Cover Page The handle http://hdl.handle.net/1887/90130

Cover Page

The handle http://hdl.handle.net/1887/90130 holds various files of this Leiden University dissertation.

Author: Witting, K.F.

Title: Exploring the Ub/UBL landscape with activity-based probes

Issue Date: 2020-05-20

Chapter 5

Generation of the UFM1 toolkit for profiling UFM1-specific

proteases and ligases

Katharina F. Witting*, Gerbrand J. van der Heden van Noort*, Christian Kofoed, Cami Talavera Ormeño, Dris el; Atmioui, Monique P.C. Mulder, and Huib Ovaa. Angew.Chem. Int. Ed. Engl.

2018 57(43): 14164-14168.

* These authors contributed equally to this work

5

Abstract

Ubiquitin-fold modifier 1 (UFM1) is a reversible post-translational modifier that is covalently attached to target proteins through an enzymatic cascade, and removed by designated proteases. Abnormalities in this process, referred to as Ufmylation, have been found to associate with a variety of human diseases. Given this, the UFM1 specific enzymes represent potential therapeutic targets, although understanding of their biological function has been hampered by the lack of chemical tools for activity profiling. To address this unmet need, we developed a diversifiable platform for UFM1 activity-based probes (ABPs) utilizing a native chemical ligation (NCL) strategy, enabling the generation of a variety of tools to profile both UFM1 conjugating and deconjugating enzymes. We demonstrate that they can be utilized both in vitro and in vivo for monitoring UFM1 enzyme reactivity opening up new research avenues.

Introduction

Post-translational modification (PTM) of proteins by chemical groups, peptides, complex molecules or even small proteins facilitates dynamic protein diversification to modulate cellular responses. Ubiquitination is one of the most common PTMs and a number of Ub-like proteins (Ubls) have been subsequently identified. Ubiquitin-fold modifier 1 (UFM1) is one of the recently identified Ubls and displays a similar tertiary structure yet has little sequence identity to Ubiquitin (Ub)

. Analogous to Ubiquitin, it is covalently attached to the lysine residues of its substrates by the sequential action of three dedicated enzymes—E1 (UBA5), E2 (Ufc1), and E3 (Ufl1) and is cleaved by UFM1 specific proteases (Ufsps)

. This process, referred to as Ufmylation, is initiated by the adenylation of the exposed C-terminal glycine of mature UFM1 and subsequent nucleophilic reaction with the active-site cysteine of UBA5.

The resulting high-energy thioester bond allows the transfer onto the catalytic site cysteine

of the E2 enzyme Ufc1, in a trans-thioesterification reaction. Lastly, the E3-like enzyme Ufl1

mediates the transfer of activated UFM1 onto the lysine residues of the protein substrates

resulting in the formation of an isopeptide linkage. In addition to releasing UFM1 from its

substrates, the UFM1 specific proteases—Ufsp1 and Ufsp2—mediate the maturation of

pro-UFM1

. Although Ufmylation has been connected to biological processes including ER

homeostasis

, vesicle trafficking

, blood progenitor development and differentiation

,

G-coupled protein receptor (GPCR) maturation[9], transcriptional control

, mitosis

, as

well as autophagy

, the underlying mechanisms remain to be studied. Furthermore,

abnormalities in the UFM1 cascade are reported to be associated with a number of human

diseases, including cancer

, diabetes

, schizophrenia

, and ischemic heart disease

and

to play a pivotal role in embryonic development and hematopoiesis

. Notwithstanding

the biochemical and structural studies of UFM1 conjugating and deconjugating enzymes that

have been undertaken

, their biological function remains enigmatic primarily due to the

lack of activity-based reagents. By contrast, diverse reagents and ABPs have been developed

for both Ub-conjugating and deconjugating enzymes

and have been expanded to Ubls

such as SUMO

and Nedd8

. This advancement of assay and activity-based reagents

has greatly propelled discoveries in the Ubiquitin field, yet such a diversifiable synthetic

platform for UFM1 needs to be developed

. While UFM1 has been prepared before using

multiple segment ligations based on KAHA chemistry, it is time consuming requiring the

incorporation of a (S)-5-oxaproline building block at multiple sites

. We first attempted

to generate UFM1 using linear solid phase peptide synthesis (SPPS) by incorporating

aggregation breakers such as pseudoproline

at permissible sites (Figure S1). Although

this linear synthesis approach yields full length UFM1, which has been utilized in a recent

study

, the synthesis wasn’t efficient, presumably due to inefficient coupling of amino acid

36 onwards (see Figure S2). To circumvent this issue, we here present a more practical two

segment native chemical ligation (NCL) approach

towards full-length UFM1 and UFM1

activity-based reagents (Figure 1). Given the increasing knowledge on the importance of

5

UFM1, our synthetic strategy gives access to valuable tools that allow the in vitro and in vivo characterization of enzymatic activity, thereby enabling insights into the dynamics of UFMylation.

Tag

O H N

Specificity Trap

H N

O OH

Ligase activity-based probe UFM1-Dha

Protease activity-based probe UFM1-PA

S

Figure 1| Schematic illustration of the UFM1 toolbox featuring activity-based probes to study the preference and selectivity of both proteases and ligases by covalently capturing active enzymes.

To improve the UFM1 synthesis, we devised a practical native chemical ligation (NCL) strategy to generate full length UFM1 based on a N-terminal peptide thioester fragment (AA 1-44) and C-terminal peptide fragment (AA 45-83) with alanine at position 45 replaced by a cysteine (Scheme 1). This methodology permitted the generation of full length UFM1 and a complete repertoire of probes in a productive manner using a minimal amount of building blocks (Scheme 1). Using SPPS and standard coupling conditions (namely 4 equiv.

Fmoc-protected amino acid, 4 equiv. PyBOP, 8 equiv. DIPEA, and double coupling cycles), we prepared the N-terminal fragment (AA 1-44) on a hyper-acid labile trityl resin to give amine (1) or functionalized its N-termini with a Rhodamine 110 fluorophore (2)

. Then, selective cleavage from the resin with 20% hexafluoro-2-propanol (HFIP) in CH

Cl

liberated the C-terminal carboxylate leaving all other protective groups in place. The C-terminal carboxylate was then converted into the mercaptomethylpropionate thioester by reaction with mercaptomethylpropionate, followed by global deprotection and HPLC purification.

Subsequently, the C-terminal fragment (A45C-83, 3) was obtained by Fmoc-based SPPS, followed by global deprotection and HPLC purification. Native chemical ligation of the

N-terminal thioester-fragment (1 or 2) and C-terminal cysteine-fragment (3) was performed under denaturing conditions in 8M Gdn·HCl containing 100 mM tris(2-carboxyethyl) phosphine (TCEP) and 100 mM mercapto-phenylacetic acid (MPAA) at pH 7.6 at 37°C.

Conversion to the full length UFM1 product (6 or 7) proceeded very efficiently in just 30 minutes. Precipitation from water and subsequent re-dissolving in Gdn·HCl followed by radical desulfurization using VA-044, glutathione and TCEP for 16 hours at 37°C resulted in formation of the full length native UFM1 as the cysteine residue at the ligation site was converted into the native alanine. Subsequent HPLC purification followed by gel-filtration yielded UFM1 and Rho-UFM1 in 85% and 79% yield, respectively.

Correct folding of purified synthetic UFM1 (Figure 2A) was verified by circular dichroism (CD) spectroscopy (Figure 2B). To further verify the correct folding and thus biochemical function we compared synthetic and recombinant UFM1 in an enzymatic reaction with UBA5 (E1).

Efficient formation of the UBA5~UFM1 thioester proved that synthetic UFM1 is processed with the same efficiency as recombinant UFM1 (Figure 2C, Figure S3).

Scheme 1. Native Chemical Ligation strategy towards native UFM1 and UFM1 activity-based probes.

5

200 220 240 260

-20 -10 0 10 20

Wavelength (nm)

(mDeg)

Expressed Synth. FL Synth. NCL

CD

B.

C.

A.

10

ExpressedSynth. FLSynth. NCL

kDa

15 25 35 55 70 100

UBA5UBA5~UFM1 62

72

kDa * * *

UFM1 10

ExpressedSynth. FLSynth. NCL

Figure 2 | Validation of synthetic UFM1. A) SDS-PAGE gel showing synthetic and expressed UFM1.

B) CD-measurements of expressed and synthetic UFM1 made by linear synthesis or native chemical ligation. Synth. FL. = UFM1 generated by linear synthesis; Synth. NCL = UFM1 generated by NCL (6).

C) UBA5 reacts with both expressed and synthetic UFM1. The asterisk (*) indicates UFM1-thioester UBA5.

Having developed a convenient UFM1 synthesis, we then focused on the generation of a UFM1-based toolbox to enable the profiling and visualization of all enzymes involved in the Ufmylation cascade (Figure 1, Scheme 1). For this purpose, we synthesized UFM1-Dha targeting the conjugating class of enzymes

and UFM1-PA to target the proteases

. To achieve this, we synthesized a C-terminal fragment lacking the last amino acid (A45C-82).

Upon cleavage from the resin with HFIP, the C-termini were equipped with either Cys(Bn)- OMe (4) or propargylamide (5). After global deprotection and HPLC purification, the desired C-terminal fragments were obtained and subsequently used in a NCL reaction as described above. ABP precursors for the UFM1 ligase probe (8) was successfully constructed by NCL followed by a radical desulfurization reaction to obtain UFM1-Cys(Bn)-OMe. This was subsequently transformed by oxidative elimination with O-mesitylenesulfonylhydroxyl- amine (MSH). Finally the methyl ester was hydrolysed, to generate the UFM1-Dha probe (8).

ABPs targeting UFM1 specific cysteine proteases (9-10) were easily constructed by native

chemical ligation as well. However, one limitation we encountered was that the remaining cysteine after NCL could not be desulphurized, as radical desulfurization conditions compromise the integrity of the propargyl warhead. But since the ligation position (AA 45) is situated outside the critical C-terminal region for UFM1 recognition by Ufsp enzymes

, we expect this small thiol group to be tolerated.

Furthermore, the routine addition of 1,4-dithiothreitol (DTT) to the enzymatic assay reaction buffers reduces any potential disulfide bridges occurring at this cysteine that might affect enzymatic processing. With these ABPs in hand, we tested their reactivity towards UFM1 ligases and proteases in cell lysates. Firstly, we assessed the reactivity of the E1 enzyme–

UBA5—towards Rho-UFM1-Dha (8). Similar to UbDha, UFM1-Dha forms an adenylate with the C-terminal glycine of UFM1. The activated methylene group of this adenylate intermediate can then undergo a nucleophilic attack of the active-site cysteine to yield either the covalent UBA5-UFM1-thioether adduct or the UBA5-UFM1~thioester

(Figure 3A). Upon reacting UBA5 with the ligase probe Rho-UFM1-Dha (8), a complex is formed that remains stable in the presence of DTT, indicative of the thioether bond formed between the active site cysteine and the dehydroalanine moiety of (8) in contrast to the thioester bond formed between UBA5 and UFM1 (7) (Figure 3B and Figure S3).

UFM1-Dha -S A.

Uba5

B.

NH S O

Uba5

OH O

S

Uba5

thioester thioether b)

a)

b) a)

6272 kDa

UBA5UBA5~UFM1 UFM1

UFM1-Dha DTT

+ +

+ + ++ -

- - - - - - --

*

*

*

UFM1 10

HN O

OH

Figure 3 | Reactivity of E1 (UBA5) towards UFM1. A) Scheme depicting the reactivity of UBA5 towards

UFM1-Dha permitting the formation of either the thioether (a) or the thioester adduct (b). B) SDS-

PAGE gel of reaction of synthetic UFM1 (6) or Rho-UFM1-Dha (8) and recombinant UBA5.

5

Furthermore, UFM1-Dha does not display cross-reactivity towards UBE1, the Ub-activating E1 enzyme, or towards the Ufm1-specific protease Ufsp1 underscoring the selectivity of this ABP (Figure S4 and S5). Next, we tested our UFM1-PA protease probe

. As expected, Rho- UFM1-PA (10) generated by NCL (containing Cys-45) showed comparable reactivity towards Ufsp1 as the Rho-UFM1-PA probe generated by linear SPPS (containing the native Ala-45), indicating that the cysteine at this position does not interfere with protease recognition, which is in agreement with literature

(Figure S6). Given that human Ufsp1 is thought to be catalytically inactive (Figure S7), we assessed the reactivity of the active murine Ufsp1 instead.

To this end, HEK293 cells transiently overexpressing Flag-tagged murine Ufsp1 or untransfected cells were lysed and incubated with Rho-UFM1-PA at 370C (Figure 4A).

Visualization by in-gel fluorescence scanning followed by immunoblotting revealed that Ufsp1 engages more readily than Ufsp2 with this ABP

(Figure 4B and Figure S8).

Immunoblotting reveals differences in UFM1 processing by these proteases. While murine Ufsp1 completely reacts with UFM1-PA after 45 min, endogenous Ufsp2 engages with UFM1- PA after prolonged incubation of nearly 6 hours (Figure 4A and B). The two UFM1 specific proteases known to date, are cysteine-proteases exhibiting a conserved papain-like fold with the classical catalytic Cys-His-Asp triad but share no sequence homology with DUBS or Ub-like protein specific proteases (ULPs) but rather constitute a new cysteine protease subfamily

. To assess whether deUbiquitinating enzymes (DUBs) and SUMO-specific proteases (SENPs) display cross reactivity towards UFM1-PA, a panel of representative deUbiquitinating and deSUMOylating enzymes were profiled. As expected, only UFM-1 specific proteases recognize and bind specifically to UFM1-PA (Figure 4 C-F and Figure S8).

Subsequently, we addressed the efficacy of UFM1-PA by monitoring UFM1 protease activity in a cellular environment, by electroporation of Rho-UFM1-PA (10) into HeLa cells ectopically expressing Flag-Ufsp1 or its catalytically inactive C53A mutant as previously described

. In-gel fluorescence analysis followed by immunoblotting revealed labeling of endogenous Ufsp2 in cells with Rho-UFM1-PA, while in Ufsp2 depleted HeLa cells reactivity was abolished (see Figure 5A, Figure S9 and Figure S10). As expected, only active Flag-Ufsp1 reacted with UFM1-PA but not it’s catalytically inactive version. Having demonstrated that our UFM1 ABP probe can be used to monitor enzymatic reactivity in living cells, we used the fluorescent ABP to visualize Ufsp reactivity by confocal microscopy. After introduction of Rho-UFM1-PA by electroporation into either unmodified or HeLa cells ectopically transfected with murine Flag-Ufsp1, probe distribution was observed both throughout the cytoplasm and the nucleus (Figure 5B). However, incorporation of Rho-UFM1-PA (10) into HeLa cells ectopically expressing murine Flag-Ufsp1 or its catalytically inactive C53A mutant, showed substantial co-localization with wild-type, but not with the catalytically incompetent enzyme (Figure 5C).

A.

Flag-Ufsp1

IB: α-Flag

Ufsp1-UFM1 Ufsp1

Flag only0’ 0.5’ 1’ 5’ 15’ 30’ 45’ 60’ 90’120’ 180’

24 34 kDa

*

B.

C.

IB: α-Flag

UCHL1 Flag-UCHL1

Rho-UFM-PA Cy5-Ub-PA

+ + +

+ +- - - -

UCHL1

*

25

+ + +

+ +- - -

-

kDa

-

WT CA

UCHL1-Ub

D.

GFP-OTUB2 Rho-UFM-PA Cy5-Ub-PA

+ + + +

+- - - -

OTUB2 + + +

+ +- - - -

IB: α-GFP

OTUB2 54

kDa *

WT CA

OTUB2-Ub

E.

F.

IB: α-SENP6 126 *

Rho-UFM-PA Rho-Ub-PA

Rho-S2-PA - + -

+ +

- -

- - -

SENP6

kDa SENP6-S2

SENP6 - 0’ 15‘ 30’ 60’ 90’ 120’ 180’ 240’ 360’ -

Ufsp2 Ufsp2-UFM1

IB: α-Ufsp2 52

kDa62

* Ufsp2

CA mutant

Ufsp2 KO

Rho-UFM-PA Rho-Ub-PA Rho-S2-PA

+

- +- -

+ - -

- - -

IB: α-SENP1

74 *

SENP1

kDa SENP1-S2

SENP1 -

Figure 4 | Assessment of UFM1-PA reactivity and specificity against a panel of cysteine protease subfamilies. A) Immunoblot showing time-dependent reactivity of Flag-Ufsp1 (murine) towards Rho- UFM1-PA (10), B) and time-dependent labeling of endogenous Ufsp2 (human). C and F) DUBs Flag- UCHL-1 and GFP-OTUB2 don’t react with UFM1-PA. D-E) Flag-SENP6 and -SENP1 react with S2-PA (=

SUMO2-PA) but are unreactive towards UFM1-PA. For fluorescence scans and actin blots see Figure S8.

5

A.

C.

B.

Rho-Ufm-PA

HeLa cells

Rho-Ufm-PA

Rho

Flag Rhodamine

zoom

Overlay Flag DAPI Rho

Flag-Ufsp1Flag-Ufsp1(CA)

zoom

zoom Zoom

α-Flag

*

Flag-Ufsp1 Flag-Ufsp1-Ufm1PA Flag-Ufsp1 Flag-Ufsp1 CA

α-actin α-Ufsp2

untransfected Ufsp2 KO

*

* * *

Ufsp2 Ufsp2-Ufm1PA

42 53 25

35 *

kDa

63

Figure 5 | Reactivity of Hela cells electroporated with Rho-UFM1-PA in the absence or presence of ectopically overexpressed murine Flag-Ufsp1. A) Immunoblots of Flag-Ufsp1 and Flag-Ufsp1(C53A) and endogenous Ufsp2 in untransfected cells following electroporation. B) Confocal images of untrans- fected HeLa cells or C) in the presence of Flag-Ufsp1 or Flag-Ufsp1 (C53A) after probe electroporation.

Cell boundaries and nuclei are indicated by a dashed line, and insets correspond to zoom-ins. Scale bars: 10µm. Quantification of colocalization (Mander’s overlap coefficient) is shown in Figure S11.

In conclusion, we present a practical native chemical ligation based synthetic approach for generating UFM1 activity based probes. Using this facile strategy, a wide variety of UFM1 ABPs equipped with fluorescent tags, as well as diverse reactive groups targeting proteases or ligases are accessible. The strategies presented here highlight a variety of assays and possibilities to utilize our ABPs in the interrogation of enzymatic activities in the UFMylation cascade. The straightforward nature of the experimental setup is expected to make them readily adoptable to comparative profiling in the presence of various perturbations, facilitating the understanding of UFM1 biology and additionally potentiate the discovery of specific UFM1 enzyme inhibitors. Based on the studies described here, we anticipate that our toolset will greatly facilitate ongoing studies on the UFM1 enzyme cascade.

Acknowledgements

We would like to thank Dr. Aimee Boyle at Leiden University for assistance with the CD measurements. This work was supported by a VICI grant to H.O. from the Netherlands Organization for Scientific Research (N.W.O.).

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Supplementary Information Chemical synthesis

Solid Phase Peptide Synthesis (SPPS)

Linear Synthesis Strategy

Solid Phase Peptide Synthesis (SPPS) was performed using standard 9-fluorenylmethoxycarbonyl (Fmoc)-based solid phase chemistry on a 25 µmol scale on an Syro II (Multisyntech) peptide synthesizer. Synthesis was initiated on an Fmoc-Val- TentagGel R TRT resin (Rapp Polymere, Cat# RA1201). Fmoc-protected amino acids were coupled using four-fold excess relative to the pre-loaded Fmoc amino acid trityl resin (0.2 mmol/g) using double couplings in N-Methyl-2-pyrrolidone (NMP) for 40 min followed by a second coupling of 60 min using PyBOP (4 eq) and DiPEA (8 eq) at room temperature. After coupling each amino acid, Fmoc deprotection was performed in 20% piperidine in NMP for 3x4/15 min in cycle 1 and 2x10/10 min for the rest of the cycles at RT. To facilitate synthesis, dipeptides were incorporated wherever possible: Fmoc-Leu-Thr(Novabiochem/Merck Millipore; Fmoc-Ile-Thr(psiMe,Mepro)-OH; Cat#8521930025), Fmoc-Gly-Ser (Novabiochem / Merck Millipore; Fmoc-Gly-Ser-(psiMe, Mepro)-OH; Cat#: 8.52200.0005), Fmoc-Gln-Thr (Novabiochem /Merck Millipore; Fmoc-Leu-Ser(psiMe,Mepro)-OH; Cat#8.52179.0005 CAS 339531-50-9), and Fmoc-Glu-Gly Novabiochem / Merck Millipore; Fmoc-Asp(OtBu)-(Dmb) Gly-OH; Cat#8521150005 CAS 900152-72-9). In the case of the UFM1-PA probe, Gly83 was omitted to allow coupling of propargylamine, which was performed as described previously[2].

Native Chemical Ligation Strategy

Solid Phase Peptide Synthesis (SPPS) was performed using standard 9-fluorenylmethoxycarbonyl (Fmoc)-based solid phase chemistry on a 25µmol scale on an Intavis MultipPep CF peptide synthesizer. For the synthesis of the N-terminal UFM1 fragment (UFM11-44), synthesis was initiated on an Fmoc-Ala-Tentagel R TRT resin (Rapp Polymere, Cat# RA1201). Fmoc-protected amino acids were coupled using four-fold excess relative to the pre-loaded Fmoc amino acid trityl resin (0.2 mmol/g) using couplings in N-Methyl- 2-pyrrolidone (NMP) for 40 min followed by a second coupling of 60 min using four-fold excess of Fmoc protected amino acid, PyBOP (4 eq) and DiPEA (8 eq) at room temperature.

After coupling each amino acid, Fmoc deprotection was performed in 20% piperidine in NMP for 3x4/15 min in cycle 1 and 2x10/10 min for the rest of the cycles at RT. To facilitate synthesis, Leu 10 and Thr 11 were coupled as a dipeptide LT (Novabiochem/Merck Millipore;

Fmoc-Ile-Thr(psiMe,Mepro)-OH; Cat#8521930025). Fmoc-L-norleucine (CHEM IMPEX INT’L,

Cat#02440; CAS 77284-32-3) was incorporated at the N-terminus to circumvent methionine

oxidation during downstream chemistry. For the C-terminal fragment of UFM1 (UFM145-

5

82), synthesis was initiated on an Fmoc-Val-Tentagel TRT resin (Rapp Polymere, Cat#RA1227) followed by double couplings and Fmoc-deprotection as described above. Dipeptides were incorporated wherever possible: Fmoc-Gly-Ser (Novabiochem/Merck Millipore; Fmoc-Gly- Ser-(psiMe,Mepro)-OH; Cat#: 8.52200.0005), Gln-Thr (Novabiochem/Merck Millipore;

Fmoc-Leu-Ser(psiMe,Mepro)-OH; Cat#8.52179.0005 CAS 339531-50-9), and Fmoc-Glu- Gly Novabiochem /Merck Millipore; Fmoc-Asp(OtBu)-(Dmb)Gly-OH; Cat#8521150005 CAS 900152-72-9). To permit native chemical ligation, Ala45 in the wild type sequence was replaced by cysteine. For the UFM1-PA and UFM1-Dha activity-based probes, Gly83 was omitted to allow coupling of propargylamine, or a S-benzyl-L-cysteine methyl ester (Cys(Bn)- OMe) in the case of UFM1-Dha.

Native Chemical Ligation reactions:

UFM1-thioester peptide (1) (1.3 mg) and cysteinyl-UFM1 peptide (3) (1.5 mg) were dissolved in 100 µL Gdn•HCl buffer (8M) and 10 µL MPAA (1M) and 10 µL TCEP (1M) were added after which the pH was adjusted to 7.6. The reaction was agitated at 370C for 30 min after which LC-MS analysis showed complete consumption of the N-terminal thioester and formation of the NCL-product.

Desulphurisation reactions:

The reaction was diluted to 10 mL in water and spun down using an Amicon spinfilter (MWCO 10 kD) to 1 mL. This procedure was repeated two times after which the remaining suspension was taken up in 4 mL (Gdn•HCl (8M)/ TCEP (1M), 4:1 v/v). Glutathione (32 mg/

mL) and VA044 (31 mg/mL) were added, the pH adjusted to 6.5 and desulphurization was accomplished by agitation overnight at 37 0C. HPLC purification followed by lyophilisation of the appropriate fractions, followed by SEC purification using 20 mM TRIS, 150 mM NaCl buffer at pH 7.6 yielded the final compounds.

RP-HPLC purifications

Shimadzu semi-preparative RP-HPLC system, equipped with a Waters C18-Xbridge 5 µm OBD (10 x 150 mm) column at a flowrate of 6.5 mL/min. using 2 mobile phases: A: MQ + 0.05% TFA, B: CH3CN + 0.05 % TFA. Gradient: 10 -> 70% B.

Gel filtration:

Size Exclusion Chromatography was performed on a Sephadex S75 10/300 column (GE Healthcare), using a 20 mM TRIS, 150 mM NaCl buffer at pH 7.6. Appropriate fractions were pooled and concentrated using an Amicon spinfilter (MWCO 10 kD) to a final concentration of ca. 1 mg/mL

LC-MS measurements:

Waters 2795 Separation Module (Alliance HT) using a Phenomenex Kinetex C18-column

(2.1x50, 2.6 μm), Waters 2996 Photodiode Array Detector (190-750 nm) and LCTTM ESI- Mass Spectrometer. Samples were run using 2 mobile phases: A = 1% CH3CN, 0.1% formic acid in water and B = 1% water and 0.1% formic acid in CH3CN. Flow rate= 0.8 mL/min, runtime= 6 min, column T= 40°C. Gradient: 0 - 95% B. Data processing was performed using Waters MassLynx Mass Spectrometry Software 4.1 (deconvolution with MaxEnt1 function).

HRMS-measurements:

High resolution mass spectra were recorded on a Waters Acquity H-class UPLC with XEVO-G2 XS Q-TOF mass spectrometer equipped with an electrospray ion source in positive mode (source voltage 3.0 kV, desolvation gas flow 900 L/hr, temperature 250°C) with resolution R = 22000 (mass range m/z = 50-2000) and 200 pg/uL Leu-Enk (m/z = 556.2771) as a lock mass.

Cysteinyl peptides:

Cys-UFM46-83 (3)

ESI MS+ (amu) calcd: 4105.70, found 4105.83, rt: 1.29 min. HRMS: [C177H295N55O55S + 3H]3+: found 1369.4028, calc. 1369.3995, [C177H295N55O55S + 4H]4+: found 1027.3020, calc. 1027.3016, [C177H295N55O55S + 5H]5+: found 822.0464, calc. 822.0428, [C177H295N55O55S + 6H]6+: found 685.2041, calc. 685.2037.

Cys-UFM46-82PA (5)

ESI MS+ (amu) calcd: 4085.71, found 4085.92, rt: 1.30 min. HRMS: [C177H295N55O55S + 3H]3+: found 1362.7411, calc. 1362.7363, [C177H295N55O55S + 4H]4+: found 1022.3049, calc. 1022.3042, [C177H295N55O55S + 5H]5+: found 818.0503, calc. 818.0449, [C177H295N55O55S + 6H]6+: found 681.8771, calc. 681.8721.

Thioester peptides:

UFM1-45S-methylpropionate (1)

ESI MS+ (amu) calcd: 4955.88, found 4956.18, rt: 1.47 min. HRMS: [C233H370N52O64S + 3H]3+: found 1652.9109, calc. 1652.9113, [C233H370N52O64S + 4H]4+: found 1239.6851, calc. 1239.6847, [C233H370N52O64S + 5H]5+: found 991.9526, calc. 991.9493, [C233H370N52O64S + 6H]6+: found 826.9634, calc. 826.9595, [C233H370N52O64S + 7H]7+:

found 708.9703, calc. 708.9664.

RHO-UFM1-45S-methylpropionate (2)

ESI MS+ (amu) calcd: 5312.22, found 5312.68, rt: 1.49 min. HRMS: [C254H382N54O68S + 3H]3+: found 1771.6080, calc. 1771.6045, [C254H382N54O68S + 4H]4+: found 1328.9521, calc. 1328.9553, [C254H382N54O68S + 5H]5+: found 1063.3638, calc. 1063.3658, [C254H382N54O68S + 6H]6+: found 886.3071, calc. 886.3062, C254H382N54O68S + 7H]7+:

found 759.8373, calc. 759.8350.

5

UFM1 derivatives:

UFM1 (6)

Isolated yield: 2.05 mg, 0.22 µmol (85.7%). ESI MS+ (amu) calcd: 8909.37, found 8911.00, rt: 1.43 min. HRMS: [C406H657N107O117 + 5H]

: found 1782.7858, calc. 1782.7856, [C406H657N107O117 + 6H]

: found 1485.8250, calc. 1485.8226, [C406H657N107O117 + 7H]

: found 1273.7136, calc. 1273.7063, [C406H657N107O117 + 8H]

: found 1114.6235, calc. 1114.6190, [C406H657N107O117 + 9H]

: found 990.8897, calc. 990.8844, [C406H- 657N107O117 + 10H]

: found 891.9011, calc. 891.8967, [C406H657N107O117 + 11H]

: found 810.9100, calc. 910.9069, [C406H657N107O117 + 12H]

: found 743.4201, calc.

743.4153, [C406H657N107O117 + 13H]

: found 686.3077, calc. 686.3070.

RHO[110]-UFM1 (7)

Isolated yield: 2.06 mg, 0.22 µmol (78.6%). ESI MS+ (amu) calcd: 9265.02, found 9266.00, rt: 1.43 min. HRMS: [C427H669N109O121 + 6H]

: found 1545.1616, calc. 1545.1693, [C427H669N109O121 + 7H]

: found 1324.5773, calc. 1324.5748, [C427H669N109O121 + 8H]

: found 1159.1305, calc. 1159.1289, [C427H669N109O121 + 9H]

: found 1030.4490, calc. 1030.4489, [C427H669N109O121 + 10H]

: found 927.5087, calc. 927.5047, [C427H- 669N109O121 + 11H]11+: found 843.2809, calc. 943.2777, [C427H669N109O121 + 12H]

: found 773.0917, calc. 773.0886, [C427H669N109O121 + 13H]

: found 713.6972, calc.

713.6978, [C427H669N109O121 + 14H]

: found 662.7930, calc. 662.7913.

UFM1-PA (9)

Isolated yield: 1.28 mg, 0.14 µmol (32.5%). ESI MS+ (amu) calcd: 8921.44, found 8921.00, rt: 1.44 min. HRMS: [C407H657N107O115S + 6H]

: found 1487.8208, calc. 1487.8197, [C407H657N107O115S + 7H]

: found 1275.4229, calc. 1275.4180, [C407H657N107O115S + 8H]

: found 1116.1199, calc. 1116.1167, [C407H657N107O115S + 9H]

: found 992.2168, calc. 992.2157, [C407H657N107O115S + 10H]

: found 893.0991, calc. 893.0949, [C407H- 657N107O115S + 11H]

: found 811.9999, calc. 811.9961, [C407H657N107O115S + 12H]

: found 744.4134, calc. 744.4138, [C407H657N107O115S + 13H]

: found 687.2245, calc.

687.2287.

RHO[110]-UFM1-PA (10)

Isolated yield: 3.04 mg, 0.33 µmol (76.7%). ESI MS+ (amu) calcd: 9277.78, found 9278.00, rt: 1.45 min. HRMS: [C428H669N109O119S + 5H]

: found 1856.3859, calc. 1856.3981, [C428H669N109O119S + 6H]

: found 1547.1647, calc. 1547.1663, [C428H669N109O119S + 7H]

: found 1326.2830, calc. 1326.2865, [C428H669N109O119S + 8H]

: found 1160.6285, calc. 1160.6267, [C428H669N109O119S + 9H]

: found 1031.7826, calc. 1031.7802, [C428H- 669N109O119S + 10H]

: found 928.7054, calc. 928.7029, [C428H669N109O119S + 11H]

: found 844.3685, calc. 844.3670, [C428H669N109O119S + 12H]

: found 774.0875, calc.

774.0871, [C428H669N109O119S + 13H]

: found 714.6212, calc. 714.6194.

CD-measurements

Validation of the structural integrity of our synthetic UFM1 was done using circular dichroism (CD). After dissolving synthetic UFM1 (40 mg/mL) in DMSO, it was diluted into ddH2O, vortexed briefly and added to 20 mM sodium phosphate buffer (pH 7.0) and buffer exchanged to 20 mM sodium phosphate buffer (pH 7.0) using a 3 kDa cut-off Amicon Ultra-15 Centrifugal Filter Unit (Millipore). In case of the expressed UFM1 and the UFM1 generated by native chemical ligation, proteins were directly buffer exchanged into 20mM sodium phosphate buffer (pH 7.0) using a 3 kDa cut-off Amicon Ultra-15 Centrifugal Filter Unit (Millipore). Final concentrations of synthetic and expressed UFM1 were determined by gel-quantification. Circular dichroism was measured using a JASCO J-815 CD Spectrometer at 25°C using samples diluted to approximately 10 µM final concentration. CD spectra were recorded ranging from 250 to 190 nm at a scan rate of 20 nm per minute and a scan width of 1 nm using a quartz cuvette with a 1 mm path length. Three cumulative measurements were averaged and plotted using Graphpad PRISM.

Protein expression and purification

UFM1 and UBA5 were expressed as N-terminal His-SUMO fusions in E. coli BL21(DE3) using autoinduction

. After reaching OD600 0.6, the temperature was lowered to 18°C and the bacteria were grown an additional 18h. Enzymes were purified using TALON beads (Clontech) equilibrated in Buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT), washed twice with Buffer A and eluted with 100mM imidazole (pH 7.5) For further purification, the enzymes were subjected to anion exchange (Resource Q, GE Healthcare) with a gradient of 1 M NaCl in Buffer A, followed by size exclusion chromatography (S75, 16/600, GE Healthcare). In the case of UFM1, the N-terminal His-SUMO tag was cleaved by incubation with 5 µM SENP2 for 1h at 4°C, followed by further purification using TALON beads (Clontech) and size exclusion chromatography using a Superdex S75 16/600 column (GE Healthcare). Pure proteins were concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at -80°C.

Constructs

Murine Ufsp1 was subcloned into a 2xFLAG-C1 vector (Clontech) at BglII/PstI restriction sites.

Similarly, human Ufsp2 amplified from cDNA was cloned into a GFP-C1 vector (Clontech) at EcoRI/XhoI sites. UFM1 and UBA5 were amplified from cDNA, and cloned into an N-terminal His

-SUMO LIC vector according to the standard protocol. The active-site cysteine (C53) in murine Ufsp1 was mutated to alanine according to the protocol of the Quik Change Site- directed Mutagenesis Kit (Invitrogen). All constructs were verified by sequencing. Human Ufsp1 was amplified from cDNA and cloned into a 2x-Flag-C1 vector (Clontech) using BglII/

PstI restriction sites.

Mammalian cell lines

HEK293T and HeLa cell lines used in this study originated from ATCC and were cultured

5

under standard conditions in DMEM (Gibco) supplemented with 10% FCS (Sigma-Aldrich) at 37°C with 5% CO

. CRISPR-mediated Ufsp2 depletion in Hela cells was performed by co- transfecting confluent HeLa with a vector harbouring the gRNA and the Cas9 and a construct conferring blasticidin resistance

. After blasticidin selection and clonal expansion, Ufsp2 depletion was verified immunoblotting against using anti-Ufsp2 antibody (1:1000 dilution, Abcam ab185965). Ufsp2 guide RNA (gRNA) was designed using the CRSIPR Design tool (http://crispr.mit.edu/), subcloned into a pX330-U6-Chimeric-BB-CBh-hSpCas9 vector (Addgene, plasmid # 42230)

. UFSP2 guide RNA designed by CRISPR Design tool (http://

crispr.mit.edu/) was subcloned into pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene

#42230, Cambridge, MA, USA), a human codon-optimized SpCas9 and chimeric guide RNA expression plasmid. All cell lines were routinely tested for mycoplasma contamination with consistently negative outcome.

Labeling of purified enzymes

Thioester-formation of His-SUMO-UBA5 and UFM1, Rho-UFM1 or UFM1-Dha were assessed by incubating 1µM of His-SUMO-UBA5 with 5µM UFM1 in labeling buffer (50 mM Bis- Tris, pH 6.5, 100 mM NaCl, 10 mM MgCl

, 0.1 mM DTT and 0.4 mM ATP) at 30°C for 30 min. Reactions were quenched by addition of 3x SDS-PAGE loading Dye without addition of reducing agents and subsequently resolved by standard SDS-PAGE gel electrophoresis.

Enzymes were visualized by in-gel fluorescence scanning (λem / λex = 480/530 nm) followed by Coomassie staining. Labeling of recombinant His

-UBE1 with Ub-Dha or UFM1-Dha was performed as described previously

.

Labeling of overexpressed enzymes in cell lysates

For overexpression of UFM1 specific proteases and DUBs, Flag-Ufsp1 and the GFP-tagged DUBs[2] were transfected into HEK293T cells using polyethylenimine (PEI, Polysciences, Inc.) according to the manufacturer’s recommendations. In case of the SENPs and the human Flag-Ufsp1, transfection of the DNA was performed using confluent HeLa cells using Effectene (Qiagen) according to the manufacturer’s instructions. After 24h, cells were scraped into ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5% NP-40, 2 mM DTT, and a Protease inhibitor tablet (Roche)), briefly sonicated on ice and clarified by centrifugation.

For all labeling reactions, 25 µL of lysate was incubated with 0.5 µg of Rho-UFM-PA at 37°C for the indicated time. Reactions were terminated by the addition of 3x SDS-PAGE loading buffer supplemented with 2-β-mercaptoethanol and boiled for 10 min at 95°C. Samples were resolved using standard SDS-PAGE gel and visualized by both fluorescence scanning (λem / λex = 480/530 nm) and by immunoblotting. Following gel transfer onto nitrocellulose membranes, immunoblotting was performed using mouse anti-Flag (1:1000 dilution, Sigma) and mouse anti-β-actin (1:10000 dilution, Sigma) and probed using fluorescent secondary antibodies anti-mouse 800 (1:10000 dilution, LiCOR, 926-3210) and anti-rabbit-800 (1:10000, LiCOR, 926-3211) and visualized on the LICOR Odyssey system v3.0.

Transfection of the UFM1 conjugating enzyme GFP-UBA5 was performed in confluent HeLa cells using Effectene (Qiagen) according to the manufacturer’s instructions. After 24h, cells were scraped into Co-IP buffer (50 mM Tris-HCl, pH 7.5, 5% glycerol, 10 mM MgCl

, 0.5%

NP-40, 1 mM DTT and Complete protease inhibitors (Roche)), resuspended and lysed by sonication on ice. Following clarification by centrifugation at maximum speed (4°C, 20 min, 20.000g), 30 µL cell lysate was incubated with 1µg Rho-UFM1, 0.4 mM ATP for 30 min at 37°C. Reactions were quenched by addition of either 3x SDS-PAGE loading buffer with or without the addition of 100mM 2-β-mercaptoethanol. Enzyme reactivity was visualized by resolving the samples using a standard SDS-PAGE gel and subsequent fluorescence scanning (λem / λex = 480/530 nm). To visualize the proportion of labelled enzyme, proteins were transferred onto nitrocellulose and immunoblotted using rabbit anti-UBA5 (1:1.000 dilution, Abcam ab177478) or HRP-Flag (1:10.000 dilution, Sigma) and probe using either fluorescent secondary antibody anti-rabbit 800 (1:10.000 dilution, LiCOR, 926-3211) and visualized on the LiCOR Odyssey system or with Super Signal West Dura Extended Duration Signal Substrate (ECL, Thermo Fisher) with subsequent visualization using the Amersham Imager AI600.

Labeling of endogenous enzymes

Cell lysates were prepared by resuspending cell pellets in three pellet volumes of HR Buffer (50 mM TRIS-HCl, pH 7.4, 5 mM MgCl

, 250 mM sucrose, 1 mM DTT) and lysed by sonication on ice. After clarification by centrifugation (20,000 rpm, 4°C, 20 min), total protein concentration was determined using Nanodrop. For cell lysate experiments, 100 µg of lysate was incubated with 0.5 µg of Rho-UFM1-PA for the indicated time at 37°C.

Reactions were quenched by the addition of 3x SDS-PAGE loading buffer supplemented with 2-β-mercaptoethanol and boiled for 10 min at 95°C. Samples were resolved by SDS- PAGE gel and probe reactivity assessed by fluorescence scanning (λem / λex = 480/530 nm).

Visualization of endogenous Ufsp2 was performed by immunoblotting flowing gel transfer to nitrocellulose membrane and probing with rabbit anti-Ufsp2 antibody (1:1000 dilution, Abcam ab185965).

Labeling with Rho-UFM1 or UFM1-Dha was performed using 30 µl cell lysates prepared as

previously described and incubated with 1 ug Rho-UFM1 or Rho-UFM1-Dha in the presence

or absence of 0.4 mM ATP for 60 minutes at 37°C. In the case of the Rho-UFM1-Dha

labeling, 1mM ATP and 1mM MgCl

were added every 20 minutes to replenish consumed

ATP. As negative controls, cell lysates were depleted of ATP by pre-incubation with Apyrase

(Sigma Aldrich) at 37°C for 20 minutes prior to Rho-UFM1-Dha addition (ATP and MgCl

were omitted). After quenching the reactions using 3x SDS-PAGE sample buffer, the samples

were resolved using a standard SDS-PAGE gel and subsequently imaged using fluorescence

scanning (λem / λex = 480/530 nm). To determine the proportion of active enzyme, the gels

were transferred to nitrocellulose membranes and probed with rabbit anti-UBA5 antibody

(1:1000 dilution, Abcam ab177478) and fluorescent secondary antibodies anti-rabbit-800

5

(1:10000, LiCOR, 926-3211).

Electroporation experiments

For electroporation experiments, 80.000 HeLa cells were seeded into a 6-well plate and transfected with murine Flag-Ufsp1 and the corresponding catalytically inactive mutant using Effectene (Qiagen), according to the manufacturer’s instructions. To facilitate the incorporation of the probe, the growth medium was replaced 4-6 h after transfection and refreshed again 1-2 h prior to electroporation. Following removal of the growth medium, cells were kept on ice for the duration of the protocol. Cells were washed twice with cold electroporation buffer (2 mM HEPES, pH 7.4, 15 mM K

HPO

/KHPO

, 250 mM mannitol, 1 mM MgCl

). 1.5 mL of a solution of Rho-UFM1 (or Rho-UFM1-PA, Rho-UFM1-Dha) in electroporation buffer (0.4 mg/mL) was added to each of the wells and electroporation was performed on ice using a Biorad GenePulser Xcell with CE and PE module Pulse Generator equipped with a Petri Pulser electroporation applicator (BTX) using the following settings:

square wave, voltage = 75V, pulse length = 3ms, pulse interval = 1.5s, number of pulses

= 5, cuvette width = 2 mm. The electroporation applicator was turned 90 degrees, and electroporation was repeated once. The probe solution was replaced by cold electroporation buffer, and cells were allowed to recover on ice for 2 min. After electroporation, cells were washed twice with ice-cold PBS and allowed to recover for 120 min under standard growth conditions. For gel-based analysis, samples were lysed using reducing SDS-PAGE loading buffer followed by brief sonication and heating at 98°C for 10 min before being separated on SDS-PAGE gel followed by visualization by fluorescence scanning (λex / λem = 480/530 nm).

Subsequently, western-blotting was performed as previously described, and membranes were probed with mouse anti-Flag (1:1000 dilution, Sigma) and mouse anti-β-actin (1:10000 dilution, Sigma).

Confocal microscopy

For microscopy experiments, the samples were fixed in 4% formaldehyde (Merck) in PBS and mounted onto glass slides (Thermo Scientific) using Prolong Gold mounting medium with DAPI (Invitrogen). Images were collected on a Leica SP8 confocal microscope equipped with HyD detectors, using a 63x oil-immersion magnification lens in combination with 2-4x digital zoom. Image processing and fluorescence intensity analysis were performed using ImageJ software and expressed in the form of Mander’s overlap coefficients calculated using JaCoP.

A.

B.

Fmoc N H

N O

O COOH LT

Fmoc N H

N O

O COOH FT

Fmoc N H

N O

O COOH IT

Fmoc N H

N O

O COOH GS

UFM1

(83AA) MSKVSFKITL TSDPRLPYKV LSVPESTPFT AVLKFAAEEF KVPAATSAII TNDGIGINPA QTAGNVFLKH GSELRIIPRD RVG

Figure S1: A) Building blocks (dipeptides) used in synthesis of UFM1. B) Amino acid sequence of UFM1.

The position of the incorporated dipeptides in the sequence are indicated bold in the sequence.

Figure S2: A) UV- traces of the FMOC deprotection during linear SPPS synthesis of UFM1. Red arrow indicates major inefficient coupling-position and start of synthesis is indicated by a black arrow. To circumvent this issue, we synthesized two fragments: a N-terminal fragment (AA 1-44) and a C-terminal fragment (AA 45-83) and ligated them using native chemical ligation (NCL). (LC-MS analysis of UFM1- PA generated by linear synthesis is shown in Figure S10).

A.

++ +- --

GFP-UBA5~UFM1 UBA5~UFM1 Fluorescence scan

Rho-UFM1

**

B.

++ +- --

GFP-UBA5~UFM1

*

IB: α-actin

IB: α-UBA5 GFP-UBA5 42

7787 10

62 82

kDa kDa

GFP-UBA5 Rho-UFM1 DTT

GFP-UBA5 Rho-UFM1 DTT

Figure S3: UBA5~Rho-UFM1 thioester formation visualized by A) in-gel fluorescence scanning and B)

corresponding immunoblots against Uba5.

5

Ub-Dha UFM1-Dha

+ +

+ +

- -

- -

DTT

- -

- - + - +

UBE1 UBE1-UbDha

kDa

120 * *

Figure S4: UBE1 does not recognize UFM1-Dha. Coomassie-stained gel showing that UBE1 reacts with Ub-Dha but not with UFM1-Dha.

B) A)

IB: α-Flag

Ufsp1 Ufsp1-UFM

* *

23 33

IB: α-actin

42

long exposure Rho-UFM1-Dha UFM1-PA

+ + +

- -

-

Fluorescent scan

UFM1-Dha

10 kDa

33

*

23

33

*

Ufsp1 Ufsp1-UFM Ufsp1-UFM

Figure S5: Competition experiments reveal preference for UFM1-PA over UFM1-Dha. A) Fluorescence scan showing that only a minor portion of ectopically overexpressed Flag-Ufsp1 reacts with Rho- UFM1-Dha (8). B) Corresponding immunoblot against the Flag-tagged protease reveals that the enzyme prefers UFM1-PA (9) over Rho-UFM-Dha (8), as becomes clear after prolonged exposure of the immunoblot. Differences in reactivity of Ufsp1 towards UFM1-PA and UFM1-Dha may be due to the unique active-site configuration of this protease[1]. Asterisks (*) denote the labeled enzymes.

WT CA

Rho-UFM1-PA (FL) Rho-UFM1-PA (NCL)

A)

+

+ +

+ - - - - - - - -

kDa

IB: α-actin IB: α-Flag

* *

Ufsp1 Ufsp1-UFM

23 33

42

B)

Fluorescence scan

Rho-UFM1-PA

10

33

** * * Ufsp1-UFM

Figure S6: Both UFM1 PA generated by native chemical ligation (NCL) and by linear synthesis (FL) react with catalytically active murine Flag-Ufsp1. FL= UFM1 generated by linear synthesis; NCL = UFM1 generated by NCL (6).

Flag-Ufsp1 (human)

IB: α-Flag Ufm1-PA _{Flag only}

- + A.

15 kDa

IB: α-actin 42

25

Figure S7: Human Ufsp1 is unreactive towards UFM1-PA as confirmed by immunoblot.

5

A.

B.

C.

D.

E.

F.

Flag-Ufsp1

IB: α-Flag

Ufsp1-UFM1 Ufsp1

Flag only0’ 0.5’ 1’ 5’ 15’ 30’ 45’ 60’ 90’ 120’180’CA

24 34 kDa

*

IB: α-actin 42

IB: α-Flag

UCHL1 Flag-UCHL1

Rho-UFM-PA Cy5-Ub-PA

+ + + + +- - --

UCHL-1

* 25

+ + + + +- - --

kDa

-

WT CA

UCHL1-Ub

IB: α-actin 42

GFP-OTUB2 Rho-UFM-PA Cy5-Ub-PA

+ + + + +-

- --

OTUB2 + + +

+ +- - --

IB: α-GFP

OTUB2 54

kDa *

WT CA

OTUB2-Ub

IB: α-actin 42

IB: α-SENP6

* 126 Rho-UFM-PA Rho-Ub-PA

Rho-SUMO2-PA- + - + + - -

- - -

SENP6

kDa SENP6-SUMO

SENP6 - -

IB: α-actin 42

Rho-UFM-PA Rho-Ub-PA Rho-SUMO2-PA

+ -

- + -

+ - -

- - -

IB: α-SENP1

74 * _SENP1

kDa SENP1-SUMO

SENP1 -

IB: α-actin 42

0’ 15‘ 30’ 60’ 90’ 120’ 180’ 240’ 360’ KO

Ufsp2 Ufsp2-UFM1

IB: α-Ufsp2 52

62

kDa *

Ufsp2

IB: α-actin 42

G.

Fluorescence scan Flag only

Flag-Ufsp1

0’ 0.5’ 1’ 5’ 15’ 30’ 45’ 60’ 90’ 120’ 180’ CA

Ufsp1-UFM1

*

Rho-Ufm-PA

Figure S8: Assessment of UFM1-PA reactivity and specificity against a panel of cysteine protease subfamilies. A) Immunoblot showing time-dependent reactivity of Flag-Ufsp1 (murine) towards Rho- UFM1-PA (10) and time-dependent labeling of endogenous Ufsp2 (human). C-D) Flag-UCHL1 and GFP- OTUB2 don’t react with UFM1-PA. E-F) Flag-SENP6 and -SENP1 are unreactive towards UFM1-PA. G) Corresponding fluorescent scans for time-dependent Flag-Ufsp1 labeling with Rho-UFM1-PA.

A.

IB: α-actin IB: α-Ufsp2

Ufsp2

kDa 52

42

Ufsp2 KO WT

Figure S9: Validation of the Ufsp2 knockout cell line (Ufsp2 KO) assessed by immunoblotting against Ufsp2 confirming the CRISPR-CAS mediated knockout.

A.

IB: α-actin IB: α-Ufsp2

Ufsp2

kDa 52

42

Ufsp2 KO WT

Figure S10: In-gel Fluorescence scans visualizing labeling of murine Flag-Ufsp1 or inactive Flag-Ufsp1 (C53A) following electroporation with Rho-UFM1-PA into HeLa cells ectopically expressing murine Flag-Ufsp1 or Flag-Ufsp1(C53A). Additionally, labeling of endogenous Ufsp2 can be detected, while no reactivity is observed in the Ufsp2 KO cells. Probe-labeled enzymes are indicated with an asterisk (*).

A.

Rho

Flag Rhodamine

zoom

Overlay Flag DAPI Rho

Flag-Ufsp1Flag-Ufsp1(CA)

zoom

zoom Zoom

0.0 0.2 0.4 0.6 0.8

Colocalization of Rho-UFM1-PA

Overlap of Rho:Flag

P < 0.0002

***

WT CA

B.

Figure S11: Reactivity of HeLa cells electroporated with Rho-UFM1-PA in the absence or presence of

ectopically expressed murine Flag-Ufsp1. A) Confocal images of HeLa cells in the presence of Flag-

Ufsp1 or Flag-Ufsp1 (C53A) after probe electroporation. B) Quantification of colocalization (Mander’s

overlap coefficient) of Rho-UFM1-PA with Flag-Ufsp1 (WT) or the mutant Flag-Ufsp1(CA) (n= 2, error

bars correspond to SD, with significance (p) calculated using a two-sided t-test).

5

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