REVIEWS New roles for old modifications: Emerging roles of N-terminal post-translational modifications in development and disease

John G. Tooley and Christine E. Schaner Tooley* Department of Biochemistry and Molecular Biology, James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, Kentucky Received 29 August 2014; Accepted 8 September 2014 DOI: 10.1002/pro.2547 Published online 00 Month 2014 proteinscience.org

Abstract: The importance of internal post-translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N-terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N-terminal PTMs are now beginning to be elucidated. New findings show that N-terminal methylation, along with Nterminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N-terminal ubiquitylation and N-terminal propionylation. Here, will discuss the recent advances in the functional studies of N-terminal PTMs, recount the new N-terminal PTMs being identified, and briefly examine the possibility of dynamic N-terminal PTM exchange. Keywords: N-terminal; methylation; acetylation; ubiquitylation; propionylation

Introduction Post-translational modification (PTM) of proteins has been documented as an important regulatory mechanism for many decades, but the intricacies of how each modification functions independently, and as part of a collective unit, has been best established for those PTMs found on histone tails.1 Work deciphering the histone “code” has led to the identification of new PTMs, new enzymes that catalyze the addition of PTMs (writers), new enzymes that remove these modifications (erasers), and novel protein families that bind PTMs and translate them into downstream signals (readers).2 Deciphering *Correspondence to: Christine E. Schaner Tooley, Department of Biochemistry & Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40202. E-mail: [email protected]

C 2014 The Protein Society Published by Wiley-Blackwell. V

these unique codes for other types of PTMs and identifying their corresponding writers, readers, and erasers are now becoming areas of expanded research focus. Occurrences of both N-terminal acetylation (on the a-amino group) and N-terminal methylation have been documented for almost 40 years.3–7 The corresponding N-terminal acetyltransferases were identified a decade later,8,9 but the first eukaryotic N-terminal methyltransferases (NRMT1 and NRMT2) were discovered only recently.10–12 Additionally, N-terminal ubiquitylation and N-terminal propionylation were only verified within the last decade,13,14 and the responsible enzymes identified within the last few years.14–16 The available enzymology for N-terminal acetylation resulted in it being the primary focus for functional studies over recent decades. However, the discoveries of

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Table I. Summary of Current Data on N-Terminal PTM N-terminal modification

Catalytic enzyme(s)

Molecular weight (kD)

Other complex members

Acetylation

26.5 20.4 39.3 27.2 19.4 27.5 25.4 32.4 18.2

Naa15p (NatA) Naa25p (NatB) Naa35p/Naa38p (NatC) Naa10p/Naa15p (NatE)

Protein degradation25,26 Protein/protein interactions30 Cytosolic sorting32 Membrane interactions33 Protein folding/aggregation33–35

Unknown

Ubiquitylation

Naa10p (NatA) Naa20p (NatB) Naa30p (NatC) Naa40p (NatD) Naa50p (NatE) Naa60p (NatF) NRMT1 NRMT2 Ube2w

Unknown

Propionylation

Naa10p (NatA)

26.5

Naa15p

Protein degradation49 Protein/DNA interactions11,50 Protein degradation13 Protein/protein interactions?16 Unknown

Methylation

NRMT1/2 and additional N-terminal PTMs have allowed for significant and rapid expansion of the field and resulted in new functional insights into this group of modifications (Table I). We will discuss both the established and novel roles for each of these modifications, underscoring recent advances in their involvement in development and disease progression.

N-terminal acetylation N-terminal acetylation is the most abundant of the N-terminal PTMs, occurring on more than 80% of eukaryotic proteins.17 It is catalyzed in the cytoplasm by the NAT family of acetyltransferases and results in a neutral charge on the a-amino group (Fig. 1).9,18,19 There are six NAT subtypes (NatA through NatF), with each having their own distinct substrate specificity. All but NatA and NatD acetylate the initiating methionine, with their catalytic specificity determined by the subsequent amino acids.20 NatD acetylates the N-terminal serine of histones H2A and H4.21 NatA methylates N-terminal serines, alanines, threonines, glycines, and valines that result from cleavage of the initiating methionine, unless the third amino acid is a proline.9,22–24 There are currently no known N-terminal deacetylases and the modification is believed to be constitutive.20 N-terminal acetylation is most commonly associated with its role in ubiquitin-mediated protein degradation and the N-end rule pathway.25,26 It was originally shown that proteins with free a-amino groups were targeted for ATP-dependent ubiquitinmediated degradation, but N-terminal acetylation could prevent this degradation.25 However, Nterminal acetylation was then also shown to play a role in promoting sequence-specific protein degradation through the N-end rule pathway.26,27 The N-end rule posits that the stability of a protein is dictated by its N-terminal sequence and modification.27 There are two branches, one responsible for the degradation of specific proteins with unacetylated alpha-amino groups (Arg/N), and one responsible for targeting N-terminally acetylated proteins (Ac/N).28 The initiating methionine (or Ala, Val, Ser, Thr, Cys,

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if the initiating methionine is cleaved) can be acetylated if the following amino acid is permissive (not Pro).24,26 The resulting “AcN-degron” is then targeted for ubiquitylation and proteasome-mediated degradation by the Doa10 E3 Ub ligase.26 In addition to dictating protein half-life, the AcN-degron can help dictate protein stoichiometry. Both Hcn1 (a subunit of the APC/C ubiquitin ligase) and Cog1 (a subunit of the Golgi-associated COG complex) are Nterminally acetylated, but their AcN-degron is blocked by binding of other subunits in the core complexes (Cut9 and Cog2/Cog3, respectively).29 If Cog1 becomes overexpressed as compared with Cog2 and Cog3, it becomes destabilized and degraded due to exposure of its AcN-degron.29 Recent studies have demonstrated additional functional roles for N-terminal acetylation outside of regulating protein stability. The E2 enzyme Ubc12 is acetylated on its N-terminal methionine.30 As acetylation removes a positive charge from the Nterminus, this allows binding of the Ubc12 Nterminal tail in the hydrophobic pocket of the E3ligase Dcn1, and is necessary for Dcn1-mediated ligation of the ubiquitin-like protein Nedd8 to Cul1.30 This is the first example of a protein that can “read” protein N-terminal acetylation, similar to bromodomain proteins that specifically bind acetylated histone residues.31 Crystal structure analysis illustrates that the acetyl group on the N-terminal tail of Ubc12 interacts directly with Dcn1 though hydrogen bonding, in addition to permitting binding in the hydrophobic pocket.30 N-terminal acetylation has also been shown necessary for proper cytosolic sorting, as aberrantly N-terminally acetylated secretory proteins mislocalize to the cytosol.32 Whether there is another N-terminal acetylation “reader,” which binds and retains proteins in the cytosol remains to be determined. Lastly, N-terminal acetylation has also been demonstrated to stabilize the helical structure of alpha-synuclein and increase its affinity for cellular membranes, modulate global protein folding and promote Sup35 prion aggregation, as well as, prevent Huntingtin (Htt) aggregation.33–35

Roles of N-Terminal PTM in Development and Disease

Figure 1. Diagram of N-terminal PTM. A: The unmodified a-amino nitrogen (red) can be neutral (shown) or positively charged depending on the surrounding pH. B, C: N-terminal acetylation and N-terminal propionylation result in a pH-independent neutral charge, with propionylation being larger by an additional methyl moiety (blue). D: N-terminal monoubiquitylation results from the direct addition of a ubiquitin moiety (green) to the a-amino group, not to an internal side chain (R). E thru H: Monomethylation, dimethylation, and trimethylation of the a-amino group. Only dimethylation of an N-terminal proline (H) and trimethylation of other N-terminal residues (G) produce a pH-independent positive charge.

The growing examples of functional roles for Nterminal acetylation signify the importance of this PTM. Indeed, loss of NatA activity results in severe developmental defects, and its misregulation is seen in a variety of cancers.36,37 Infants with mutations in ARD1, the gene encoding the catalytic subunit of the human NatA complex, develop Ogden syndrome, a lethal X-linked disorder exhibiting premature aging, craniofacial abnormalities, hypotonia, developmental delays, and cardiac arrhythmias.36,37 Overexpression of ARD1 has been found in both lung and colorectal cancers, is sufficient to drive cellular transformation, and is correlated with poor survival.38,39 However, overexpression of ARD1 in breast cancer correlates with smaller tumors, less metastasis, and better prognosis.40 In these cases, ARD1 appears to be preventing cell migration and promoting autophagy.40,41 Besides the NatA complex, human NatB and NatD also appear to regulate different aspects of cell growth and apoptosis.37 NatB subunits have been

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found overexpressed in a subset of hepatocellular carcinomas, while NatD shows reduced expression in the same type of cancer.42,43 Due to the large number of N-terminally acetylated substrates, the opposing affects of Nat misregulation are likely celltype and context dependent. The challenge in the field now will be deciphering which substrates are responsible for promoting the observed phenotypes. In lieu of the recently discovered roles for Nterminal acetylation in stabilization of alphasynuclein, Sup35 amyloid formation, and prevention of Htt aggregation, it will also be interesting to determine the role of this PTM in the progression of neurodegenerative orders such as Parkinson’s disease, Creutzfeldt-Jakob disease, and Huntington’s disease.33–35

N-terminal methylation N-terminal methylation of proteins has similarly been documented for many decades, though functional studies on its role in protein regulation have

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just recently begun. In contrast to N-terminal acetylation, N-terminal trimethylation (or dimethylation of an N-terminal proline) produces a permanent positively charged a-amino group that is insensitive to pH (Fig. 1).44 It also abolishes the nucleophilicity of the a-amino nitrogen.44 N-terminal methylation is catalyzed in the nucleus by the NRMT (N-terminal RCC1 Methyltransferase) family of N-terminal methyltransferases, NRMT1 and NRMT2.10–12,45 NRMT1 is ubiquitously expressed, while NRMT2 has low, tissue-specific expression.12 There are no known N-terminal demethylases. NRMT1 and NRMT2 methylate a similar Nterminal consensus sequence, with NRMT1 functioning as a trimethylase and NRMT2 as a monomethylase.12 The consensus sequence for the enzymes was originally thought to be specific for an X-Pro-Lys Nterminal sequence (X being any amino acid besides Leu, Ile, Trp, Asp, or Glu), but has subsequently been expanded to accept most uncharged polar or nonpolar amino acids at the second position and either Lys or Arg in the third.45 Data bank analysis with this expanded consensus predicts more than 300 targets for N-terminal methylation. Many predicted targets have subsequently been confirmed including: regulator of chromatin condensation 1 (RCC1), SET/TAF-I/PHAPII, retinoblastoma protein (RB), DNA damage-binding protein 2 (DDB2), and centromere protein B (CENPB).11,45–47 Many proven and predicted targets are also ribosomal proteins and myosin light chain regulatory subunits.10,11,45,48 Like N-terminal acetylation, N-terminal methylation was believed to be a general regulator of protein degradation.44 It was discovered as a blocking group preventing digestion of the N-terminal peptide of Crithidia oncopelti cytochrome c557.49 It is also commonly found on proteins that contain an exposed N-terminal tail.44 While N-terminal methylation on an exposed tail can protect from cellular aminopeptidases, it is also ideally situated to regulate protein interactions. In fact, N-terminal methylation has now been demonstrated to function as a regulator of protein/DNA interactions.50 Loss of N-terminal methylation of RCC1, results in its decreased binding affinity for DNA and mislocalization from chromatin.11,50 Similarly, loss of N-terminal methylation of CENPB and DDB2 results in their decreased binding to centromeric CENP-B box DNA and sites of DNA damage, respectively.46,47 It is hypothesized that the resulting positively charged N-terminus interacts with the negatively charged phosphate groups of the DNA backbone to strengthen the protein/DNA interaction.50 N-terminal methylation is also found at sites of interaction between proteins in multisubunit complexes, and is therefore predicted to additionally mediate hydrophilic protein/protein interactions, although this prediction remains to be experimentally verified.44

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Levels of N-terminal methylation increase in response to various cellular stressors, including increased cell density, heat shock, and arsenite treatment, and loss of N-terminal methylation produces a variety of corresponding phenotypes.47,51,52 In yeast, expression of a nonmethylatable mutant of the ribosomal protein Rpt1 results in decreased cellular growth and increased sensitivity to oxidative stress.48 Depletion of yeast NRMT1 (TAE1) produces defects in both translation efficiency and fidelity.53 In human cells, the mislocalization of RCC1 from chromatin due to loss of N-terminal methylation disrupts the Ran-GTP gradient and results in multipolar spindle formation and the production of aneuploid cells (Fig. 2).11,50 The diminished ability of DDB2 to accumulate at foci of DNA damage after loss of its N-terminal methylation results in compromised ATM activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity to UV exposure.46 Given the accumulated phenotypes seen with loss of NRMT1 expression, it is not surprising that NRMT1 misregulation has been observed in a variety of cancers. NRMT1 is underexpressed in testicular seminomas and the stroma surrounding breast tumors.54,55 However, in both colorectal cancers and lymphomas, NRMT1 has been found overexpressed as compared with normal tissue.56–58 Similar to NatA, the indication of a role for both NRMT1 upregulation and downregulation in tumorigenesis signifies the involvement of different substrates in different tissues. In fact, we have seen that in breast cancer cell lines, NRMT1 loss induces cell growth, while its overexpression can slow proliferation (CST unpublished data). However, in colon cancer cell lines, both overexpression and knockdown enhance cellular growth (JGT unpublished data). We also see that NRMT1 loss makes breast cancer cells more susceptible to chemicals inducing DNA damage. Additionally, NRMT1 knockout mice exhibit phenotypes associated with defective DNA repair, including reduced size, female-specific infertility, liver degeneration, and premature death (CST unpublished data). These observations correspond to the phenotypes seen with loss of N-terminal methylation of DDB2.46 There are a variety of additional NRMT substrates involved in maintaining genomic integrity and the DNA repair response, and they are likely also playing a role in promoting the observed phenotypes.11,45 It will now be interesting to tease out exactly what substrates are responsible for driving the different types of tumorigenesis and the developmental defects seen with loss of NRMT1 and to determine if independent roles exist for NRMT2.

N-terminal ubiquitylation and propionylation N-terminal ubiquitylation is a more recently discovered PTM, and its role in proteasome-mediated

Roles of N-Terminal PTM in Development and Disease

Figure 2. N-terminal methylation regulates RCC1 binding to DNA. A: During interphase, N-terminally methylated RCC1 is chromatin bound and produces the nuclear Ran-GTP gradient needed to maintain nuclear transport. In mitosis, N-terminally methylated RCC1 remains bound to chromatin. With breakdown of the nuclear membrane, this keeps the concentration of Ran-GTP highest around the chromatin and helps correctly position the spindle poles. B: Loss of N-terminal methylation of RCC1 (due either to loss of NRMT1 or mutations in the consensus sequence) has no known affect on nuclear transport, as the nuclear membrane remains intact and Ran-GTP stays in the nucleus. However, during mitosis, disruption of RCC1 binding to DNA due to loss of N-terminal methylation results in RCC1 diffuse throughout the cell, disruption of the Ran-GTP gradient, and multipolar spindle formation.11,50

degradation was discovered through work with the cell cycle regulator p21.13 It was observed that deletion of all internal lysines of p21 was not sufficient to prevent its ubiquitylation, and subsequent analysis found that it was actually the a-amino group of the N-terminal methionine that was ubiquitylated.13,59 Interestingly, wild type p21 had higher ubiquitylation levels than p21 with deleted internal lysines, but their rates of degradation were indistinguishable.13 This signifies an independent role for N-terminal ubiquitylation in proteasome-mediated protein degradation. This new “non-traditional” mode of protein ubiquitylation is distinct from the N-end rule pathway.60 In the N-end rule pathway, the modified or unmodified N-terminal residue is a recognition site for the ubiquitin ligase, which binds and subsequently ubiq-

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uitylates internal lysines.61 Here, the a-amino group itself is ubiquitylated, with ligase binding presumably somewhere downstream.62 The first ubiquitinconjugating enzyme (E2) responsible for N-terminal ubiquitylation, Ube2w, was recently discovered.15,16 Ube2w specifically mono-ubiquitylates the N-termini of its substrates, including SUMO-2, CHIP, Tau, and ataxin-3.15,16 In the case of SUMO-2, N-terminal monoubiquitylation primes the substrate for polyubiquitylation on lysine 63 by the Ubc13-UEV1 E2 heterodimer.16 This indicates monoubiquitylation of the a-amino group, could also serve to attract readers (i.e., other ubiquitin-conjugating enzymes) distinct from those that can bind N-terminal acetyl or methyl marks. Interestingly, removal of ubiquitin from the Ube2w substrate CHIP is catalyzed by another Ube2w substrate, the deubiquitinating

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enzyme (DUB) ataxin-3.63 It remains to be tested if ataxin-3 is actually removing the N-terminal ubiquitylation on CHIP. If so, ataxin-3 could be the first identified enzyme capable of removing an Nterminal PTM. In addition to the previously mentioned substrates, a variety of other proteins have been shown to be N-terminally ubiquitylated.60 These include the myogenic transcriptional switch protein MyoD, the signaling kinase ERK3, the human papillomavirus 16 oncoprotein E7, the latent membrane proteins LMP1 and LMP2A of the Epstein Barr Virus, and the developmental regulator Id2.59,60,64–68 Before Ube2w was discovered to be an N-terminal E2, it was shown responsible for monoubiquitylating BRCA1 and the Fanconi anemia proteins FANCL and FANCD2.69–71 However, the site of ubiquitylation on these proteins was not clearly defined, and it is possible they too are N-terminally ubiquitylated.16 As N-terminal ubiquitylation is just emerging as a significant PTM, there are likely many more undiscovered substrates. However, the undefined consensus sequence makes prediction through data bank analysis complicated. The known targets have little sequence conservation, and other than being prevented by N-terminal acetylation, N-terminal ubiquitylation appears somewhat promiscuous.60 Given the important roles of the already known targets of N-terminal ubiquitylation, significant roles of this N-terminal PTM in human disease and development are sure to emerge. The newest N-terminal PTM to be discovered is propionylation. It was first suggested after comprehensive analyses of N-terminal PTMs from human Nt2/d1 and HeLa cells.72,73 While less than a dozen proteins with this modification were identified, these reports verified for the first time the existence of Nterminal propionylation in vivo and suggested there was an enzyme capable of propionylating the aamino group of proteins.72,73 The enzyme turned out to be the N-terminal acetyltransferase complex NatA, and this was the first demonstration that Nterminal acetyltransferases can also catalyze Nterminal propionylation.14 Although the other Nat complexes were not tested, the N-terminal sequences of previously identified substrates of N-terminal propionylation predict that most subclasses of the Nat family will exhibit this catalytic function.14 While there is not yet a proven N-terminal “depropionylase,” internal histone propionylation can be removed by histone deacetylases, so a hypothetical N-terminal deacetylase could potentially remove both modifications.74 Although they are catalyzed by the same enzyme, N-terminal acetylation is extremely abundant and N-terminal propionylation is extremely rare.14 It is hypothesized that this stoichiometry is regulated by the availability of Acetyl-CoA and

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Prop-CoA in the cell, as the concentration of AcetylCoA is more than 20 times greater than that of Prop-CoA.14,75 It will now be interesting to determine if N-terminal acetylation and N-terminal propionylation of the same residue result in different functional readouts. N-terminal propionylation adds one additional methyl moiety compared with acetylation (Fig. 1). This increases the hydrophobicity and bulkiness of the modification and has the possibility to either disrupt or enhance PTM-dependent interactions.

Emerging topics The discovery of new N-terminal PTMs and Nterminal modifying enzymes in the last decade has significantly advanced the field of N-terminal PTMs and demonstrated that, similar to histone PTMs, these PTMs have functional relevance outside of simply regulating protein stability. There is evidence that N-terminal acetylation, methylation, and ubiquitylation can also regulate either protein/DNA or protein/protein interactions. As histone acetylation and histone methylation can be “read” by distinct bromodomain and chromodomain-containing proteins, it will now be interesting to determine if Nterminal modifications can be recognized by their own set of “reader” proteins.31,76 Given that Nterminal acetylation and methylation differentially alter the charge and hydrophobicity of the Nterminus, one can envision how these single modifications could dictate protein interactions, with methylation promoting charged, hydrophilic interactions and acetylation alternatively promoting interactions with hydrophobic pockets. It will also be interesting to see, if like histones, N-terminal modifications are dynamic and interchangeable. Due to the absence of any known Nterminal deacetylases, demethylases, or DUBs, it is currently believed that these modifications are static and irreversible. However, it was believed for many years that histone methylation was irreversible, and this was shown incorrect with the identification of the first histone demethylase in 2004.77 It was also thought that because N-terminal methylation requires a proline in the second position after methionine cleavage, and N-terminal acetylation is specifically inhibited by a proline in that position, these two modifications were mutually exclusive.10,11,24 However, the determination of an expanded Nterminal methylation consensus sequence has demonstrated that proline is not absolutely required in the second position.45 In fact, the first protein that can be either N-terminally acetylated or Nterminally methylated, myosin regulatory light chain 9 (MYL9), has just been identified.45 It will now be exciting to see if these modifications are interchangeable on the same molecule, and correspondingly, identify the proteins responsible for

Roles of N-Terminal PTM in Development and Disease

Figure 3. Model of dynamic N-terminal modification exchange. Given that the N-terminal acetyltransferases are cytoplasmic and the N-terminal methyltransferases are nuclear, we propose a model where proteins that have a permissive consensus sequence are acetylated in the cytoplasm and this modification promotes hydrophobic (/) interactions and cytoplasmic functions (i.e., filopodia formation). However, as substrates are needed for nuclear functions, they are imported into the nucleus (potentially deacetylated there) and methylated by the N-terminal methyltransferases. The resulting positive charge of N-terminal methylation would strengthen DNA binding and other hydrophilic interactions and promote nuclear functions, such as transcriptional regulation. As the need for nuclear function diminishes, substrates would be exported back into the cytoplasm (potentially demethylated) and reset for another round of acetylation.

removing them. We propose a model where, at least for acetylation and methylation, these modifications can interchange on the same molecule in a localization dependent manner (Fig. 3). Substrates with a permissive consensus sequence would be acetylated in the cytoplasm by the Nat family of acetyltransferases. This would strengthen hydrophobic interactions and promote cytoplasmic functions. However, for proteins that have dual cytoplasmic and nuclear roles, a signal for their nuclear function would result in nuclear import, deacetylation (by a putative N-terminal deacetylase), and methylation by NRMT1 and NRMT2. The now positively charged N-terminus is poised to promote protein/DNA binding and other hydrophilic interactions. Upon completion of the substrate’s nuclear function, it would export to the cytoplasm, be demethylated (by a putative N-terminal demethylase), and be poised for another round of N-terminal acetylation.

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