Fig 1.
SneRING is a predicted RING domain ligase.
(A) Multiple alignment of the RING domains of SneRING (SNE_A12920), some bacterial relatives (Sim-KFB93: Simkaniaceae bacterium RefSeq:QVL56959; Sim-QNJ27: Simkaniaceae bacterium RefSeq:MDJ0651792; Chl-HKST: Chlamydiia bacterium Uniprot: A0A960X5Q0), and sequences of the two best DALI hits (pdb:3LRQ (TRIM37 from Homo sapiens) and pdb:2CKL (RING2 from Mus musculus)). Residues invariant or conserved in at least 50% of the sequences are shown on black and grey background, respectively. Residues involved in the coordination of Zn1 and Zn2 are highlighted in red and magenta, respectively. (B) Alphafold model of SneRING shows the N-terminal RING-like domain (blue) and a C-terminal domain of unknown function (DUF, green). The Zn1 and Zn2 ions coordinated by the RING-like domain are shown in red and magenta, respectively. (C) Alphafold model of the RING domain of SneRING (blue) in contact with UbcH5b (grey). The Zn1 and Zn2 ions coordinated by the RING-like domain are shown in red and magenta, respectively. The predicted contact residues Glu-22 (E22) and Ser-19 (S19) and their interaction partners are labeled.
Fig 2.
SneRING shows ubiquitination activity in an in vitro assay.
(A) Purified recombinant SneRING was mixed with recombinant E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme (UbcH5b), ubiquitin, and ATP for the indicated periods. A reaction without the ligase served as a control. Samples were analyzed by SDS-PAGE and western blot, using an antibody against ubiquitin. (B) In vitro autoubiquitination reaction of wildtype SneRING and SneRING-ligase with mutated E2 enzyme interacting region (SneRING S19R_E22R) was performed for 5 h as in A and analyzed by SDS-PAGE and western blot, using anti-ubiquitin antibodies. (C) In vitro autoubiquitination reaction of wildtype SneRING was performed as in A in combination with indicated E2 enzymes for 5 h, followed by SDS-PAGE and western blot analysis using ubiquitin antibody. (D) SneRING was incubated with E2 enzyme UbcH5b or UBE2T in an in vitro autoubiquitination assay as described in A. Samples were taken at indicated time points and tested by immunoblot using the primary antibody against ubiquitin. Poly-Ub, polyubiquitin; Di-Ub, diubiquitin; Mono-Ub, monoubiquitin.
Fig 3.
SneRING generates K6-, K11-, K48-, and K63-linked ubiquitin chains that can be digested by specific DUBs.
(A) An in vitro autoubiquitination reaction, as in Fig 2A, was incubated for 5 h, after which a UbiCRest assay was performed using the indicated chain type-specific DUBs for 1 h at 37 °C. All DUBs were present at a final concentration of 1.5 µM, except OTUB1, which was present at 3 µM. Ubiquitin chain cleavage was analyzed by SDS-PAGE and western blot using a primary antibody against ubiquitin and SneRING as a control. (B) An in vitro autoubiquitination reaction, as in A, was incubated with Cezanne (K11-linked ubiquitin chain-specific DUB) and AMSH (K63-linked ubiquitin chain-specific DUB). Degradation of ubiquitin chains was detected by SDS-PAGE and western blot using anti-ubiquitin antibody. Poly-Ub, polyubiquitin; Di-Ub, diubiquitin; Mono-Ub, monoubiquitin. (C, D) A 5 h in vitro autoubiquitination reaction of SneRING with UbcH5b (C) or UBE2T (D) was analyzed by mass spectrometry. The graphs show log10 intensities of GlyGly-modified residues for the different linkage types, as determined by MaxQuant.
Fig 4.
SneRING localizes to the host cell cytosolic, light membranes, and inner mitochondrial membrane after overexpression.
(A) U2OS cells were transfected with a FLAG-tagged version of the SneRING using the transfection reagent PEI MAX. 72 h post-transfection and 2 days p.i., cells were fixed and stained using DAPI (blue channel), and a primary antibody against FLAG followed by the fluorophore-coupled secondary antibody (green channel). Images were taken using laser scanning confocal microscopy. The scale bar represents 30 µm. (B) U2OS cells were transfected as in A and, 36 h post-transfection, analyzed by SDS-PAGE and western blot using antibodies against the FLAG-tag and tubulin. (C) U2OS cells were transfected with FLAG-SneRING as in A, and 48 h post-transfection, the cells were separated by differential centrifugation into a crude mitochondrial (Mito), light membrane (LM), and cytosolic (Cyt) fraction. Total cell lysate of non-transfected cells (Control) and transfected cells (Total) served as controls. The samples were analyzed by SDS-PAGE and western blot using antibodies against Mic60, Calnexin, Tubulin, GAPDH, and FLAG. (D) Hek293T cells were transfected with FLAG-SneRING using the calcium-phosphate method. After 48 h of expression, mitochondria were isolated and then incubated in isotonic buffer (-Swelling) or in hypotonic buffer (+Swelling) to rupture the outer mitochondrial membrane, combined with the treatment using 50 µg/mL of protease K (PK). For complete solubilization, mitochondria were treated with 1% Triton X-100 (1% Triton) and PK and precipitated using 72% trichloroacetic acid. Samples were analyzed by SDS-PAGE and western blot, using antibodies against Calnexin, Tom70, CPOX, Mic60, Tim44, and FLAG-tag. (E) Mitochondria as in D were either sonicated in a high-salt buffer (500 mM NaCl pH 7.6) or subjected to carbonate extraction (100 mM Na2CO3 pH 10.8 or 100 mM Na2CO3 pH 11.5). Pellet (P) and supernatant (S) fractions were separated by ultracentrifugation, while untreated total samples (T) served as controls. Samples were analyzed by SDS-PAGE and western blot using antibodies against Hsp60, Sam50, Mic60, and FLAG. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tom70, translocase of the outer mitochondrial membrane 70; CPOX, coproporphyrinogen oxidase; Mic60, mitochondrial contact site and cristae organizing system 60; Tim44, translocase of the inner mitochondrial membrane 44; Sam50, sorting and assembly machinery 50.
Fig 5.
SneRING interacts with mitochondrial and ER proteins.
(A, B) A FLAG-tagged version of the SneRING was overexpressed in U2OS cells using PEI MAX transfection. 24 h later, cells were infected with Sne at an MOI of 1. Non-transfected cells served as a control. 48 h p.i., samples were collected and immunoprecipitation was performed using FLAG-magnetic beads, followed by mass spectrometry analysis. The graphs show identified proteins, with significance (-log10p-value calculated by two-tailed T-test, n = 3) plotted against the log2 fold change (log2FC) of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to non-transfected/infected controls (U2OS+Sne) (A) or of transfected/infected U2OS cells (U2OS+Sne + SneRING) relative to transfected/non-infected controls (U2OS+SneRING) (B). Enriched proteins are labeled in red, reduced proteins are shown in blue, and grey represents unchanged proteins. Only host cell proteins are shown. (C) Samples were prepared, and immunoprecipitation was performed as in A. Input and elution fractions were analyzed by SDS-PAGE and western blot using antibodies against Sne heat shock protein SnGroEL (star indicates that the signal represents a cross-reactive band observed at 130 kDa), Mic60, CKAP4, Erlin2, PHB, Cox5a, Tom70, and FLAG. Mic60, mitochondrial contact site and cristae organizing system 60; CKAP4, cytoskeleton‐associated protein 4; Erlin2, ER Lipid Raft Associated 2; PHB, Prohibitin; Cox5a, cytochrome c oxidase subunit 5A; Tom70, translocase of the outer mitochondrial membrane 70.
Fig 6.
SneRING ubiquitinates CKAP4 in vitro.
(A, B) A reaction containing purified recombinant SneRING, recombinant E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme (UbcH5b), ubiquitin, and ATP was mixed with the respective substrate (purified recombinant GST (A) or His-CKAP4 (B)) for the indicated periods at 37 °C. Reactions without the ligase and/or substrate served as controls. Samples were analyzed by SDS-PAGE and western blot, using antibodies against SneRING, GST, ubiquitin, and CKAP4. Asterisks indicate ubiquitinated CKAP4. Ponceau staining of the western blot membranes is shown for the loading control.