Delineating Crosstalk Mechanisms of the Ubiquitin Proteasome System That Regulate Apoptosis

Abstract

Regulatory functions of the ubiquitin-proteasome system (UPS) are exercised mainly by the ubiquitin ligases and deubiquitinating enzymes. Degradation of apoptotic proteins by UPS is central to the maintenance of cell health, and deregulation of this process is associated with several diseases including tumors, neurodegenerative disorders, diabetes, and inflammation. Therefore, it is the view that interrogating protein turnover in cells can offer a strategy for delineating disease-causing mechanistic perturbations and facilitate identification of drug targets. In this review, we are summarizing an overview to elucidate the updated knowledge on the molecular interplay between the apoptosis and UPS pathways. We have condensed around 100 enzymes of UPS machinery from the literature that ubiquitinates or deubiquitinates the apoptotic proteins and regulates the cell fate. We have also provided a detailed insight into how the UPS proteins are able to fine-tune the intrinsic, extrinsic, and p53-mediated apoptotic pathways to regulate cell survival or cell death. This review provides a comprehensive overview of the potential of UPS players as a drug target for cancer and other human disorders.

Keywords: DUBs; E3 ligases; apoptosis; ubiquitin proteasome system; ubiquitination.

Figure 1

The ubiquitin pathway. Ubiquitination is the covalent addition of a 76-amino acid ubiquitin tag to the N-terminal lysine of the substrate protein. This process requires the sequential actions of three enzymes, an activating enzyme (E1) that forms an ATP-dependent labile thioester linkage with the carboxyl-terminal group of ubiquitin through its cysteine thiol group, thereby activating the C-terminus of ubiquitin for nucleophilic attack; a conjugating enzyme (E2) that transiently carries the activated ubiquitin molecule as a thiol ester; and an ubiquitin ligase (E3) that finally transfers the activated ubiquitin from the E2 to the ε-amino group of acceptor lysine residue of the substrate.

Figure 2

Different types of Ubiquitination and specific function of Ubiquitin linkages. Ubiquitination process can be broken down into three groups, based on how the substrate protein gets tagged by ubiquitin molecule(s). Modification of the substrate with the formation of an isopeptide bond between the C-terminus of the activated ubiquitin and a single substrate lysine leads to the monoubiquitination, when multiple substrates' lysine residues modify with a single ubiquitin molecule each, that results into the multi-monoubiquitination and polyubiquitin chains using one of the seven available lysine residues (K6, K11, K27, K29, K33, K48, or K63) of one ubiquitin molecule attached via an isopeptide bond to the C-terminus of another ubiquitin molecule add further complexity to ubiquitin-encoded protein fate. K48-linked polyubiquitinated proteins are known to be highly unstable and call for degradation via 20S proteasome both for normally folded or misfolded proteins otherwise. K29- and K33-branched mixed chains have shown to regulate AMP-activated protein kinase-related kinases. Polyubiquitination in vivo through K29/K33-linked mixed chains blocks their kinase activation by interfering with phosphorylation of the activation-loop residues. K29-branched ubiquitin chains also shown to promote the proteasomal and lysosomal degradation of proteins, whereas K63-branched polyubiquitination majorly plays a key role in various non-degradative processes such as regulation of endocytosis, DNA repair, protein kinase activation, signal transduction, intracellular trafficking of membrane proteins, and stress responses. K63 mediated linkages also known to facilitate the autophagic degradation of substrate proteins and their associated cellular materials, such as damaged mitochondria and invading pathogens. Monoubiquitination and multi-monoubiquitination have been implicated in non-proteasomal regulatory functions like proteins translocation to the nucleus, cytoskeleton and endocytic machinery, virus budding, DNA repair, or modulating enzymatic activity and protein-protein interactions. Majority of the ubiquitin attachment on the protein appears to be at lysine residue, although N-terminal methionine (M1) and cysteine modifications have also been reported. Formation of a peptide bond between the N-terminal methionine residue of one ubiquitin molecule and the C-terminal glycine of the next in the chain results into the linear ubiquitin chains. Linear ubiquitin chains i.e. M1-linked chains primarily play pivotal roles in inflammatory and immune responses. Linear ubiquitin chain is formed by LUBAC (linear ubiquitin chain assembly complex), a multisubunit member of RBR family of E3 ligases. The complex is made of three enzymes: HOIP, HOIL1, and SHARPIN.

Figure 3

Regulation of Intrinsic pathway by E3 ligases and DUBs. Multiple signals such as DNA damage, unfolded protein response, cytokine deprivation, free radical generating compounds, removal of nutrients, oxygen or growth factors, and endoplasm reticulum stress etc. contributes in the activation of intrinsic or the mitochondrial apoptotic pathway. Following stress, two proapoptotic Bcl-2 (B-cell lymphoma 2) proteins- Bax (Bcl-2 associated X protein) and Bak (Bcl-2 homologous antagonist killer) are activated upon structural changes. They move to the mitochondria, homodimerizes, and generate pores onto the mitochondrial surface, which enhances the membrane permeabilization and cytochrome c then starts to releases. In the presence of ATP, cytochrome c associates with Apaf-1, leading to self-oligomerization of Apaf-1 via CED-4 domains. This leads to the exposure of CARDs of Apaf-1. When these CARDs domain of Apaf-1 and caspase-9 binds, an apoptosome is formed. Dimerization of procaspase-9 forms an active caspase-9, which further activates the caspase-3 and 7 to commit cell death. Bcl-2 family proteins are categorized as antiapoptotic [Bcl-2, Bcl-xl (Bcl-extra-large), Bcl-w, Mcl-1 (Myeloid leukemia cell differentiation 1), Bcl-b and A1], proapoptotic (Bax, Bak, and Bok (Bcl-2 related ovarian killer)] and regulatory BH3-only proteins [Bad (Bcl-2 antagonist of cell death), Bik (Bcl-2-interacting killer), Bid (BH3-interacting domain death agonist), Bim (Bcl-2 interactor mediator of cell death), Bmf (Bcl-2-modifying factor), Noxa, and Puma (p53 upregulated modulator of apoptosis)], Antiapoptotic proteins suppress the apoptotic signals by limiting Bax and Bak thus blocking cytochrome-c release. In case of cellular stress, Bim and Noxa counteract the effect of antiapoptotic proteins. BH3-only proteins release Bax-Bak from inhibition thus promoting Momp (Mitochondrial outer membrane potential) and apoptosis. The IAPs (inhibitors of apoptosis) are the negative regulators of the intrinsic pathway. XIAP (X-linked Inhibitor of Apoptosis Protein) is one of the IAP to be seen upregulated in cancer and is an inhibitor of caspase. Activation of the intrinsic apoptotic pathway leads to the release of Smac/Diablo (Second mitochondrial activator of caspases or direct IAP binding protein with low pI), Omi/HtrA2 (Omi stress-regulated endoprotease or High-temperature requirement A2) and Arts (Apoptosis-related protein in the TGF-beta signaling pathway) into the cytosol. Smac/Diablo and Omi/HtrA2, when released into cytosol upon stimuli, use their IBM (IAP-binding motif) domain to bind to the BIR2 and BIR3 domains of the IAPs. They prevent the XIAPs ability to inhibit caspases and thus mediate apoptosis. Arts interact with residues of the BIR3 domain of XIAP distinct from Smac/Diablo and Omi/HtrA2 suggesting it to antagonize XIAP in a different way. Several E3 ligase and DUBs involved at different stages of the pathway are shown as red ovals.

Figure 4

Illustration of E3 ligases and DUBs that regulate extrinsic pathway of Apoptosis. Signaling molecule such as Fas ligand, TNF (Tumor necrosis factor) and TRAIL (TNF related apoptosis-inducing ligand) bind to the transmembrane death receptors like FasR, TNFR, death receptor 4/5, respectively to induce the extrinsic apoptosis pathways. TNF extrinsic apoptosis pathway is initiated by binding of TNF ligand to it two cognate surface receptors, TNF-R1 and TNF-R2. Ligand binding to TNF-R1 results in the dissociation of the SODD (silencer of death domains) from the cytoplasmic tail of TNF-R1 and this facilitates recruitment of adaptor protein TRADD (TNFR1-associated death domain protein). This association further leads to the recruitment of FADD (Fas-associated death domain protein), TRAF2 (TNFR-associated factor 2), TRAF5, RIP (receptor-interacting protein), and c-IAP1/2 (cyto inhibitor of apoptosis proteins) and forms a DISC (death-inducing signaling complex). DISC mediates the enrolment of additional proteins such as the initiator caspase, procaspase-8, which, then proteolytically cleaved, releases an active form of caspase-8, which further activates the effector caspase-3 and 7 and leads to the apoptosis. Activation of caspase-8 is regulated by c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein), a catalytically inactive homolog of caspase 8 that interacts with procaspase-8. Several IAPs (inhibitors of apoptosis) also regulate apoptosis by interacting with TRAF2. Similarly, when Fas protein binds to FasL, it promotes the recruitment of FADD adaptor protein that has DD (Death domain) and DED (Death effector domain) that associate with caspase-8 and 10 and forms a DISC (Hongmei, 2012). Later, apoptosis is preceded just same as in TNF signaling. Several ubiquitin ligases and DUBs that are involved in the pathway and critically regulating the apoptosis are shown as red ovals.

Figure 5

Illustration of E3 ligases and DUBs that regulate TNF induced NF-κB pathway. TNFR signaling also results in the activation of cell survival via activation of NF-kB pathway by recruiting TRADD, TRAF1, and TRAF2, which further enroll RIP1 and c-IAP1/2. TRAF2 is very pivotal in activating the IKK complex (IKKα and IKKβ) and recruits it to the TNF-R1 complex upon phosphorylation of these proteins by TAK2/3 proteins of TAK1 kinase complex. IKKα and IKKβ kinases, phosphorylate Ser32 and Ser36 in IκB isoforms (IκBα, IκBβ) that leads to their ubiquitin-dependent degradation and entry of NF-κB transcription factors p50, p65 (RelA) heterodimer into the nucleus upon activation leading to transcription of genes such as c-FLIP, c-IAP, Bcl-xL, Bcl-2 etc. Red oval indicates the E3 ligases and DUBs involved at various stages.

Figure 6

Pictorial representation of p53 pathway regulated by E3 ligases and DUBs. p53 is a transcription factor involved in apoptosis in response to stress signals. p53-p300 interaction is inhibited by MDM2, thus reducing p53 activity. p53-mediated apoptosis signaling is regulated by many E3 ligases and DUBs shown as red ovals.