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Decrypting Ufmylation: How Proteins Are Modified with UFM1

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Decrypting Ufmylation: How Proteins Are Modified with UFM1 biomolecules Review Decrypting UFMylation: How Proteins Are Modified with UFM1 Sayanika Banerjee, Manoj Kumar and Reuven Wiener * Department of Biochemistry and Molecular Biology, The Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel; [email protected] (S.B.); [email protected] (M.K.) * Correspondence: [email protected]; Tel.: +972-2-6757327 Received: 19 September 2020; Accepted: 9 October 2020; Published: 14 October 2020 Abstract: Besides ubiquitin (Ub), humans have a set of ubiquitin-like proteins (UBLs) that can also covalently modify target proteins. To date, less is known about UBLs than Ub and even less is known about the UBL called ubiquitin-fold modifier 1 (UFM1). Currently, our understanding of protein modification by UFM1 (UFMylation) is like a jigsaw puzzle with many missing pieces, and in some cases it is not even clear whether these pieces of data are in the right place. Here we review the current data on UFM1 from structural biology to biochemistry and cell biology. We believe that the physiological significance of protein modification by UFM1 is currently underestimated and there is more to it than meets the eye. Keywords: UFM1; ubiquitin-like proteins; UFMylation; conjugating enzymes; UBA5; UFC1; UFL1 1. Overview Most ubiquitin-like proteins (UBLs) modify target molecules, mainly proteins, and this is required for their physiological functions. This therefore implies that understanding a UBL’s physiological functions requires answers to the following two major questions: (1) what are the mechanisms by which the UBL modifies its targets and how this is regulated and (2) how is this modification translated to a physiological outcome. In this review, we try to answer these two questions based on the current data on ubiquitin-fold modifier 1 (UFM1). Specifically, we tackle each question from different directions to give a comprehensive picture of this modification, while pointing out the knowledge gaps and the future challenges in the UFM1 field. 2. Ubiquitin-Fold Modifier 1 (UFM1) Since the discovery of ubiquitin in the late 1970s, eight distinct UBLs (not including the different members of the SUMO and ATG8 families) have been identified in humans [1,2], with the last one, ubiquitin-fold modifier 1 (UFM1), being discovered in 2004 [3]. The premature UFM1 comprises 85 amino acids; upon maturation it undergoes catalytic cleavage by the deufmylating enzymes UFSP1 and UFSP2, which remove the last two amino acids, generating a mature UFM1 with a C-terminal Gly [3–6]. This resembles the maturation process of Ub, which is translated as a fusion protein with several Ub repeats or as fusions with ribosomal proteins, and following cleavage by deubiquitinating enzymes, a mature Ub with 76 amino acids is generated [7]. Superposition of the UFM1 structure with Ub’s structure provides an RMSD of 1.6 Å, and similar to Ub, UFM1 possesses a β-grasp fold with 4 β-stands and α-helices [8,9]. Despite sharing the same fold as Ub, UFM1 has only 21.7% sequence identity and 47% similarity to Ub. Like Ub and other UBLs, UFM1 ends with a C-terminal Gly that, upon conjugation to target protein, forms an isopeptide bond. However, in contrast to Ub and other UBLs that have two Gly residues at the C-terminus, UFM1 has a Val residue instead of the first Gly, Biomolecules 2020, 10, 1442; doi:10.3390/biom10101442 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1442 2 of 14 Biomolecules 2020, 10, x 2 of 14 thereforefirst Gly, possessing therefore possessing a unique C-terminal a unique sequenceC-terminal of Val-Gly.sequence Both of Val-Gly. in Ub and Both UFM1, in Ub an and Arg residueUFM1, an is locatedArg residue N-terminally is located to the N-terminally Gly-Gly or theto Val-Glythe Gly- sequence,Gly or the respectively. Val-Gly sequence, This therefore respectively. implies thatThis treatmenttherefore ofimplies a UFM1-modified that treatment protein of a withUFM1-modified trypsin (cleaves protein C-terminally with trypsin to Arg) (cleaves leaves C-terminally a signature ofto Val-GlyArg) leaves that isa linkedsignature to a of target Val-Gly protein’s that is Lys linked side chainto a target via an protein’s isopeptide Lys bond. side chain Currently, via an commercial isopeptide antibodiesbond. Currently, against commercia tri-peptidel ofantibodies Gly-Gly against that is linked tri-peptide to the of Lys Gly-Gly side chain that is are linked used to in proteomicsthe Lys side tochain identify are used Ub-modified in proteomics proteins. to identify Accordingly, Ub-mod theified development proteins. Accordingl of antibodiesy, the against development the unique of signatureantibodies of against Val-Gly the that unique is linked signature to the Lysof Val-Gly side will that significantly is linked to benefit the Lys proteomics side will researchsignificantly on proteinbenefit modificationproteomics research by UFM1. on protein modification by UFM1. OneOne hallmark hallmark of of Ub Ub is is the the hydrophobic hydrophobic patch patch comprising comprising L8, L8, I44, I44, and and V70 V70 [ 10[10].]. This This surface surface has has beenbeen shown shown to to be be required required for for Ub Ub function function and and signaling. signaling.Moreover, Moreover, it it is is needed needed for for the the activation activation of of UbUb by by its its cognate cognate E1, E1, UBA1 UBA1 [ 11[11].]. InIn the the case case of of UFM1, UFM1, the the hydrophobic hydrophobic patch patch is is partially partially conserved conserved withwith I50 I50 and and I77 I77 superimposed superimposed byby I44I44 andand V70V70 ofof Ub, Ub, respectively. respectively. Accordingly,Accordingly, these these residues residuesare are inin the the interface interface of of UFM1 UFM1 that that interacts interacts with with the the adenylation adenylation domaindomain ofof thethe UFM1UFM1 activatingactivating enzyme,enzyme, UBA5UBA5 [12[12].]. However,However, UFM1UFM10s′s electrostaticelectrostatic surfacesurface didiffersffers fromfrom UbUb (Figure(Figure1 ).1). Consequently, Consequently, UFM1 UFM1 hashas a a pI pI of of 9.6 9.6 that that is is significantly significantly higher higher than than that that of of Ub Ub (6.56), (6.56), suggesting suggesting that that at at physiological physiological pH pH UFM1,UFM1, in in contrast contrast to to Ub, Ub, is is positively positively charged. charged. FigureFigure 1. 1.Comparison Comparison betweenbetween electrostaticelectrostatic potentialpotential surfacessurfaces ofof ubiquitin-foldubiquitin-fold modifiermodifier 11 (UFM1)(UFM1) (PDB(PDB id id 5IA7) 5IA7) and and ubiquitin ubiquitin (Ub) (PDB(Ub) id(PDB 1UBQ). id 1UBQ Upper). panel: Upper cartoon panel: representation cartoon representation of the orientation of the oforientation the indicated of protein;the indicated lower panel: protein; the correspondinglower panel: electrostaticthe corresponding surface. elec Thetrostatic electrostatic surface. potential The surfaceelectrostatic was calculated potential usingsurface UCSF was ca Chimeralculated [13 using]. UCSF Chimera [13]. UbUb does does not not only only modifymodify targettarget proteinsproteins withwith thethe additionaddition ofof aa singlesingle UbUb (mono-ubiquitination),(mono-ubiquitination), butbut can can also also add add Ub Ub chains chains that that are are linked linked via via ubiquitin’s ubiquitin’s own own lysines. lysines. Indeed, Indeed, over over the the last last few few years years itit has has been been shown shown that that Ub Ub generates generates not not only only di difffferenterent types types of of homotypic homotypic chains chains but but also also mixed mixed Ub Ub chainschains comprising comprising di differentfferent linkages linkages as as well well as as branched branchedchains chains [14[14].]. However,However, inin thethe casecase ofof UFM1,UFM1, whichwhich has has six six lysines lysines at at positions positions 3, 3, 7, 7, 19, 19, 34, 34, 41, 41, and and 69, 69, to to date date only only K69-linked K69-linked UFM1 UFM1 chains chains have have beenbeen reported reported [ 15[15].]. Therefore,Therefore, whether whether UFM1 UFM1 generates generates other other types types of of chains chains is is not not yet yet clear. clear. TheThe UFM1UFM1 sequencesequence isis highly highly conserved conserved withwith 80.6%80.6%sequence sequenceidentity identityand and88.2% 88.2%of of similarity similarity betweenbetween human human and andCaenorhabditis Caenorhabditis elegans elegans.. In In humans, humans, UFM1 UFM1 is is encoded encoded by by a a single single gene geneUFM1 UFM1that that undergoesundergoes alternative alternative splicing. splicing. Interestingly,Interestingly, while while the the canonical canonical isoform isoform of of UFM1 UFM1 encodes encodes a a protein protein comprisingcomprising 8585 aminoamino acids,acids, thethe mRNAmRNA isis 31683168 bpbp withwith aa veryvery longlong 3-UTR3-UTR ofof moremore thanthan 28002800 bpbp [[3].3]. InIn contrast, contrast, the the other other isoform isoform is is 846 846 bp bp and and generatesgenerates aa proteinprotein withwith 103103 aminoamino acidsacids [[16].16]. Currently,Currently, littlelittle is is known known about about the the physiological physiological roles roles of of UFM1 UFM1 alternative alternative splicing. splicing. Biomolecules 2020, 10, 1442 3 of 14 Biomolecules 2020, 10, x 3 of 14 3. UFM1 Conjugation 3. UFM1 Conjugation UFM1 is conjugated to its target proteins via a three-enzyme cascade involving E1, E2, and E3 UFM1 is conjugated to its target proteins via a three-enzyme cascade involving E1, E2, and E3 enzymes. Similar to other UBLs and in contrast to Ub, which has tens of E2s and hundreds of E3s, enzymes. Similar to other UBLs and in contrast to Ub, which has tens of E2s and hundreds of E3s, UFM1 has only one E2 and one E3 [1]. First, the non-canonical E1, UBA5, activates the C-terminus of UFM1 has only one E2 and one E3 [1]. First, the non-canonical E1, UBA5, activates the C-terminus of UFM1 through consecutive adenylation and thioesterification reactions. UBA5 then transfers UFM1 UFM1 through consecutive adenylation and thioesterification reactions.
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