Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System

Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System D Ho¨ller and I Dikic, University Hospital, Frankfurt, Germany Published by Elsevier Inc.

Glossary Autophagy Cellular process governing the removal, degradation, and recycling of cytosolic material that involves the ‘eating’ of damaged proteins and organelles by specialized membrane structures. Coupled monoubiquitination Mechanism of monoubiquitin conjugation to Ub receptors that requires a functional UBD. Immunoproteasome Specialized form of the proteasome dedicated to the generation of peptides for presentation by MHC class I. Mitophagy Removal of damaged mitochondria by autophagy.

Abbreviations AMSH CUE DSB DUB EGFR GAT

GLUE

Associated molecule with the SH3 domain of STAM Coupling of ubiquitin conjugation to endoplasmic reticulum degradation Double-strand break Deubiquitinating enzyme Epidermal growth factor receptor GGA (Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding protein) and Tom (target of Myb) GRAM-like ubiquitin binding in EAP45

Ubiquitination: An Inducible and Reversible Posttranslational Modification

Proteasome Cellular machinery for regulated Ub-dependent destruction of soluble proteins composed of lid (regulatory particle, 19S) and base (core particle, 20S). Signalosome Multiprotein complex of dynamic composition that transduces intracellular signals. Ub receptor Protein harboring one or more Ub-binding domains facilitating the selective recognition of proteins modified with monoUb or polyUb of different linkage types. Ubiquitination Enzymatic cascade governing the covalent attachment of ubiquitin to the e-amino group of a lysine present in a substrate protein.

HECT NZF PTEN RING UBA UBAN UBD UBM UBP UIM USP VHS

Homologous to E6-AP carboxyl terminus Npl4 Zn-F (zinc finger) Phosphatase and tensin homologue Really interesting new gene Ubiquitin-associated domain Ubiquitin binding in ABIN and NEMO Ubiquitin-binding domain Ubiquitin-binding motif Ubiquitin-specific processing protease Ubiquitin-interacting motif Ubiquitin-specific protease Vps (vacuolar sorting protein) 27/Hrs/STAM

Ubiquitin (Ub) is a highly conserved globular protein of 76 amino acids. Its flexible C-terminal tail carries a characteristic RGG motif that can be covalently linked to lysine residues of substrate proteins, thereby changing their biochemical and biophysical properties. The conjugation process is both inducible and reversible and requires the coordinated action of three enzymes: the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2), and the Ub-ligating enzyme (E3) (Figure 1). In the first step, E1 activates the C-terminal Gly (G) of Ub by forming an E1
Ub thioester in an ATP-dependent manner. The activated Ub is then transferred from E1 to the active cysteine of E2 forming an E2
Ub thioester. In the last step, E2
Ub cooperates with the E3 Ub ligase to covalently couple Ub to the substrate protein by forming an isopeptide bond between the e-amino group of a lysine and the C-terminal G of Ub. To a lesser extent, also serine, threonine, cysteine, and the N-terminal amino group of the substrate protein serve as acceptor sites for Ub. The E3 ligase is the only component of

the conjugation machinery that directly binds to the substrate and is therefore responsible for substrate specificity. The high number of E3 ligases (compared to E2 and E1 enzymes) found in the human proteome reflects this crucial role: around 600 E3 ligases cooperate with approximately 25 E2 and 2 E1 enzymes. E3 ligases can be divided in three major groups, RING-type, HECT-type, and U-box-type. The most prevalent E3 ligases contain a zinc-finger domain called RING (really interesting new gene) that has an essential role in Ub transfer but does not possess catalytic activity itself. The RING-type E3 ligases rather function to bridge E2
Ub and the substrate. Instead, the HECT (homologous to E6-AP carboxyl terminus)-type ligases themselves form a thioester with Ub transferred from the E2 enzyme and catalyze the transfer to the substrate without further help of E2. The U-box E3 ligases are characterized by a modified RING motif and act in a similar way as RING E3s. The ubiquitination of a protein is induced by extra- or intracellular signals (resulting, e.g., from environmental changes, stress, or cell damage) and regulated in several ways. The most common mechanism involves the phosphorylation of the substrate, thereby creating a recognition signal for

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Ub + ATP E1

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Ub recycling 26S proteasome Substrate (a)

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‘Writers’ >600 E3 ligases Ub Protein

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‘Erasers’ >100 DUBs

Figure 1 Ubiquitin conjugation and deconjugation: writer–reader–eraser. (a) Ub conjugation occurs through the sequential action of Ub-activating (E1), Ub-conjugating (E2), and Ub-ligating enzymes (E3) in dependence on ATP. The initial steps involve formation of thioester bond between active site cysteins of E1 and E2 and carboxyl terminal group of Ub. There are two types of E3 ligase enzymes. The HECT (homologous to E6AP C-terminus) E3s facilitate Ub transfer onto substrate via forming of thioester intermediate with Ub, while RING (really interesting new gene) E3s recruit E2 in proximity of the substrate and facilitate the transfer of Ub from E2 to the target proteins. (b) Ub networks are based on E1–E2–E3 enzymes (writers) that modify the substrate protein with a specific Ub code and DUB enzymes that remove Ub chains from the substrate and recycle Ub molecules (erasers), whereas Ub modifications are decoded by UBD-containing proteins (readers).

binding of an E3 ligase. This happens, for example, during downregulation of ligand-activated epidermal growth factor receptor (EGFR). Transphosphorylation at position Tyr1045 allows docking of RING E3 ligase c-Cbl that mediates multiubiquitination of the receptor and thus ensures its lysosomal sorting. On the other hand, the activity of the E3 ligase itself can be regulated by phosphorylation, for example, it can

release E3 ligase’s autoinhibitory conformation and allow E2 binding. In order to ubiquitinate the EGFR, c-Cbl needs to be activated by phosphorylation. This modification releases the autoinhibited conformation and allows E2 binding. Another example is the activation of Itch through JNK Ser/Thr phosphorylation. On the other hand, phosphorylation can inhibit certain E3 ligases or destroy binding sites on substrates.

Cell Adaptation, Injury and Death | Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System

Importantly, ubiquitination is also a reversible process. Around 100 deubiquitinating enzymes (DUBs) have been identified in the human genome that cleave Ub from the modified protein and recycle it to the cellular pool. Moreover, Ub is made as a precursor protein consisting of multiple Ub proteins or Ub fused to the amino terminus of ribosomal proteins. DUBs play an essential role in cleaving these precursor proteins to generate free Ub. Five different subfamilies of DUBs are known: ubiquitinspecific proteases (USP) (forming the largest family), Ub carboxy-terminal hydrolases, Machado–Joseph disease protein domain proteases, ovarian tumor (otubain) proteases, and JAB1/MPN/Mov34 metalloenzyme motif proteases. Many DUBs harbor Ub-binding domains (UBDs), such as the zinc finger USP (Zn-USP) domain, the Ub-interacting motif (UIM), and the Ub-associated (UBA) domain. These motifs facilitate the recognition and the recruitment of ubiquitinated substrates to ensure specificity. Quite frequently, E3 and DUB activities are present in the same protein complex or even in the same protein (e.g., A20), allowing the editing of Ub modifications. Among all known posttranslational modifications, ubiquitination is the most versatile one because Ub contains itself seven lysines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) that allow the formation of homotypic and a great variety of heterotypic chains. It has been shown that different types of linkages trigger specific cellular answers: the attachment of a single Ub molecule (monoubiquitination) serves as sorting signal during endocytic trafficking and lysosomal degradation of cell-surface proteins. Lys48-linked chains label proteins for proteasomal degradation, Lys63-linked chains are implicated in the assembly of signaling complexes, and Lys11 chains promote proteasomal degradation of targets during cell cycle progression and in endoplasmic reticulum-associated degradation (ERAD). The cellular functions of many other types of ubiquitination have not yet been studied in much detail, yet mass spectrometry of cellular Ub conjugates has revealed that each possible linkage type exists in the cell (Figure 2).

Reading the Ub Code: Ub Receptors In order to translate the various Ub tags encoded by specific linkages into the appropriate cellular response, the cell has evolved UBDs that are able to distinguish between different Ub modifications. The UBDs are present in a wide variety of proteins that are implicated in diverse cellular signaling pathways. To date, more than 20 families of UBDs have been identified, with more to come: UBDs containing single or multiple alpha-helices (UBA, UIM, DUIM, MIU, CUE, GAT, VHS, and UBAN), zinc fingers (NZF, ZnF-UBP, ZnF-A20, and UBZ), pleckstrin-homology fold (GLUE and PRU), UBC-like (UEV and UBC), or other structures (SH3, UBM, PFU, and Jab1/MPN). Although the UBDs are structurally diverse and exhibit linkage specificity in vivo, most of them share a common mode of binding to Ub engaging a hydrophobic patch centered on Ile44 of Ub. Importantly, different types of chains adopt specific conformations in solution. K48-linked di- and tetra-Ub chains have a closed conformation, with the hydrophobic patch surfaces around Ile44 buried at the interdomain interface (interactions with UBD-containing proteins

Ub Lys

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Monoubiquitination: endocytosis, protein transport, DNA repair, histone regulation

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Figure 2 Ub linkage types and proteolytic/nonproteolytic functions. Different types of Ub conjugates can regulate various signaling events, such as receptor endocytosis, cell cycle progression, differentiation, DNA repair, apoptosis, immune response, or protein degradation by Ub–proteasomal system (UPS). The keys to these diverse functions are Ub receptors (UBD-containing proteins) that are able to discriminate the various types of Ub tags and link them to the appropriate cellular pathway.

still occur since this conformation is dynamic, oscillating between the open and packed structure). K63-linked chains exhibit an extended conformation with fully exposed hydrophobic patches. UBDs discriminate different chain types by sensing the spatial distribution and positioning of individual Ub units of a chain. This is possible because Ub receptors often dimerize or oligomerize or contain multiple UBDs that cooperate in chain-specific binding. Only a small subset of UBDs interacts directly with the linker region and does not rely on linkage-specific avidity. Notably, the microenvironment in which the interaction happens plays a crucial role for linkage-specific binding of UBDs. While in one protein complex a UBD shows a preference for K63-chains, the same UBD binds preferentially to K48 chains in another complex. Moreover, interaction affinity and specificity can be sometimes modulated by other posttranslational modifications such as phosphorylation.

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A characteristic feature of Ub/UBD interactions is that they are very weak (Kd in the micromolar range) and the question arose how low-affinity Ub/UBD interactions can support a specific and efficient signaling network. Kds are mostly determined in vitro using the isolated UBD rather than the integral Ub-receptor functioning in cells. In vivo avidity due to oligomerization of Ub receptors and multior polyubiquitinated substrates, the presence of multiple UBDs in the Ub receptor (different UBDs in the same protein or multiplication of the same UBD within protein) plays a central role. Moreover, proteins harboring a UBD often utilize a secondary binding site present in the ubiquitinated protein or both proteins are part of the same large protein complex (signalosome). In the case of ubiquitinated membrane proteins, often, membrane lipids serve as secondary binding site. Such a bipartite interaction not only provides increased affinity but also ensures specificity. On the other hand, it has been proposed that the dynamic Ub-based networks indeed benefit from low-affinity interactions because this facilitates an efficient and rapid reconstruction of protein complexes during adaptation to extra- and intracellular signals or during the passage of the endosomal cargo along multiple sorting complexes.

Removal of Proteins by the Proteasome The most abundant and best-studied type of ubiquitination is K48-linked polyubiquitination (of 4 or more Ub molecules) that directs the regulated destruction of the substrate by the proteasome. The Ub–proteasome system (UPS) is the major cellular degradation system for soluble proteins and regulates many fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction. The 26S proteasome, a massive 1.5 MDa proteolytic machine, is the final executer of protein destruction (Figure 3). It is composed of the barrel-shaped 20S core particle (CP;
700 kDa), where degradation occurs, and the 19S regulatory particle (RP;
900 kDa), which caps the CP on one or both sides and provides an access portal for substrates to the catalytic core. The 19S RP contains subunits (Rpn10 and Rpn13) that are equipped with UBDs and function to directly recruit polyubiquitinated substrates to the proteasome. Another subunit possesses deubiquitinating activity (Rpn11) and removes Ub from the substrates, while the six AAA þ ATPase subunits (Rt1–6) function to unfold the protein substrates and deliver them into the proteolytic chamber formed by the 20S CP. In addition to these subunits, a number of proteins reversibly associate with the RP,

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Figure 3 Structure proteasome. The 26S proteasome contains a central, barrel-like core particle (the 20S proteasome, CP) composed of four stacked seven-membered rings. A six-membered ring of AAA ATPase proteins binds to one or both outer a-rings and, together with two non-ATPase subunits, forms the base, while nine other subunits comprise the adjoining lid. In turn, the base and lid comprise the 19S regulatory particle (19S, RP), which functions in the recognition of ubiquitylated substrates and their deubiquitylation, unfolding, and transfer into the central chamber of the 20S proteasome. Within the 20S proteasome, subunits b1, b2, and b5 of both adjacent b-rings expose their proteolytically active sites, exhibiting postglutamyl peptide-hydrolyzing (PGPH), trypsin-like, and chymotrypsin-like cleavage specificity, respectively. Under conditions of acute immune or stress response, these three b subunits can be substituted during de novo proteasome biosynthesis for the interferon-g-inducible subunits b1i, b2i, and b5i. This results in the replacement of standard 20S proteasomes with immunoproteasomes, which have different cleavage specificities.

Cell Adaptation, Injury and Death | Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System

including E3 ligases, DUBs (such as Usp14), and shuttling factors (e.g., Rad23) that deliver polyubiquitinated substrates to the proteasome. The 20S CP contains three different proteolytically active sites utilizing an N-terminal threonine residue as the active nucleophile: postglutamyl peptide-hydrolyzing and trypsin- and chymotrypsin-like cleavage specificity. The relative importance of each active site for the degradative process depends on the individual substrate. The composition of the proteasome is modular and dynamic and can be altered in response to physiological demands, for example, in response to environmental insults, such as oxidative stress or immune signaling that implicates the activation of the immunoproteasome, a specific proteasome variant that exhibits different proteolytic properties than the constitutive proteasome and essentially contributes to the acquisition of adaptive immunity. Hematopoietic cells or cells exposed to inflammatory cytokines or pathogens express three immunosubunits (b1i, b2i, and b5i), which form the catalytic centers of immunoproteasomes. They exhibit enhanced ability to generate antigenic peptides appropriate for MHC class I presentation on the cell surface. This process is strictly required to efficiently prime CD8(þ) T cells and to initiate an adaptive immune response. Whereas an age-related increase in the concentration of immunoproteasome subunits could reflect a state of constant inflammation or cell stress, downregulation of immunoproteasomes has been found to serve as an immune surveillance escape mechanism in several types of tumors. Another successful oncogenic mechanism relying on insufficient antigen processing was observed in human cervical carcinoma and melanoma cells that express a nonfunctional variant of the immunoproteasome subunit b5i. Despite the initial reasonable concern that inhibition of the proteasome could be lethal for all cell types (due to accumulation of damaged proteins), the targeting of the proteasome using bortezomib (Velcade, PS-341 or originally synthesized as MG-341) in anticancer therapies has emerged as a successful strategy. Bortezomib was approved as the first proteasome inhibitor for the treatment of multiple myeloma. Since then, it has been also successfully used for treatment of various malignancies. Generally, UPS inhibitors induce apoptosis more efficiently in strongly proliferating cells. One of the major mechanisms involves the upregulation of NOXA, which is a proapoptotic protein. Another important mechanism of bortezomib is through suppression of the nuclear factor-kB (NF-kB) signaling pathway resulting in the downregulation of its antiapoptotic target genes.

Cell-Surface Proteins are Routed to the Lysosome for Degradation Whereas cytosolic soluble proteins are degraded by the proteasome, many cell-surface proteins such as the G-proteincoupled receptors or receptor tyrosine kinases (RTKs) follow a different route involving clathrin-mediated endocytosis and Ub-dependent endolysosomal sorting. Upon ligand binding, these receptors are activated and transmit their signals into the cell, leading to cell growth, differentiation, migration, etc. To avoid overstimulation of the cells, negative effector proteins

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are recruited concomitantly to mediate receptor downregulation. Among these negative regulators are E3 ligases (such as members of the Cbl family) that attach multiple single Ub molecules (multiubiquitination) and K63-linked di-Ub to the intracellular part of the receptor. These modifications are recognized by multiple UIMs present in a series of endocytic adaptor proteins, such as Eps15, epsin, and the ESCRT complex, that sort the activated receptor toward the lysosome for proteolytic destruction. Notably, during endosomal trafficking, the receptor continues to emit compartment-specific signals until the receptor is permanently inactivated in the lysosome. Receptors that are deubiquitinated by endosomal DUBs (such as AMSH or UBPY) escape lysosomal targeting and are recycled back to the plasma membrane where they can be activated once again by extracellular signals. Impaired Ub-mediated downregulation of cell-surface receptors leads to uncontrolled cellular growth, migration, or differentiation, depending on the activated pathway.

Nonproteolytic Functions of Ubiquitin The regulated proteasomal degradation of cytosolic proteins was the first identified role of ubiquitination and has been studied extensively. However, in recent years, more and more nonproteolytic functions of Ub have been described that play a crucial role in many physiological and pathophysiological processes.

Ub Links Proteins to Specific Cellular Compartments The cell is a highly compartmentalized factory. Each compartment is endowed with a specific function that requires the presence of a certain set of proteins. Sometimes, for example, in response to stimuli, proteins need to be removed from a compartment or delivered to it. Mono- and K63-linked diubiquitination of proteins has been shown to direct the targeting of proteins to specific cellular compartments. The importance of Ub-dependent protein localization has been recently shown for Ras proteins (NRas, HRas, and K-Ras) that play a central role in transducing signals that control cell proliferation, differentiation, motility, and survival. Ras proteins can dynamically localize to different cellular membranes and their localization determines the signaling outcome. Rabex-5 is an E3 ligase that mono- and diubiquitinates Ras proteins and thereby promotes their endosomal association and endosomal-specific signaling. Yet, the Ub receptor that traps ubiquitinated Ras proteins at endosomal membranes needs to be identified. Just as Ub-dependent localization is important for the physiological function of many proteins, deregulated ubiquitination can have detrimental effects. PTEN (phosphatase and tensin homologue) is one of the genes most frequently inactivated in human cancers. Monoubiquitination regulates its nuclear translocation and the ubiquitin-acceptor lysines (Lys289 and Lys13) are mutated in some types of tumors. Failure to attach monoUb leads to exclusion of PTEN from the nucleus and has been associated with a cancer-susceptibility syndrome (Cowden syndrome) and advanced stages of tumorigenesis.

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Cell Adaptation, Injury and Death | Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System

Ub-Dependent Assembly of Signaling Complexes (Signalosomes) By the attachment of Ub, new interaction surfaces are created that allow the remodeling of protein complexes endowing them with specific functions. In particular, during DNA repair and NF-kB signaling, the dynamic assembly and disassembly of multiprotein complexes play a crucial role. The NF-kB pathway is a ubiquitous stress response that relies on the Ub-dependent assembly of signalosomes to activate the NF-kB family of transcription factors (see details later). In a similar way, protein machineries that coordinate the repair of damaged DNA require different types of Ub modifications to assemble and function efficiently. Proteins involved in DNA damage repair pathways contain one or more UBDs that facilitate their rapid recruitment to sites of DNA lesions. DNA double-strand breaks (DSBs), produced during normal cellular metabolism and DNA replication, through ionizing radiation or chemical mutagens, are among the most toxic DNA lesions. The cell responds to DSBs by the rapid assembly of repair factors at the break site. This process, which is initiated by ATM-mediated phosphorylation of histone variant H2AX, involves several waves of nonproteolytic ubiquitination and SUMOylation (SUMO is a Ub-like molecule that acts as a protein modifier as well). The E3 ligase BRCA1 is an important component of the repair machinery and is frequently mutated in familial breast cancer and ovarian cancer. The same multimeric complex also contains Rap80 and recruitment of the whole complex to DSBs is critically dependent on two UIMs present in Rap80. These UIMs sense lys63-linked Ub conjugates attached to histones H2A and H2AX around damage sites by the E3 ligases RNF8 and RNF168. Insufficient ubiquitination is associated with cancer predisposition and immunodeficiency.

Ub Regulates the Activity of Enzymes and Ub Receptors Monoubiquitination can directly regulate the activity of enzymes. For example, the oncogenic GTPase K-Ras is activated by monoubiquitination. The site-specific attachment of Ub significantly impaired the response of K-Ras to GAPs (i.e., the deactivation pathway) and thus increased the amount of GTPbound (activated) RAS and its binding to downstream effectors. Vice versa, mutation of lysine residue that is modified by Ub (Lys147) decreased K-Ras tumorigenicity in vivo. The activity of Ub receptors is regulated by monoubiquitination as well. Their ability to noncovalently interact with Ub is a prerequisite for the covalent attachment of the Ub molecule, which has coined the term ‘coupled monoubiquitination.’ Different mechanisms of coupled monoubiquitination have been described: E3-dependent and E3-independent. The E3dependent coupled monoubiquitination is carried out by HECT-type E3 ligases that undergo autoubiquitination (such as Nedd4). The covalently attached Ub facilitates the interaction of E3 with the UBD of the Ub receptor and the activated Ub is transferred from the catalytic cysteine on a lysine of the Ub receptor. In the E3-independent process, the Ub-loaded E2-conjugating enzyme is directly recruited to the UBD of the Ub receptor and mediates the transfer of the Ub. The consequence of coupled monoubiquitination is in both cases the intramolecular binding of the UBD to the attached Ub

molecule and thus the functional inactivation of the Ub receptor. In this way, coupled monoubiquitination serves to finetune Ub-mediated cellular responses by regulating the availability/activity of effector proteins that link the Ub signal to a certain cellular response pathway. In the following section, we are going to describe different examples of how ubiquitination specifically participates in cell adaptation mechanisms and cover diseases that result from aberrations in the UPS.

The UPS in Stress Adaptation Both Ub and proteasome activity are required for cells to cope with various types of stresses. A decline in proteasome activity, either age-related or induced by chemicals, sensitizes cells to stresses.

Oxidative Stress One of the common insults encountered by cells is oxidative stress, damage caused by reactive oxygen species (ROS). Excessive oxidants can damage basically all cellular components, such as proteins, DNA, and lipids, and cause dysfunction of cells. Oxidative damage has been linked with several diseases, including cancer, diabetes, and cardiovascular and neurodegenerative diseases, and also with aging. The defense mechanisms against oxidative damage include antioxidants and antioxidant enzymes that quench ROS, molecular chaperones that help to repair oxidized proteins, and proteolytic systems that eliminate irreparably damaged proteins. While there is no doubt that the proteasome is the major degradation system for oxidized proteins, it is a matter of debate to which degree ubiquitination is required for this process. Several findings imply that Ub is involved in restoring protein homeostasis during recovery from oxidative stress. For example, the accumulation of ubiquitinated species of oxidized proteins due to the use of proteasome inhibitors indicates that these Ub conjugates are destined for proteasomal degradation. Moreover, mild oxidative stress upregulates the Ub-conjugating machinery (E1, E2, and E3 enzymes) and promotes formation of Ub conjugates. However, a great number of oxidized proteins can directly bind to the proteasome and be degraded independently of ubiquitination. Enhanced proteasomal degradation of regulatory proteins, such as IkB, in response to mild oxidative stress also plays a role in coping with oxidative stress by activating the NF-kB pathway. Notably, the Ub conjugation machinery and the proteasome are themselves targets of oxidative stress. E1, E2s, HECT-type E3s, and DUBs have a cysteine residue in their active sites that reacts with oxidized glutathione (GSSG, produced upon exposure to oxidative stress), thus (reversibly) blocking their binding to Ub. Moreover, the proteasome is indirectly impaired by nondegradable cross-linked protein aggregates generated after extensive oxidative insults.

Adaptation to Low Oxygen Though oxidative stress can cause significant damage for an organism, oxygen is essential for eukaryotic life. The cellular

Cell Adaptation, Injury and Death | Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System

response to changes in oxygen tension during normal development or pathological processes, such as cardiovascular disease and cancer, is regulated by the transcription factor, hypoxia-inducible factor (HIF), that transactivates a variety of genes to trigger adaptive responses under hypoxia (compromised oxygen tension). Under normoxia (normal oxygen tension), HIF is hydroxylated and recognized by the von Hippel–Lindau tumor suppressor protein (pVHL). pVHL is the recognition component of the elongin BC/Cul2/pVHL Ub–ligase complex that mediates polyubiquitination and proteasomal degradation of hydroxylated HIF. Under hypoxia, HIF is not hydroxylated, escapes pVHL-mediated degradation, and triggers the cellular adaptive response to hypoxia, which includes transcriptional activation of SLC2A1 (glycolysis), VEGFA (angiogenesis), and EPO (erythropoiesis). Notably, while this pathway plays an important physiological role, it can be also exploited by tumors where the lack of oxygen and nutrients often represents the major growth-limiting factor. In fact, germline mutations of VHL cause the von Hippel–Lindau hereditary cancer syndrome, and somatic mutations of this gene have been linked to the development of sporadic hemangioblastomas and clear-cell renal carcinomas. These tumors are typically highly vascularized and profit from a rich supply of nutrients and oxygen.

NF-kB: Master Regulator of Stress Response and Inflammation The major pathway for triggering stress responses and immune cell activation upon pathogen invasion controls the transcription factor NF-kB, which regulates the transcription of numerous proinflammatory and cell-survival genes. On the other hand, excessive NF-kB signaling (e.g., due to chronic infections with pathogens or genetic aberrations in regulatory pathway components) has been linked to both inflammatory and malignant diseases, such as B-cell lymphoma, myeloma, and colon cancer, as well as neurodegenerative diseases. On the molecular level, the NF-kB pathway is tightly regulated by proteolytic and nonproteolytic ubiquitination. In resting cells, NF-kB proteins are kept inactive in the cytoplasm in association with inhibitory IkB proteins. Activation of the canonical pathway requires ubiquitination and subsequent proteasomal degradation of IkB. This liberates NF-kB that can enter the nucleus to exert its function as transcription factor controlling the expression of numerous genes coordinating defense mechanisms or survival pathways. However, in order to be ubiquitinated and degraded, IkB needs to be phosphorylated. This happens in response to a wide variety of extracellular stimuli including proinflammatory cytokines (such as TNF-a and IL-1b), and pathogen-associated molecular patterns (such as bacterial lipoprotein and lipopolysaccharide), that induce IkB phosphorylation through the IkB kinase (IKK) complex. IKK contains a regulatory subunit IKKg/NEMO (NF-kB essential modulator), which plays a central role in this process. NEMO is a substrate for linear and K63-linked polyubiquitination and also binds to polyUb chains composed of various linkages. Interfering with either ubiquitination of NEMO or NEMOs Ub-binding ability is sufficient to block the activation of the pathway, highlighting the crucial role of Ub modifications in NF-kB signaling. The linear Ub chains on

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NEMO are conjugated by a RING-type ligase known as LUBAC (linear Ub assembly complex), which is composed of HOIP (catalytic core of the enzyme) and HOIL1 and Sharpin (accessory proteins). Mutations in Sharpin or HOIL genes in mice or humans, respectively, lead to phenotypes characteristic of deficiencies in the NF-kB pathway. Also, the noncanonical pathway, which is activated by a subset of TNF superfamily ligands, such as lymphotoxin (LT)-b, B-cell-activating factor (BAFF), and the CD40 ligand, requires the UPS for activation. The phosphorylation of p100 by the noncanonical IKK promotes ubiquitination and processing of p100 to p52 by the UPS. Together with RelB, p52 regulates target genes involved in several biological processes, including B-cell survival and lymphoid organogenesis.

Failed Adaptation: Consequences of Defective UPS Being the major regulator of timely and selective protein turnover, the Ub system is implicated in virtually every physiological process and its deregulation is associated with the inability of cells to respond properly to internal and external changes and insults. In particular, alterations or mutations affecting not only E3 ligases but also DUBs are linked to a number of human diseases, including cancer, neurodegeneration, diabetes, immune deficiencies, or autoimmunity.

Cancer Tumors arise when cells fail to regulate cellular signaling pathways that control cell proliferation, differentiation, and apoptosis. This is generally caused by mutations in genes that express key proteins involved in these pathways and genomic instability is regarded as one hallmark of cancer. E3 ligases directly affect tumorigenesis as they regulate the stability of oncogenes or tumor suppressors by mediating their proteasomal or lysosomal degradation. Prominent examples are MDM2, which targets the tumor suppressor p53, and SCF and APC/C ligases, which control cell cycle progression and mitotic exit, respectively, by controlling the stability of cyclin/ CDK complexes and of the cell cycle inhibitor p27. Also, members of the Cbl family of E3 ligases are proto-oncogenes that play a well-established role in the downregulation of activated RTKs through mediating their lysosomal sorting and degradation. But also, nonproteolytic ubiquitination plays a role both in cancer development and the prevention of cancer. Several DNA repair pathways that ensure genomic integrity upon internal or environmental insults are dependent on monoubiquitination or atypical Ub chains (as described earlier).

Autoimmunity The failure of an organism to recognize its own constituent parts as self (¼self tolerance) allows an immune response against its own cells, which may cause autoimmune diseases such as diabetes mellitus type 1, systemic lupus erythematosus, rheumatoid arthritis, and allergies. Excessive NF-kB activation is one well-studied cause for the aberrant immune cell activation in these diseases. A20 is a Ub-editing enzyme that restricts

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Cell Adaptation, Injury and Death | Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System

signaling duration and intensity of several molecules involved in the NF-kB pathway and specific polymorphisms in the A20 genomic locus predispose humans to autoimmune disease. In B cells, A20 restricts autoantibody production and germinal center B-cell numbers. Site-specific proteasome inhibitors could be especially useful as immunosuppressive agents in autoimmune diseases by modulating antigen processing and MHC class I-restricted antigen presentation.

Diabetes Type 2 (Failed Adaptation to Elevated Glucose Levels) Diabetes type 2 is a metabolic disorder, the primary manifestation of which is an elevated blood glucose level; however, numerous other metabolic abnormalities such as obesity are concomitant. Elevation of blood glucose, for example after a meal, triggers the release of insulin from the b-cells in the pancreas. The circulating insulin has two major effects: to stimulate glucose uptake into cells and to suppress glucose production by the liver. Diabetes type 2 arises from insulin resistance due to impaired insulin signaling in target tissues. Defects in the Ub system can affect the cellular response to insulin at several levels: ligand-induced downregulation of the insulin receptor, decreased downstream signaling by insulin receptor substrate (IRS) proteins, and impaired biological response to insulin. The most relevant insulin resistance seems to result from the downregulation of IRS proteins, which is mediated by SOCS1 and SOCS3 that function as substrate recognition modules in cullin–RING E3 ligases. Notably, SOCS1 and SOCS3 are induced by inflammatory factors supporting the notion that inflammation participates in the pathogenesis of diabetes and obesity. Moreover, the muscle-specific E3-ligase MG53/TRIM72 has been shown to target both the insulin receptor and IRS1 for Ub-dependent degradation. MG53/TRIM72 is overexpressed in insulin resistance models, and its depletion blocked diet-induced systemic insulin resistance.

Neurodegenerative Diseases The by-products of mitochondrial energy-generating pathways are toxic free radicals that can cause oxidative stress if their amount exceeds the neuronal capacity to quench them. Dysfunctional mitochondria therefore need to be eliminated by a Ub-dependent process called mitophagy. The protein kinase PINK1 (PTEN-induced putative kinase 1) senses mitochondrial fidelity and recruits Parkin, a Ub E3 ligase, selectively to impaired mitochondria. Parkin labels the damaged organelle by attaching polyUb of different linkage types to several outer mitochondrial membrane proteins and thus primes it for autophagic elimination (mitophagy). The fundamental importance of this pathway is illustrated by the fact that genetic loss of either PINK1 or Parkin leads to early-onset Parkinson’s disease (PD). PD is characterized by a progressive loss of dopaminergic neurons from the substantia nigra, leading to muscle tremors, stiffness, and bradykinesia. Besides oxidative stress, the formation of protein aggregates is a hallmark of neurodegenerative diseases, including PD, Alzheimer’s disease, or Huntington’s disease. A characteristic feature of aggresomes is that they contain significant amounts of ubiquitinated proteins. In fact, aberrations in Ub-dependent degradation systems have been linked to aggresome formation and neurodegeneration. While the UPS is the main degradation pathway for soluble misfolded proteins, it is unable to eliminate aggregated proteins because they cannot be unfolded and inserted into the proteasomal core particle. The cell is cleared from aggresomes by a process called autophagy instead. Autophagy governs the process of self-digestion of bulky cytosolic material aiming at the removal, degradation, and recycling of damaged proteins and organelles. p62 and the related NBR1 are adaptor proteins equipped with UBA domains that sequester Ubmodified cargo and link it via the LC3-interacting region to autophagic membranes.

Muscle Atrophy

Conclusions

Muscle wasting or atrophy occurs in a variety of physiological or pathological circumstances, including inactivity or muscle disuse, fasting, and diseases such as cancer and diabetes. In its initial stage, muscle breakdown has beneficial effects since it provides the organism with free amino acids that can be reused for energy production or protein synthesis. However, when this catabolic state is maintained over a long period of time, it becomes detrimental and even life-threatening. The central mechanism in muscle wasting is increased intracellular proteolysis, primarily due to the activation of the UPS. The musclespecific E3 ligases Trim32, Atrogin1, and Murf1 are induced by insulin resistance, NF-kB in response to several disease states, stress, or inflammatory cytokines such as TNFa and are responsible for the ubiquitination and degradation of the different components of skeletal muscle. Since the proteasome is a central component in muscle wasting, it represents an attractive drug target to block excessive muscle loss. Though studies in humans are not yet completed, many studies in animals have demonstrated that proteasome inhibitors efficiently slow down muscle wasting.

The Ub signaling networks are intimately linked to virtually every cellular process and are often deregulated in the pathogenesis of human diseases. Moreover, accumulated evidence indicates that cross talk between the Ub system and other posttranslational modifications (including protein phosphorylation, acetylation, or methylation) provides higher layer of complexity in regulating cell life, injury, and death. Thus, multiple efforts are focused on the development of efficient therapeutic methods to target the Ub system that has a potential for novel treatments of neurodegenerative disease, cancer, immunologic disorder, and microbial infection.

Further Reading Part 1. Ubiquitination: An Inducible and Reversible Posttranslational Modification Hershko, A., Ciechanover, A., 1998. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479.

Cell Adaptation, Injury and Death | Cell Adaptation, Injury, and Death: Ubiquitin–Proteasome System

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