also regulates vasodilation through the eNOS pathway (reviewed in Levin, 2011).
Extra-nuclear signaling additionally cross-talks with nuclear estrogen receptor signaling and is required to control ER protein stability (La Rosa et al, 2012)
Recent data have demonstrated that the membrane ESR1 can interact with various endocytic proteins to traffic and signal within the cytoplasm. This receptor intracellular trafficking appears to be dependent on the phyical interaction of ESR1 with specific trans-membrane receptors such as IGR-1R and beta 1-integrin (Sampayo et al, 2018)

Many signaling pathways rely on the activation of nuclear factor kappa B (NFkB), which is critical for the induction of the appropriate cellular function in response to various stimuli such as inflammatory cytokines, microbial products or various types of stress (Lawrence T 2009; Hoesel B and Schmid JA 2013). The NFkB family of transcription factors is kept inactive in the cytoplasm by inhibitor of kappa B (IkB) family members (Oeckinghaus A and Ghosh S 2009). Canonical NFkB activation depends on the phosphorylation of IkB by the I kappa B kinase (IKK) complex, which contains two catalytic subunits named IKK alpha, IKK beta and a regulatory subunit named NFkB essential modulator (NEMO or IKBKG) (Rothwarf DM et al. 1998). Phosphorylation of IkB leads to K48-linked ubiquitination and proteasomal degradation of IkB, allowing translocation of NFkB factor to the nucleus, where it can activate transcription of a variety of genes participating in the immune and inflammatory response, cell adhesion, growth control, and protection against apoptosis (Collins T et al. 1995; Kaltschmidt B et al. 2000; Lawrence T 2009).

IKBKG is encoded by an X-linked gene. Null alleles of the gene are lethal in hemizygous males, whereas hypomorphic alleles typically result in the impaired NFkB signaling in patients with a broad spectrum of clinical phenotypes in terms of both developmental defects and immunodeficiency (Döffinger R et al. 2001; Hanson EP et al. 2008). Several categories of mutations affecting IKBKG have been reported in humans (Döffinger R et al. 2001; Vinolo E et al. 2006; Fusko F et al. 2008). The first category of these mutations consists of hypomorphic mutations typically involving the zinc finger domain and nearby C-terminal regions and causing hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID) in males (Jain A et al. 2001; Shifera AS 2010). The second category consists of amorphic mutations causing incontinentia pigmenti (IP) in females and, generally, prenatal death in males (Aradhya S et al. 2001; Fusco F et al. 2004). The third category is composed of hypomorphic mutations involving the stop codon causing anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), osteopetrosis and lymphedema (OL-EDA-ID) in males (Döffinger R et al. 2001). Also some patients with a defective IKBKG gene can develop immunodeficiency without ectodermal dysplasia (Orange JS et al. 2004). This module describes several EDA-ID-associated hypomorphic IKBKG mutations that have been reported to affect inflammatory responses initiated by toll like receptors (TLR).

Under basal conditions, mixed lineage kinase domain-like protein (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3) are pre-associated in the cytoplasm (Meng Y et al. 2021). Upon induction of necroptosis the RIPK3:MLKL complex is recruited to the necrosome where RIPK3 phosphorylates and activates MLKL (Meng Y et al. 2021). Studies in human cell lines suggest that phosphorylated MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014). Its trafficking via a Golgi-microtubule-actin-dependent mechanism facilitates plasma membrane translocation, where membrane disruption causes cell death (Samson AL et al. 2020). However, MLKL also exerts non-necroptotic functions such as regulation of endosomal trafficking or MLKL-induced activation of the NLRP3 inflammasome (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). In addition, MLKL was shown to mediate myelin breakdown in diabetic neuropathy (Guo J et al. 2022). Activation of MLKL triggers its K63-linked ubiquitination (Yoon S et al. 2022). Conjugation of K63-linked polyubiquitin (pUb) chains in the N-terminal HeLo domain of phosphorylated MLKL targets MLKL to endosomes where MLKL is involved in endosomal compartment trafficking and generation of intraluminal and extracellular vesicles (EV) (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). Endosome-bound MLKL is excluded from the cell within EV (Yoon S et al. 2022). Mass spectroscopic and western blot assays found that HECT-type E3 ubiquitin-protein ligase Itchy homolog (ITCH) is released within EVs alongside MLKL from human epithelial HT-29 cells in response to combined treatment with the cytokine TNF, the inhibitor of apoptosis proteins (IAP) antagonist BV6, and the caspase inhibitor z-VAD-fmk (TBZ) (Yoon S et al. 2017; 2022). Overexpression of ITCH also dramatically enhanced the association of MLKL with ESCRT proteins. Further, ITCH was shown to interact with MLKL to catalyze K63-linked pUb of MLKL at K50 targeting it to the endosomal membrane (Yoon S et al. 2022). Overexpression of ITCH in TBZ-treated HT-29 cells enhanced the ubiquitination of MLKL, but not of K50R MLKL and also enhanced the association of MLKL with ESCRT proteins (Yoon S et al. 2017; 2022). These data suggest that ITCH catalyzes K63-linked polyubiquitination of MLKL at K50 targeting it to the endosomal membrane, where MLKL is no longer involved in necroptosis but instead has a role in vesicle-mediated trafficking. Further, K63-linked polyUb-MLKL contributes to the host defense against pathogenic bacteria by targeting intracellular bacteria, such as Listeria monocytogenes, Yersinia enterocolitica and Escherichia coli, to endosomal-lysosome trafficking system (Yoon S et al. 2022).

This Reactome event shows ITCH-mediated K63-linked polyubiquitination of MLKL at K50.

Exogenous stimuli provoke assembly of the receptor-interacting serine/threonine protein kinase RIPK1:RIPK3 oligomeric complex, termed the necrosome, which acts as a platform for recruiting and activating mixed lineage kinase domain-like protein (MLKL), the terminal effector pseudokinase in the necroptotic signaling pathway (reviewed by Murphy JM 2020). RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2018; Samson AL et al. 2020). Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014). Its trafficking via a Golgi-microtubule-actin-dependent mechanism facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020). However, MLKL also exerts non-necroptotic functions such as regulation of endosomal trafficking or MLKL-induced activation of the NLRP3 inflammasome (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). Activation of MLKL triggers its K63-linked ubiquitination (Yoon S et al. 2022). Conjugation of K63-linked polyubiquitin (pUb) chains in the N-terminal HeLo domain of phosphorylated MLKL targets MLKL to endosomes where MLKL is involved in endosomal compartment trafficking and generation of intraluminal and extracellular vesicles (EV) (Yoon S et al. 2017; Shlomovitz I et al. 2020; Yoon S et al. 2022). Endosome-bound MLKL is excluded from the cell within EV (Yoon S et al. 2022). Mass spectroscopic and western blot assays found that HECT-type E3 ubiquitin-protein ligase Itchy homolog (ITCH) is released within EVs alongside MLKL from human epithelial HT-29 cells in response to combined treatment with the cytokine TNF, the inhibitor of apoptosis proteins (IAP) antagonist BV6, and the caspase inhibitor z-VAD-fmk (TBZ) (Yoon S et al. 2017; 2022). Further, ITCH co-immunoprecipitated with MLKL upon co-expression of tagged proteins in human embryonic kidney 293T (HEK293T) cells (Yoon S et al. 2022). ITCH also associated with MLKL in MLKL knocked-down HT-29 cells that inducibly expressed wild-type MLKL. Immunocytochemistry assay showed that MLKL colocalized with ITCH in both the early and the late endosomes in TBZ-treated HT-29 cells. Mutagenesis analysis using HEK293T cells revealed that ITCH binds the pseudokinase domain of MLKL. WW domain of ITCH facilitates this binding. ITCH was shown to catalyze K63-linked pUb of MLKL at K50 targeting it to the endosomal membrane (Yoon S et al. 2022).

NOTCH2 is activated by binding Delta-like and Jagged ligands (DLL/JAG) expressed in trans on neighboring cells (Shimizu et al. 1999, Shimizu et al. 2000, Hicks et al. 2000, Ji et al. 2004). In trans ligand-receptor binding is followed by ADAM10 mediated (Gibb et al. 2010, Shimizu et al. 2000) and gamma secretase complex mediated cleavage of NOTCH2 (Saxena et al. 2001, De Strooper et al. 1999), resulting in the release of the intracellular domain of NOTCH2, NICD2, into the cytosol. NICD2 traffics to the nucleus where it acts as a transcriptional regulator. For a recent review of the cannonical NOTCH signaling, please refer to Kopan and Ilagan 2009, D'Souza et al. 2010, Kovall and Blacklow 2010. CNTN1 (contactin 1), a protein involved in oligodendrocyte maturation (Hu et al. 2003) and MDK (midkine) (Huang et al. 2008, Gungor et al. 2011), which plays an important role in epithelial-to-mesenchymal transition, can also bind NOTCH2 and activate NOTCH2 signaling.

In the nucleus, NICD2 forms a complex with RBPJ (CBF1, CSL) and MAML (mastermind). The NICD2:RBPJ:MAML complex activates transcription from RBPJ binding promoter elements (RBEs) (Wu et al. 2000). NOTCH2 coactivator complexes directly stimulate transcription of HES1 and HES5 genes (Shimizu et al. 2002), both of which are known NOTCH1 targets. NOTCH2 but not NOTCH1 coactivator complexes, stimulate FCER2 transcription. Overexpression of FCER2 (CD23A) is a hallmark of B-cell chronic lymphocytic leukemia (B-CLL) and correlates with the malfunction of apoptosis, which is thought be an underlying mechanism of B-CLL development (Hubmann et al. 2002). NOTCH2 coactivator complexes together with CREBP1 and EP300 stimulate transcription of GZMB (granzyme B), which is important for the cytotoxic function of CD8+ T cells (Maekawa et al. 2008).

NOTCH2 gene expression is differentially regulated during human B-cell development, with NOTCH2 transcripts appearing at late developmental stages (Bertrand et al. 2000).

NOTCH2 mutations are a rare cause of Alagille syndrome (AGS). AGS is a dominant congenital multisystem disorder characterized mainly by hepatic bile duct abnormalities. Craniofacial, heart and kidney abnormalities are also frequently observed in the Alagille spectrum (Alagille et al. 1975). AGS is predominantly caused by mutations in JAG1, a NOTCH2 ligand (Oda et al. 1997, Li et al. 1997), but it can also be caused by mutations in NOTCH2 (McDaniell et al. 2006).


Hajdu-Cheney syndrome, an autosomal dominant disorder characterized by severe and progressive bone loss, is caused by NOTCH2 mutations that result in premature C-terminal NOTCH2 truncation, probably leading to increased NOTCH2 signaling (Simpson et al. 2011, Isidor et al. 2011, Majewski et al. 2011).

Expression of heme oxygenase 1 (HMOX1) is regulated by various indicators of cell stress, while HMOX2 is expressed constitutively. Both catalyze the breakdown of heme into biliverdin (BV), carbon monoxide (CO), and ferrous iron. Biliverdin is immediately reduced to bilirubin (BIL). Both bilirubin and carbon monoxide can localize to different compartments and outside the cell. Cytoprotection by HMOX1 is exerted directly by HMOX1 and by the antioxidant metabolites produced through the degradation of heme. Additionally, due to the reactive nature of labile heme, its degradation is intrinsically protective.

HMOX1 confers cytoprotection against cell death in various models of lung and vascular injury by inhibiting apoptosis, inflammation, and immune cell proliferation. It binds to the NACHT domain of NLRP3 inflammasome, blocking its activation. In mouse it directly binds STAT3 to control the generation of pathogenic Th17 cells during neutrophilic airway inflammation. It also blocks phosphorylation of STAT3 by PTK6 and co-inhibits Socs3, a negative feedback factor of Stat3 activation, as well as RORγt, thereby decreasing Th2 and Th17 immune responses, and alleviating airway inflammation.

The beneficial effects of the three products generated by HMOX1 differ not only in their inherent molecular mechanisms, but also in their downstream cellular targets. To date, this is the only enzymatic system known to exhibit such characteristics. Iron is a vital component of many biological systems and is capable of producing hydroxyl radicals via fenton chemistry. For this reason, iron is sequestered by the storage multimer ferritin and to prevent oxidative damage while maintaining the iron pool. On the other hand, the protective effects of bilirubin and CO are broadly recognized, which has led to their consideration as therapeutics for a range of diseases. Bilirubin has been recognized as one of the most potent antioxidants in nature, and moderate increases of its serum level have been shown in numerous large-scale population and epidemiological studies to have a protective effect against cardiovascular and metabolic disease. These effects are mediated by bilirubin scavenging of superoxide anions and reactive nitrogen species (RNS), and by activating the transcription factor PPAR-alpha.

CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. CO possesses intriguing signaling properties affecting numerous critical cellular functions including but not limited to inflammation, cellular proliferation, and apoptotic cell death. Binding of CO with key ferrous hemoproteins serves as a posttranslational modification that regulates important processes as diverse as aerobic metabolism, oxidative stress, and mitochondrial bioenergetics. The most important of these is the mitochondrial cytochrome c oxidase (Cco). By locally blocking mitochondrial respiration the main source of reactive oxygen species (ROS) in the cell is switched off. Additionally CO enables efficient reduction of methemoglobin (MetHb) by H2O2, thus preventing the generation of free heme in hemorrhagic diseases and malaria (Origassa and Câmara, 2013; Morse et al, 2009; Ryter et al, 2006; Cooper and Brown, 2008; Hinds and Stec, 2008).

Mitochondrial proteases participate in proteostasis, the regulation of proteins to maintain a functional proteome, by degrading unfolded, unassembled, and oxidatively damaged proteins (reviewed in Ng et al. 2021, Song et al. 2021). Degradation of mitochondrial proteins by proteases also serves to regulate transcription by TFAM, oxidative phosphorylation by electron carriers, lipid translocation by PRELID1 and STARD7, and mitochondrial fission and fusion by OPA1 and OMA1 (reviewed in Ahola et al. 2019). Because of the bacterial origin of mitochondria, they contain a number of bacterial type proteases, including LONP1 in the matrix, CLPP:CLPX (CLPXP) in the matrix, HTRA2 (OMI) in the intermembrane space, AFG3L2 in the mitochondrial inner membrane and protruding into the matrix, and YME1L1 in the mitochondrial inner membrane and protruding into the intermembrane space (reviewed in Deshwal et al. 2020, Szczepanowska and Trifunovic 2022).
The hexameric LONP1 complex, which is homologous to Lon proteases of eubacteria such as E. coli, binds substrate proteins in the matrix and inner membrane, unfolds them in an ATP-dependent mechanism, and degrades them (reviewed in Gibellini et al. 2020). LONP1 also acts as an ATP-dependent chaperone that is independent of its protease function (reviewed in Gibellini et al. 2020).
Like LONP1, the CLPXP complex unfolds matrix proteins in an ATP-dependent reaction and degrades them, however, the ATPase/unfolding function and the protease function are performed by separate subunits, with CLPX hexamers unfolding substrate proteins and translocating them to CLPP tetradecamers for processive degradation (reviewed in Mabanglo et al. 2021, Mabanglo and Houry 2022).
AFG3L2 (m-AAA+) forms either homohexamers or heterohexamers with its paralog SPG7 (Paraplegin) that are anchored in the mitochondrial inner membrane and protrude into the matrix (reviewed in Patron et al. 2018, Steele and Glynn 2019, Zhang and Mao 2020). The substrate protein enters the central channel formed by the ATPase domains of AFG3L2 and is unfolded and translocated to the pore formed by the protease domains, where it is degraded (reviewed inZhang and Mao 2020).
Like AFG3L2, YME1L1 (YME1L, i-AAA+) is a homohexameric complex that is anchored in the mitochondrial inner membrane, however, YME1L1 protrudes into the intermembrane space where it unfolds substrate proteins of the intermembrane space and inner membrane in an ATP-dependent reaction and then degrades them (reviewed in Steele and Glynn 2019, Ohba et al. 2020, Zhang and Mao 2020).
HTRA2 (OMI) forms soluble trimeric complexes in the intermembrane space that degrade substrate proteins, notably amyloid precursor proteins that are translocated to the intermembrane space and inner membrane. HTRA2 released from mitochondria into the cytosol also participates in regulating apoptosis (reviewed in Vande Walle et al. 2008).
Mutations in mitochondrial proteases cause diseases, such as spastic paraplegia (SPG7), ataxia (AFG3L2), and Parkinson's Disease (HTRA2), that typically have neurological symptoms among others (reviewed in Su et al. 2019, Gomez-Fabra Gala and Vogtle 2021).

After the human respiratory syncytial virus A (hRSV A) enters host cells, an initial round of transcription and translation of virally-encoded mRNAs ensues, which is followed by genome replication.

The negative sense, single-stranded RNA (-ssRNA) genome of the human respiratory syncytial virus (RSV) A is 15.2 kb long and contains 10 genes that encode 11 proteins. The 10 genes, going from the 3' end to the 5' end of the -ssRNA are: 1C (NS1), 1B (NS2), N, P, M, SH, G, F, M2, and L. Except for the M2 gene, each gene encodes one protein. The two overlapping open reading frames (ORFs) of the M2 gene encode proteins M2-1 and M2-2.

The N gene encodes the nucleoprotein, which forms decameric and hendecameric (11-fold) rings around which viral genomic RNA is packaged. The L and P genes encode the large polymerase subunit and the phosphoprotein polymerase cofactor subunit, respectively, of the RNA-dependent RNA polymerase complex (RdRP) (reviewed in Battles and McLellan 2019). The L protein contains three conserved enzymatic domains: the RNA-dependent RNA polymerase (RdRp) domain, the polyribonucleotidyl transferase (PRNTase or capping) domain, and the methyltransferase (MTase) domain (reviewed in Sutto-Ortiz et al. 2023). The M2-1 product of the M2 gene is a transcription processivity factor, while the M2-2 product of the M2 gene is a nonstructural protein that regulates the switch between transcription and genome replication. The SH, G, and F genes encode three proteins that are embedded in the viral envelope: small hydrophobic protein, attachment protein, and fusion protein, respectively. The secreted isoform of G protein (sG) mediates immune evasion. The NS1 and NS2 genes encode nonstructural proteins that function together to inhibit apoptosis and interferon response in infected cells. For review, please refer to Battles and McLellan 2019.

The genomic -ssRNA and the antigenome RNA are encapsidated as they are synthesized, and the association of the nascent RNA with the N protein is likely what causes the replicating polymerase to be processive, with the processivity being further augmented by the M2-1 processivity factor (reviewed in Fearns and Deval 2016). The C-terminal arm of the N protein, known to interact with the P protein subunit of RdRP complex, extends above the plane of N decamers. The interaction between N and P proteins may allow the RdRP complex to distort the helical conformation of the nucleocapsid during RNA synthesis. A long beta-hairpin in the N-terminal region of the N protein may be the site of contact with the catalytic L subunit of the RdRP complex. The proposed model for RNA synthesis in RSV is that the RdRP complex induces a hinge movement of the N-terminal region with respect to the C-terminal region of the N protein that allows the polymerase to thread through the template RNA without the need to disassemble the nucleocapsid (Tawar et al. 2009). The hinge movement would enable 11 bases available for readout at a time (Tawar et al. 2009), consistent with the accumulation of abortive transcripts 9-11 nucleotides in length in P protein phosphorylation mutants that impair transcript elongation (Dupuy et al. 1999).

The M2-2 protein regulates the shift from positive to negative sense RNA synthesis. While the mechanism has not been fully elucidated, M2-2 was shown to directly bind to the L protein and to inhibit positive sense RNA synthesis (reviewed in Noton and Fearns 2015).

For review, please refer to Collins et al. 2013.

Angiogenin (ANG) cleaves within or near the anticodon of specific tRNAs including but not limited to: tRNA Arg ACG (Fu et al. 2009), tRNA Arg CCG (Fu et al. 2009), tRNA Glu CTC (Fu et al. 2009), tRNA Gly CCC (Fu et al. 2009), tRNA Gly GCC (Fu et al. 2009), tRNA Met CAT (Fu et al. 2009, Su et al. 2019), tRNA Pro AGG (Yamasaki et al. 2009), tRNA Pro TGG (Yamasaki et al. 2009), tRNA Val AAC (Fu et al. 2009), tRNA Ala AGC (Su et al. 2019), tRNA Ala CGC (Su et al. 2019), tRNA Ala TGC (Su et al. 2019), tRNA Asp GTC (Su et al. 2019), tRNA Glu TTC (Su et al. 2019), tRNA His GTG (Su et al. 2019), tRNA Leu CAG (Su et al. 2019), tRNA Leu TAG (Su et al. 2019), tRNA Lys TTT (Su et al. 2019), tRNA Ser GCT (Su et al. 2019), tRNA Ser CGA (Su et al. 2019), tRNA Val CAC (Su et al. 2019), tRNA Val TAC (Su et al. 2019) (also Lee and Vallee 1989, Saxena et al. 1992, Emara et al. 2010, Ivanov et al. 2011). The products are a 5' fragment of about 30-35 nt and a 3' fragment of about 40 nt known as tRNA halves or stress-induced tRNA fragments (tiRNAs) (Emara et al. 2010). As a result of ANG cleavage, the 5’ tRNA halves contain 5' monophosphates (Emara et al. 2010) and 3' cyclic monophosphates (Shigematsu et al. 2018), while the 3’ tRNA halves contain 5' hydroxyl groups (Shigematsu et al., 2018). ANG cleaves tRNA in response to biological conditions such as exposure to sex hormones and stresses such as starvation, oxidative stress, and virus infection (Fu et al. 2009, Emara et al. 2010, Ivanov et al. 2011, Wang et al. 2013, Honda et al. 2015, Selitsky et al. 2015), but several tRNA halves are still produced after stress in ANG knockout cells (Su et al. 2020). The 5' tiRNAs inhibit translation by displacing eIF4F from the m(7)G caps of mRNAs (Emara et al. 2010, Ivanov et al. 2011). The 3’ tiRNAs protect cells against stress-induced apoptosis by interacting with cytochrome C (inferred from mouse homologs in Saikia et al, 2014). The products of ANG have modifications present on mature tRNAs (Drino et al. 2020); therefore, the cleavage is believed to occur in the cytosol (Yamasaki et al. 2009, reviewed in Lyons et al. 2018) perhaps as ANG is translocated from receptors on the plasma membrane through the cytosol to the nucleus.

PTEN (phosphatase and tensin homolog deleted in chromosome 10) is a tumor suppressor gene that is deleted or mutated in a variety of human cancers. TP53 (p53) stimulates PTEN transcription (Stambolic et al. 2000, Singh et al. 2002). PTEN, acting as a negative regulator of PI3K/AKT signaling, affects cell survival, cell cycling, proliferation and migration. PTEN regulates TP53 stability by inhibiting AKT-mediated activation of TP53 ubiquitin ligase MDM2, and thus enhances TP53 transcriptional activity and its own transcriptional activation by TP53. Beside their cross-regulation, PTEN and TP53 can interact and cooperate to regulate survival or apoptotic phenomena (Stambolic et al. 2000, Singh et al. 2002, Nakanishi et al. 2014).
In response to UV induced DNA damage, PTEN transcription is stimulated by binding of the transcription factor EGR1 to the promoter region of PTEN (Virolle et al. 2001).
PTEN transcription is also stimulated by binding of the activated nuclear receptor PPARG (PPARgamma) to peroxisome proliferator response elements (PPREs) in the promoter of the PTEN gene (Patel et al. 2001), binding of the ATF2 transcription factor, activated by stress kinases of the p38 MAPK family, to ATF response elements in the PTEN gene promoter (Shen et al. 2006) and by the transcription factor MAF1 (Li et al. 2016).
NR2E1 (TLX) associated with transcription repressors binds the evolutionarily conserved TLX consensus site in the PTEN promoter. NR2E1 inhibits PTEN transcription by associating with various transcriptional repressors, probably in a cell type or tissue specific manner. PTEN transcription is inhibited when NR2E1 forms a complex with ATN1 (atrophin-1) (Zhang et al. 2006, Yokoyama et al. 2008), KDM1A (LSD1) histone methyltransferase containing CoREST complex (Yokoyama et al. 2008), or histone deacetylases HDAC3, HDAC5 or HDAC7 (Sun et al. 2007).
Binding of the transcriptional repressor SNAI1 (Snail1) to the PTEN promoter represses PTEN transcription. SNAI1-mediated repression of PTEN transcription may require phosphorylation of SNAI1 on serine residue S246. Binding of SNAI1 to the PTEN promoter increases in response to ionizing radiation and is implicated in SNAI1-mediated resistance to gamma-radiation induced apoptosis (Escriva et al. 2008). Binding of another Slug/Snail family member SNAI2 (SLUG) to the PTEN gene promoter also represses PTEN transcription (Uygur et al. 2015).
Binding of JUN to the AP-1 element in the PTEN gene promoter (Hettinger et al. 2007) inhibits PTEN transcription. JUN partner FOS is not needed for JUN-mediated downregulation of PTEN (Vasudevan et al. 2007).
Binding of the transcription factor SALL4 to the PTEN gene promoter (Yang et al. 2008) and SALL4-medaited recruitment of the transcriptional repressor complex NuRD (Lu et al. 2009, Gao et al. 2013), containing histone deacetylases HDAC1 and HDAC2, inhibits the PTEN gene transcription. SALL4-mediated recruitment of DNA methyltransferases (DNMTs) is also implicated in transcriptional repression of PTEN (Yang et al. 2012).
Binding of the transcription factor MECOM (EVI1) to the PTEN gene promoter and MECOM-mediated recruitment of polycomb repressor complexes containing BMI1 (supposedly PRC1.4), and EZH2 (PRC2) leads to repression of PTEN transcription (Song et al. 2009, Yoshimi et al. 2011).

TP53 is the most frequently mutated tumor suppressor gene, with mutations present in more than 50% of human tumors and germline mutation in TP53 being underlying cause of the cancer-predisposing Li-Fraumeni syndrome (reviewed in Monti et al. 2020). The TP53 gene maps to chromosomal band 17p13 and encodes a transcription factor that contains four functional domains. A transactivation domain (TAD) involves amino acid residues 1-61 and is involved in interaction with components of the transcription machinery. A DNA binding domain (DBD) involves amino acid residues 94-290 and interacts with specific DNA target sequences called p53 response elements. A C-terminal domain (CTD) involves residues 357-393 and regulates DNA binding (reviewed in Monti et al. 2020). A tetramerization domain (TD) involves amino acids 325-355 and is needed for the formation of TP53 homotetramers. TP53 is considered the “guardian of the genome” (Lane 1992) as it is activated by DNA damage to initiate, depending on the amount of damage, cell cycle arrest, senescence or apoptosis (reviewed in Reinhardt and Schumacher 2012). In addition, TP53 regulates the expression of DNA repair genes, and is involved in the regulation of metabolism and autophagy (reviewed in Monti et al. 2020).
Most cancer-derived TP53 mutations are missense mutations that affect the central DNA binding domain of TP53 (amino acid residues 94-312). Eight hotspot amino acid substitutions in this region (R175H, G245S, R248Q, R248W, R249S, R273H, R273S and R282W) are found in close to 30% of TP53-mutated cancers. Based on their functional impact, TP53 mutations can be classified as 1) loss-of-function (LOF), 2) partial LOF (which may involve temperature sensitivity); 3) wild type-like (WT-L) or super-transactivating (ST) mutants; 4) mutants with altered specificity (AS), which are active or partially active on some but inactive on other TP53 target genes; 5) dominant-negative (DN) mutants, able to tetramerize with and inhibit the activity of the wild type TP53 protein. Some of the TP53 mutants, especially in the category of ST and AS mutants, are gain-of-function (GOF) mutants, able to interact with novel target genes and/or novel components of the transcriptional machinery (reviewed in Monti et al. 2020, and Gencel-Augusto and Lozano 2020).
Due to the complex function of WT-L, ST, AS and DN mutants of TP53, we have so far focused on annotating LOF mutants of TP53 which are unable to oligomerize due to mutations in the TD. Although accounting for a small percent of TP53 mutants, TD mutant are therefore considered to be completely defective in transcriptional activity, with no possibility of AS, DN and GOF effects (Chène and Bechter 1999, reviewed in Chène 2001, and Kamada et al. 2016). However, when overexpressed, some missense TD mutants of TP53 can form homotetramers and heterotetramers with the wild type TP53 which are partially functional and some extent of AS, DN and GOF effects may not be excluded for those mutants (Atz et al. 2000, reviewed in Chène 2001). In addition, the synthetic mutant p153(1-320) which consists of the first 320 amino acids and lacks the TD and CTD, while unable to tetramerize, can form stacked oligomers at the recombinant target gene promoter and induce transcription at a low level. Stacked oligomers are formed through interactions that involve amino acid residues outside the TD, which are facilitated by the presence of a target DNA sequence (Stenger et al. 1994).