A family of unconventional deubiquitinases with modular chain specificity determinants

Abstract

Deubiquitinating enzymes (DUBs) regulate ubiquitin signaling by trimming ubiquitin chains or removing ubiquitin from modified substrates. Similar activities exist for ubiquitin-related modifiers, although the enzymes involved are usually not related. Here, we report human ZUFSP (also known as ZUP1 and C6orf113) and fission yeast Mug105 as founding members of a DUB family different from the six known DUB classes. The crystal structure of human ZUFSP in covalent complex with propargylated ubiquitin shows that the DUB family shares a fold with UFM1- and Atg8-specific proteases, but uses a different active site more similar to canonical DUB enzymes. ZUFSP family members differ widely in linkage specificity through differential use of modular ubiquitin-binding domains (UBDs). While the minimalistic Mug105 prefers K48 chains, ZUFSP uses multiple UBDs for its K63-specific endo-DUB activity. K63 specificity, localization, and protein interaction network suggest a role for ZUFSP in DNA damage response.

Fig. 1

ZUFSP and Mug105 are related to UFM1/Atg8 proteases. a Structurally correct alignment of the catalytic domains of human ZUFSP (this work), mouse UFSP2 (3OQC) and human ATG4A (2P82). The S. pombe Mug105 sequence was added by sequence similarity to ZUFSP. Invariant and conservatively replaced residues are shown on black or gray background, respectively. Catalytic residues are highlighted in blue. b Conservation of the four UBZ-like zinc fingers of ZUFSP, in comparison to the structurally characterized UBZ finger (3WUP). c Conservation of the MIU domain of ZUFSP, in comparison to other human MIU domains

Fig. 2

ZUFSP and Mug105 are ubiquitin-specific proteases with different chain specificity. a Activity assays with Ub-/UbL-AMC substrates shown as released fluorescence (RFU) over time (min) with ZUFSP or Mug105. Shown RFU values are the means of triplicates. b Fluorescent scan of a suicide probe reaction of ZUFSP or Ufsp2 with Cy5-Ub-PA (arrow) and Rho-Ufm1-PA (arrowhead). Asterisks (*) mark the shifted band after reaction. c Suicide probe reaction of ZUFSP or ATG4B with Ub-PA and LC3B-PA. Asterisks (*) mark the shifted band after reaction. d, e Linkage specificity analysis with ZUFSP. A panel of di-ubiquitin (d) or tetra-ubiquitin (e) chains was treated with ZUFSP for the indicated time points. f Time course of cleavage of K63-linked Ub₆₊ chains by full-length ZUFSP. g Linkage specificity analysis with Mug105. A panel of Ub₂ was treated with Mug105 for the indicated time points. h Suicide probe reaction with Mug105 and K48-diUb-VME, K63-diUb-VME or Ub-PA. Arrows mark the shifted bands after reaction

Fig. 3

Crystal structure of ZUFSP^232-578 in covalent complex with Ub-PA. a Overview of the crystal structure in cartoon representation. The catalytic core of ZUFSP is shown in gray, ubiquitin in blue. The MIU region on helix α1 of ZUFSP is colored green, the zUBD region in cyan. The putative S1’ ubiquitin-binding α2/α3 helices are shown in red. The catalytic triad is shown as sticks and colored orange. b Structural superposition of the catalytic domain of ZUFSP (blue) and UFSP2 (3OQC, cyan) in two perspectives. RMS distance is 3.65 Å over 200 residues. c Magnification of the active site of ZUFSP (gray). The catalytic triad is colored orange, putative components of the oxyanion hole in green and Trp-423, closing the substrate binding groove directly next to the active site, is in dark pink. Ubiquitin is shown in blue color. The active sites of Ufsp2 (cyan) and papain (violet) are superimposed. Structurally equivalent residues of Ufsp2 and ZUFSP are shown as sticks. Important residues of ZUFSP (black), Ufsp2 (cyan) and papain (violet) are labeled. Important ubiquitin residue labels contain asterisks. In the available structure (3OQC) of UFSP2, the catalytic cysteine was mutated to a serine, indicated here as ‘S294

Fig. 4

ZUFSP UBDs contribute to chain cleavage and specificity. a Schematic representation of ZUFSP domain architecture. UBZ-like zinc fingers (Z), MIU domain (M), novel ZUFSP ubiquitin-binding domain (zUBD) and α2/α3 region are shown as boxes. b Activity of ZUFSP FL and truncations lacking the UBDs against K63-linked Ub₆₊ chains. Positions of the truncated ZUFSP proteins are indicated by arrows. c Comparison of Mug105 and ZUFSP^310-578 activity against ubiquitin-AMC. The shown RFU values are the mean of triplicates. d Chain specificity of ZUFSP catalytic core (ZUFSP^310-578) compared to full-length Mug105. Both DUBs were tested against K48- and K63-linked Ub₂ for the indicated time points. e Pull-down analysis of ZUFSP (full-length and two N-terminal truncations) against a mixture of K63-linked Ub₄ and Ub₅ chains

Fig. 5

Determinants of chain specificity. a Recognition of ubiquitin C terminus by the catalytic core of ZUFSP. Ubiquitin (blue) and ZUFSP (gray/green) shown in cartoon representation with key residues highlighted as sticks. Blue and black residue labels refer to ubiquitin and ZUFSP, respectively. Salt bridges are indicated by dotted lines. b Activity of C-terminus recognition mutants (ZUFSP^232–578 D406A or E428A) against K63-linked Ub₄ was compared to ZUFSP^232–578 and inactive ZUFSP (ZUFSP^232–578 C360A). c Activity of mutants described in b against RLRGG-AMC. The RFU values shown are the means of triplicates. d Structural superposition of ubiquitin-binding interfaces to zUBD (cyan, this work) and the MIU domain of Rabex-5 (2FIF, orange). Orientation of the two helical ubiquitin-binding domains differ by 20°. e SeqLogo representation of the consensus sequences for the MIU motif (top) and the zUBD derived from the ZUFSP family as shown in Supplementary Fig. 1 (bottom). f Magnification of the interaction interface between ubiquitin and zUBD. Relevant residues are shown as sticks and labeled black in case of ubiquitin and cyan in case of zUBD. Electrostatic interactions are indicated as dotted lines. g Activity of a MIU mutant (ZUFSP^148–578 L240A/Q241A) and two zUBD mutants (ZUFSP^148–578E256A and E259A) on K63-linked Ub₄, in comparison to wild-type ZUFSP^148-578. h Activity time course of the α2/3-deletion mutant ZUFSP^{148–578; Δ-α2/3} on K63-linked Ub₄ chains, compared to activity of the parental ZUFSP^148–578 construct

Fig. 6

ZUFSP localization and interaction network. a FLAG-tagged versions of inactive ZUFSP^C360A (left) and active ZUFSP (middle, right) were expressed in HEK293T cells and co-precipitating proteins quantified by mass spectrometry. Log2 enrichment ratios relative to uninduced/untransfected controls are plotted against log10 signal intensity. The right panel shows the results after nuclease treatment. Consistently enriched proteins are labeled in color, red for DNA-dependent and blue for DNA-independent enrichment. The bait ZUFSP is off-scale and hence not shown; the x/y coordinates are 9.7/11.1 (left), 11.0/11.2 (middle), and 10.5/11.6 (right panel). b FLAG-tagged ZUFSP was immunoprecipitated from HEK293T cells in the presence or absence of nuclease. Coimmunoprecipitated endogenous RPA32 was visualized with α-RPA32 4E4. c Electrophoretic mobility shift assay (EMSA) comparing the DNA-binding preferences of the full-length ZUFSP to the N-terminal truncations (ZUFSP^148–578 and ZUFSP^232–578). All constructs were tested against a panel of oligonucleotides previously tested for SSBP1 binding, including ssDNA, dsDNA, short hairpin (OriL), and long hairpin (OriL+6). d Localization of ZUFSP N-terminally fused to mVenus or C-terminal fused to eGFP (green) was visualized in fixed U2OS cells. Cells are shown in phase contrast (PhaCo) and nuclei are stained with DAPI (blue). Scale bar = 10 µm. e Localization of mVenus-tagged ZUFSP to sites of NIR laser-induced DNA damage in U2OS cells (top panels), as compared to eGFP alone (bottom panels). Images were taken immediately before (left) and 10 s after 800 nm laser irradiation (right). Scale bar = 10 µm