Binding surface mapping of intra- and interdomain interactions among hHR23B,ubiquitin, and polyubiquitin binding site 2 of S5a

hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.     

Structure of hRpn10 Bound to UBQLN2 UBL Illustrates Basis for Complementarity between Shuttle Factors and Substrates at the Proteasome

The 26S proteasome is a highly complex 2.5-MDa molecular machine responsible for regulated protein degradation. Proteasome substrates are typically marked by ubiquitination for recognition at receptor sites contributed by Rpn1/S2/PSMD2, Rpn10/S5a, and Rpn13/Adrm1. Each receptor site can bind substrates directly by engaging conjugated ubiquitin chains or indirectly by binding to shuttle factors Rad23/HR23, Dsk2/PLIC/UBQLN, or Ddi1, which contain a ubiquitin-like domain (UBL) that adopts the ubiquitin fold. Previous structural studies have defined how each of the proteasome receptor sites binds to ubiquitin chains as well as some of the interactions that occur with the shuttle factors. Here, we define how hRpn10 binds to the UBQLN2 UBL domain, solving the structure of this complex by NMR, and determine affinities for each UIM region by a titration experiment. UBQLN2 UBL exhibits 25-fold stronger affinity for the N-terminal UIM-1 over UIM-2 of hRpn10. Moreover, we discover that UBQLN2 UBL is fine-tuned for the hRpn10 UIM-1 site over the UIM-2 site by taking advantage of the additional contacts made available through the longer UIM-1 helix. We also test hRpn10 versatility for the various ubiquitin chains to find less specificity for any particular linkage type compared to hRpn1 and hRpn13, as expected from the flexible linker region that connects the two UIMs; nonetheless, hRpn10 does exhibit some preference for K48 and K11 linkages. Altogether, these results provide new insights into the highly complex and complementary roles of the proteasome receptor sites and shuttle factors.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Ubiquitin ligase activity of c-Cbl guides the epidermal growth factor receptor into clathrin-coated pits by two distinct modes of Eps15 recruitment

We have demonstrated previously that c-Cbl requires the presence of a functional ubiquitin interacting motif (UIM) in Eps15 to mediate epidermal growth factor receptor (EGFR) endocytosis. Both the ubiquitin ligase activity of c-Cbl and the UIM of Eps15 were necessary for plasma membrane recruitment of Eps15 and entry of ligand-bound EGFR into coated pits and vesicles containing Eps15. This is consistent with a scenario in which ubiquitin moieties appended to activated EGFR complexes act as docking sites for Eps15 and thereby recruit receptors into clathrin coated pits. Here, we have investigated which additional structural features of c-Cbl are required for this process. We find that c-Cbl can guide ligand-bound EGFR into the Eps15 internalization route by two distinct mechanisms. These are either dependent on the phosphotyrosine binding domain of c-Cbl that directly binds to the EGFR or on the region C-terminal of the Ring finger, which allows for indirect binding to an alternative site on the receptor. No strict requirement exists for either ubiquitin modified EGFR or the Cbl binding ubiquitination substrate CIN85 as docking site for the UIM of Eps15. Only in the phosphotyrosine binding-dependent pathway, the EGFR is ubiquitinated and may serve as a site of recruitment for Eps15. Only in this pathway, Eps15 is tyrosine-phosphorylated, but this appears unrelated to its capacity to participate in EGFR internalization.     

A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes

The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.     

A ubiquitin-interacting motif from Hrs binds to and occludes the ubiquitin surface necessary for polyubiquitination in monoubiquitinated proteins

The ubiquitin-interacting motif (UIM) is a short, approximately 20 residue, structural element, which is present in, but not limited to, the proteins involved in endocytotic and proteasomal degradation. UIMs facilitate endocytotic vesicular sorting of the monoubiquitinated proteins and may be important for the targeting of the polyubiquitinated proteins to the proteasome. Using heteronuclear NMR backbone and side-chain chemical shift mapping of the ubiquitin interaction surface, the UIM from the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, specifically binds to the ubiquitin hydrophobic surface using UIM's well-conserved central helical LALAL motif. Molecular modeling of the ubiquitin:UIM_Hrs complex suggests that binding occurs through a specific interaction of Leu263 and Leu267 of the UIM_Hrs with two ubiquitin hydrophobic patches located in close proximity to the ubiquitin major polyubiquitination site, Lys48. Intramolecular binding of ubiquitin to a UIM in monoubiquitinated proteins would render Lys48 unavailable for further ubiquitination, thus, explaining the absolute requirement of UIMs for monoubiquitination. Two leucines, Leu265 and Leu269, located on the opposite face of UIM_Hrs can also interact, albeit less favorably than Leu263 and Leu267, with the ubiquitin hydrophobic patches, suggesting a possible mode for polyubiquitin:UIM binding and apparent preference of UIMs for polyubiquitins.     

Structural determinants for the binding of ubiquitin-like domains to the proteasome

HHR23A, a protein implicated in nucleotide excision repair, belongs to a class of proteins containing both a ubiquitin-like (Ubl) domain and one or more ubiquitin-associated (UBA) domains, suggesting a role in the ubiquitin-proteasome pathway as well. The Ubl domain binds with high affinity to the second ubiquitin-interacting motif (UIM) of the S5a subunit of the proteasome. Here we present the solution structures of the HHR23A Ubl domain, the second UIM of S5a (UIM-2), and the Ubl:S5a-UIM-2 complex. The HHR23A Ubl domain is structurally similar to ubiquitin. The S5a UIM forms an alpha-helix with an unexpected hairpin loop that contributes to the binding interface with Ubl. The molecular determinants of the Ubl-proteasome interaction are revealed by analysis of the structures, chemical shift mapping, mutant binding studies and sequence conservation.     

Molecular mechanisms of coupled monoubiquitination

Many proteins contain ubiquitin-binding domains or motifs (UBDs), such as the UIM (ubiquitin-interacting motif) and are referred to as ubiquitin receptors. Ubiquitin receptors themselves are frequently monoubiquitinated by a process that requires the presence of a UBD and is referred to as coupled monoubiquitination. Using a UIM-containing protein, eps15, as a model, we show here that coupled monoubiquitination strictly depends on the ability of the UIM to bind to monoubiquitin (mUb). We found that the underlying molecular mechanism is based on interaction between the UIM and a ubiquitin ligase (E3), which has itself been modified by ubiquitination. Furthermore, we demonstrate that the in vivo ubiquitination of members of the Nedd4 family of E3 ligases correlates with their ability to monoubiquitinate eps15. Thus, our results clarify the mechanism of coupled monoubiquitination and identify the ubiquitination of E3 ligases as a critical determinant in this process.     

Rad23 Interaction with the Proteasome Is Regulated by Phosphorylation of Its Ubiquitin-Like (UbL) Domain

Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.     

The effects of the polyglutamine repeat protein ataxin-1 on the UbL-UBA protein A1Up

The ataxin-1 interacting ubiquitin-like protein (A1Up) contains an amino-terminal ubiquitin-like (UbL) region, four stress-inducible, heat shock chaperonin-binding motifs (STI1), and an ubiquitin-associated domain (UBA) at the carboxyl terminus of A1Up. Although proteins that have both an UbL and UBA domain are thought to play a crucial role in proteasome-mediated activities, few are characterized, except for hHR23A/B. Similar to other UbL-containing proteins, the UbL of A1Up is essential for the interaction of A1Up with the S5a subunit of the 19S proteasome. Importantly, the interaction with the 19S proteasome was disrupted in the presence of the polyglutamine repeat protein, ataxin-1. The UbL domain of A1Up is ubiquitinated by both Lys(48)-linked and Lys(63)-linked chains. Intact A1Up is stable, suggesting that ubiquitination of A1Up is important for degradation-independent targeting of A1Up to the 19S proteasome. The UBA domain of A1Up binds polyubiquitin chains and has a role in the stability of A1Up and in the subcellular localization of A1Up. When the UBA domain was deleted, the localization of A1Up was entirely cytoplasmic, and it co-localized with the proteasome. Interestingly, the interaction between A1Up and mutant ataxin-1-(82Q) increased the half-life of A1Up, whereas nonpathogenic wild-type ataxin-1-(30Q) or ataxin-1-(82Q)-A776 did not.     

Ubiquitin-interacting Motifs Confer Full Catalytic Activity,but Not Ubiquitin Chain Substrate Specificity,to Deubiquitinating Enzyme USP37

Ubiquitin-specific proteases (USPs) consist of a family of deubiquitinating enzymes with more than 50 members in humans. Three of them, including USP37, contain ubiquitin-interacting motifs (UIMs), an ∼20-amino acid α-helical stretch that binds to ubiquitin. However, the roles of the UIMs in these USP enzymes remain unknown. USP37 has three UIMs, designated here as UIMs 1, 2, and 3 from the N-terminal side, between the Cys and His boxes comprising the catalytic core. Here, we examined the role of the UIMs in USP37 using its mutants that harbor mutations in the UIMs. The nuclear localization of USP37 was not affected by the UIM mutations. However, mutations in UIM2 or UIM3, but not UIM1, resulted in a significant decrease in USP37 binding to ubiquitinated proteins in the cell. In vitro, a region of USP37 harboring the three UIMs also bound to both Lys⁴⁸-linked and Lys⁶³-linked ubiquitin chains in a UIM2- and UIM3-dependent manner. The level of USP37 ubiquitination was also reduced by mutations in UIM2 or UIM3, suggesting their role in ubiquitination of USP37 itself. Finally, mutants lacking functional UIM2 or UIM3 exhibited a reduced isopeptidase activity toward ubiquitinated proteins in the cell and both Lys⁴⁸-linked and Lys⁶³-linked ubiquitin chains. These results suggested that the UIMs in USP37 contribute to the full enzymatic activity, but not ubiquitin chain substrate specificity, of USP37 possibly by holding the ubiquitin chain substrate in the proximity of the catalytic core.     

The N-terminal Regulatory Domain of Cyclin A Contains Redundant Ubiquitination Targeting Sequences and Acceptor Sites

Cyclin A is destroyed during mitosis by the ubiquitin-proteasome system. Like cyclin B, a destruction box (D-box) motif is required for the destruction of cyclin A. However, Cyclin A degradation is more complicated than cyclin B because cyclin A’s D-box motif is more extensive and proteolysis involves complex signaling in some organisms. In this study, we found that in addition to the D-box, the region between residues 123-157 also contributed to the ubiquitination and degradation of human cyclin A. Indeed, removal of the bulk of the N-terminal regulatory domain was needed to completely stabilize cyclin A and eliminate ubiquitination. A putative second RxxL motif around residue 138 played only a minor role in cyclin A degradation. To distinguish between sequences recognized by the ubiquitination machinery and the ubiquitin acceptor sites per se, we utilized a novel approach involving in vitro cleavage of cyclin A after ubiquitination. We found that several lysine residues proximal to the D-box (Lys37, Lys54, and Lys68) were ubiquitin acceptor sites. Cyclin A lacking the three lysine residues was degraded slower than the wild-type protein. Although these lysines were normally used, ubiquitination could shift to other cryptic sites when the preferred sites were unavailable, suggesting the exact positions of the ubiquitin chains also contributed to degradation. Together, these data revealed that ubiquitination does not occur randomly on cyclin A and open up questions on the precise function of the D-box.     

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.     

Rad23 promotes the targeting of proteolytic substrates to the proteasome

Rad23 contains a ubiquitin-like domain (UbL(R23)) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23 Delta rpn10 Delta mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10 Delta mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbL(R23) caused stabilization of a model substrate, indicating that an unregulated UbL(R23)-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23 Delta rpn10 Delta mutant phenotype required both UbL(R23) and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.     

Ubiquitination of p53 at multiple sites in the DNA-binding domain

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.     

Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates

Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin-activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha-lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha-lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added.     

2',5'-oligoadenylate synthetase gene in chicken: gene structure, distribution of alleles and their expression

We have cloned the gene for chicken 2',5'-oligoadenylate synthetase (ChOAS) by the method of polymerase chain reaction with use of ChOAS cDNA sequence. The ChOAS gene is composed of five introns and six exons containing all of the sequence of the ChOAS cDNA from the start to the stop codon. The first five exons of ChOAS gene which encode the OAS catalytic domain have a similar structure to HuOAS1 gene including the exon-intron boundaries. However, the length of introns of ChOAS gene is only 1/7 of those of HuOAS1 gene. The sixth exon of the ChOAS gene encodes the ubiquitin-like (UbL) domain of two consecutive sequence (UbL1 and UbL2) homologous to ubiquitin. ChOAS encoded in a single copy gene has at least two alleles, OAS(*)A and OAS(*)B. The differences between these two alleles are in the sixth exon of the gene; a 96-nucleotide sequence in the UbL1 portion of OAS(*)A is deleted from OAS(*)B. No OAS(*)B gene was detected in nine lines of chickens tested other than Leghorns. Almost the same levels of ChOAS-A and -B proteins induced physiologically in erythrocytes were detected in infant chickens (2-week-old), but in grown-up chickens (6-month-old) the level of erythrocyte OAS-B was markedly reduced in most of B/B chickens. Thus, the UbL domain of ChOAS is responsible for the maintenance of the OAS level in the tissue.     

Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting

Hrs has an essential role in sorting of monoubiquitinated receptors to multivesicular bodies for lysosomal degradation, through recognition of ubiquitinated receptors by its ubiquitin-interacting motif (UIM). Here, we present the structure of a complex of Hrs-UIM and ubiquitin at 1.7-A resolution. Hrs-UIM forms a single alpha-helix, which binds two ubiquitin molecules, one on either side. These two ubiquitin molecules are related by pseudo two-fold screw symmetry along the helical axis of the UIM, corresponding to a shift by two residues on the UIM helix. Both ubiquitin molecules interact with the UIM in the same manner, using the Ile44 surface, with equal binding affinities. Mutational experiments show that both binding sites of Hrs-UIM are required for efficient degradative protein sorting. Hrs-UIM belongs to a new subclass of double-sided UIMs, in contrast to its yeast homolog Vps27p, which has two tandem single-sided UIMs.     

The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system. Direct evidence for ubiquitination at the N-terminal residue

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.