Degradation of Some Polyubiquitinated Proteins Requires an Intrinsic Proteasomal Binding Element in the Substrates

Lysine 48-linked polyubiquitin chains usually target proteins for 26 S proteasomal degradation; however, this modification is not a warrant for destruction. Here, we found that efficient degradation of a physiological substrate UbcH10 requires not only an exogenous polyubiquitin chain modification but also its unstructured N-terminal region. Interestingly, the unstructured N-terminal region of UbcH10 directly binds the 19 S regulatory complex of the 26 S proteasome, and it mediates the initiation of substrate translocation. To promote ubiquitin- dependent degradation of the folded domains of UbcH10, its N-terminal region can be displaced by exogenous proteasomal binding elements. Moreover, the unstructured N-terminal region can initiate substrate translocation even when UbcH10 is artificially cyclized without a free terminus. Polyubiquitinated circular UbcH10 is completely degraded by the 26 S proteasome. Accordingly, we propose that degradation of some polyubiquitinated proteins requires two binding interactions: a polyubiquitin chain and an intrinsic proteasomal binding element in the substrates (likely an unstructured region); moreover, the intrinsic proteasomal binding element initiates substrate translocation regardless of its location in the substrates.     

Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat

The glycine-alanine (GA) repeat of the Epstein-Barr virus nuclear antigen-1 inhibits in cis ubiquitin-dependent proteolysis in mammalian cells through a yet unknown mechanism. In the present study we demonstrate that the GA repeat targets an evolutionarily conserved step in proteolysis since it can prevent the degradation of proteasomal substrates in the yeast Saccharomyces cerevisiae. Insertion of yeast codon-optimised recombinant GA (rGA) repeats of different length in green fluorescent protein reporters harbouring N-end rule or ubiquitin fusion degradation signals resulted in efficient stabilisation of these substrates. Protection was also achieved in rpn10delta yeast suggesting that this polyubiquitin binding protein is not required for the rGA effect. The conserved effect of the GA repeat in yeast opens the possibility for the use of genetic screens to unravel its mode of action.     

Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein

In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.     

Cooperation between an intrinsically disordered region and a helical segment is required for ubiquitin-independent degradation by the proteasome

The 26 S proteasomal complex, which is responsible for the bulk of protein degradation within the cell, recognizes its target substrates via covalently linked polyubiquitin moieties. However, a small but growing number of proteasomal substrates are degraded without a requirement for ubiquitinylation. One such substrate is the pyrimidine biosynthetic enzyme thymidylate synthase (EC 2.1.1.45), which catalyzes the synthesis of TMP and is the sole de novo source of TTP for DNA replication and repair. Previous work showed that intracellular proteolysis of human thymidylate synthase is directed by a degron at the polypeptide's N-terminal end, composed of an intrinsically disordered region (IDR) followed by a highly conserved amphipathic α-helix (hA). In the present report, we show that the hA helix does not function simply as an extension or scaffold for the IDR; rather, it provides a specific structural component that is necessary for degradation. Furthermore, its helical conformation is required for this function. We demonstrate that small domains from heterologous proteins can substitute for the IDR and the hA helix of human thymidylate synthase, indicating that the degradation-promoting function of these regions is not sequence-specific. The results, in general, indicate that cooperation between intrinsically disordered domains and α-helical segments is required for ubiquitin-independent degradation by the proteasome. There appears to be little sequence constraint on the ability of these regions to function as degron constituents. Rather, it is the overall conformation (or lack thereof) that is critical.     

VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome

The 26S proteasome recognizes a vast number of ubiquitin-dependent degradation signals linked to various substrates. This recognition is mediated mainly by the stoichiometric proteasomal resident ubiquitin receptors S5a and Rpn13, which harbor ubiquitin-binding domains. Regulatory steps in substrate binding, processing, and subsequent downstream proteolytic events by these receptors are poorly understood. Here we demonstrate that mammalian S5a is present in proteasome-bound and free states. S5a is required for efficient proteasomal degradation of polyubiquitinated substrates and the recruitment of ubiquitin-like (Ubl) harboring proteins; however, S5a-mediated ubiquitin and Ubl binding occurs only on the proteasome itself. We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association. Multiubiquitination events within the VWA domain can further regulate S5a association. Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.     

Stabilization signals: a novel regulatory mechanism in the ubiquitin/proteasome system

The turnover of cellular proteins is a highly organized process that involves spatially and temporally regulated degradation by the ubiquitin/proteasome system. It is generally acknowledged that the specificity of the process is determined by constitutive or conditional protein domains, the degradation signals, that target the substrate for proteasomal degradation. In this review, we discuss a new type of regulatory domain: the stabilization signal. A model is proposed according to which protein half-lives are determined by the interplay of counteracting degradation and stabilization signals.     

Selective targeting of proteins within secretory pathway for endoplasmic reticulum-associated degradation

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity.     

Recognition of the polyubiquitin proteolytic signal

Polyubiquitin chains linked through Lys48 are the principal signal for targeting substrates to the 26S proteasome. Through studies of structurally defined, polyubiquitylated model substrates, we show that tetraubiquitin is the minimum signal for efficient proteasomal targeting. The mechanism of targeting involves a simple increase in substrate affinity that is brought about by autonomous binding of the polyubiquitin chain. Assigning the proteasomal signaling function to a specific polymeric unit explains how a single ubiquitin can act as a functionally distinct signal, for example in endocytosis. The properties of the substrates studied here implicate substrate unfolding as a kinetically dominant step in the proteolysis of properly folded proteins, and suggest that extraproteasomal chaperones are required for efficient degradation of certain proteasome substrates.     

Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.     

NF-kappaB dictates the degradation pathway of IkappaBalpha

IkappaB proteins are known as the regulators of NF-kappaB activity. They bind tightly to NF-kappaB dimers, until stimulus-responsive N-terminal phosphorylation by IKK triggers their ubiquitination and proteasomal degradation. It is known that IkappaBalpha is an unstable protein whose rapid degradation is slowed upon binding to NF-kappaB, but it is not known what dynamic mechanisms control the steady-state level of total IkappaBalpha. Here, we show clearly that two degradation pathways control the level of IkappaBalpha. Free IkappaBalpha degradation is not controlled by IKK or ubiquitination but intrinsically, by the C-terminal sequence known as the PEST domain. NF-kappaB binding to IkappaBalpha masks the PEST domain from proteasomal recognition, precluding ubiquitin-independent degradation; bound IkappaBalpha then requires IKK phosphorylation and ubiquitination for slow basal degradation. We show the biological requirement for the fast degradation of the free IkappaBalpha protein; alteration of free IkappaBalpha degradation dampens NF-kappaB activation. In addition, we find that both free and bound IkappaBalpha are similar substrates for IKK, and the preferential phosphorylation of NF-kappaB-bound IkappaBalpha is due to stabilization of IkappaBalpha by NF-kappaB.     

alpha-Synuclein protofibrils inhibit 26 S proteasome-mediated protein degradation: understanding the cytotoxicity of protein protofibrils in neurodegenerative disease pathogenesis

The impaired ubiquitin-proteasome activity is believed to be one of the leading factors that contribute to Parkinson disease pathogenesis partially by causing alpha-synuclein aggregation. However, the relationship between alpha-synuclein aggregation and the impaired proteasome activity is yet unclear. In this study, we examined the effects of three soluble alpha-synuclein species (monomer, dimer, and protofibrils) on the degradation activity of the 26 S proteasome by reconstitution of proteasomal degradation using highly purified 26 S proteasomes and model substrates. We found that none of the three soluble alpha-synuclein species impaired the three distinct peptidase activities of the 26 S proteasome when using fluorogenic peptides as substrates. In striking contrast, alpha-synuclein protofibrils, but not monomer and dimer, markedly inhibited the ubiquitin-independent proteasomal degradation of unstructured proteins and ubiquitin-dependent degradation of folded proteins when present at 5-fold molar excess to the 26 S proteasome. Together these results indicate that alpha-synuclein protofibrils have a pronounced inhibitory effect on 26 S proteasome-mediated protein degradation. Because alpha-synuclein is a substrate of the proteasome, impaired proteasomal activity could further cause alpha-synuclein accumulation/aggregation, thus creating a vicious cycle and leading to Parkinson disease pathogenesis. Furthermore we found that alpha-synuclein protofibrils bound both the 26 S proteasome and substrates of the 26 S proteasome. Accordingly we propose that the inhibitory effect of alpha-synuclein protofibrils on 26 S proteasomal degradation might result from impairing substrate translocation by binding the proteasome or sequestrating proteasomal substrates by binding the substrates.     

Mutant p62/SQSTM1 UBA domains linked to Paget’s disease of bone differ in their abilities to function as stabilization signals

We show that the ubiquitin-associated domain (UBA) of human p62/sequestosome-1 (SQSTM1) can delay degradation of proteasome substrates in yeast. Taking advantage of naturally occurring mutant UBA domains that are linked to Paget’s disease of bone (PDB), we found that three of the four mutant UBA domains tested in this study were able to inhibit proteasomal degradation, albeit not to the same extent as the wild-type domain. Interestingly, the stability measured as the fraction of folded protein, and not the ubiquitin binding properties, of the PDB-associated UBA domains correlated with their protective effects. These data suggest that the protective effect of UBA domains depends on their structural integrity rather than ubiquitin binding capabilities.     

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.     

Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system

We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2(VHL) substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2(VHL) or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC(50) = 1.7 μM) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.     

5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal

5-Azacytidine- and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs. The 5-aza-CdR-induced degradation, which occurs in the nucleus, could be blocked by proteasomal inhibitors and required a functional ubiquitin-activating enzyme. The drug-induced degradation occurred even in the absence of DNA replication. Treatment of cells with other nucleoside analogs modified at C-5, 5-fluorodeoxyuridine and 5-fluorocytidine, did not induce the degradation of DNMT1. Mutation of cysteine at the catalytic site of Dnmt1 (involved in the formation of a covalent intermediate with cytidine in DNA) to serine (CS) did not impede 5-aza-CdR-induced degradation. Neither the wild type nor the catalytic site mutant of Dnmt3a or Dnmt3b was sensitive to 5-aza-CdR-mediated degradation. These results indicate that covalent bond formation between the enzyme and 5-aza-CdR-incorporated DNA is not essential for enzyme degradation. Mutation of the conserved KEN box, a targeting signal for proteasomal degradation, to AAA increased the basal level of Dnmt1 and blocked its degradation by 5-aza-CdR. Deletion of the catalytic domain increased the expression of Dnmt1 but did not confer resistance to 5-aza-CdR-induced degradation. Both the nuclear localization signal and the bromo-adjacent homology domain were essential for nuclear localization and for the 5-aza-CdR-mediated degradation of Dnmt1. Polyubiquitination of Dnmt1 in vivo and its stabilization upon treatment of cells with a proteasomal inhibitor indicate that the level of Dnmt1 is controlled by ubiquitin-dependent proteasomal degradation. Overexpression of the substrate recognition component, Cdh1 but not Cdc20, of APC (anaphase-promoting complex)/cyclosome ubiquitin ligase reduced the level of Dnmt1 in both untreated and 5-aza-CdR-treated cells. In contrast, the depletion of Cdh1 with small interfering RNA increased the basal level of DNMT1 that blocked 5-aza-CdR-induced degradation. Dnmt1 interacted with Cdh1 and colocalized in the nucleus at discrete foci. Both Dnmt1 and Cdh1 were phosphorylated in vivo, but only Cdh1 was significantly dephosphorylated upon 5-aza-CdR treatment, suggesting its involvement in initiating the proteasomal degradation of DNMT1. These results demonstrate a unique mechanism for the selective degradation of DNMT1, the maintenance DNA methyltransferase, by well-known DNA-hypomethylating agents.     

Pathway choice between proteasomal and autophagic degradation

Efficient degradation of abnormal or aggregated proteins is crucial to protect the cell against proteotoxic stress. Selective targeting and disposal of such proteins usually occurs in a ubiquitin-dependent manner by proteasomes and macroautophagy/autophagy. Whereas proteasomes are efficient in degrading abnormal soluble proteins, protein aggregates are typically targeted for degradation by autophagic vesicles. Both processes require ubiquitin-binding receptors, which are targeted to proteasomes via ubiquitin-like domains or to phagophores (the precursors to autophagosomes) via Atg8/LC3 binding motifs, respectively. The use of substrate modification by ubiquitin in both pathways raised the question of how degradative pathway choice is achieved. In contrast to previous models, proposing different types of ubiquitin linkages for substrate targeting, we find that pathway choice is a late event largely determined by the oligomeric state of the receptors. Monomeric proteasome receptors bind soluble substrates more efficiently due to their higher affinity for ubiquitin. Upon substrate aggregation, autophagy receptors with lower ubiquitin binding affinity gain the upper hand due to higher avidity achieved by receptor bundling. Thus, our work suggests that ubiquitination is a shared signal of an adaptive protein quality control system, which targets substrates for the optimal proteolytic pathway.     

The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.     

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 with a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 can constitute a mechanistic link between DNA damage and the ribosomal stress pathway, and a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.     

Blm10 protein promotes proteasomal substrate turnover by an active gating mechanism

For optimal proteolytic function, the central core of the proteasome (core particle (CP) or 20S) has to associate with activators. We investigated the impact of the yeast activator Blm10 on proteasomal peptide and protein degradation. We found enhanced degradation of peptide substrates in the presence of Blm10 and demonstrated that Blm10 has the capacity to accelerate proteasomal turnover of the unstructured protein tau-441 in vitro. Mechanistically, proteasome activation requires the opening of a closed gate, which allows passage of unfolded proteins into the catalytic chamber. Our data indicate that gate opening by Blm10 is achieved via engagement of its C-terminal segment with the CP. Crucial for this activity is a conserved C-terminal YYX motif, with the penultimate tyrosine playing a preeminent role. Thus, Blm10 utilizes a gate opening strategy analogous to the proteasomal ATPases HbYX-dependent mechanism. Because gating incompetent Blm10 C-terminal point mutants confers a loss of function phenotype, we propose that the cellular function of Blm10 is based on CP association and activation to promote the degradation of proteasome substrates.