EMBO/soda-nel huggingface dataset:
Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode (2016)
Figure 3

Figure 3 - Molecular features of C3b-bound MCP and SPICE and comparison with C3b-FH. A. Superposition of MCP and FH domains CCP3-4 (shown as blue and orange cartoons, resp.) on the surface of C3b. B. Cartoon representation of the superposition of domains CCP3-4 of MCP (blue), FH (orange) and SPICE (green). Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4, MCP and SPICE show a rotation of approximately 100° in the position of CCP4 compared to FH. C. The reported mutagenesis data on MCP CCP3 and CCP4 (data from Liszewki et al. (Liszewski et al, 2000)) are consistent with the crystal structure. Shown are two orientations of the surface of MCP CCP3-4 as in the crystal structure of the C3b-MCP complex, highlighting the contact sites with the C3b platform (purple), the MCP mutations identified as critical for C3b binding (red) and those not affecting the interaction (blue). The GB24 antibody epitope is shown as yellow contour on the MCP surface. D. Model of the putative positioning of the flexible CCP1-2 domains of MCP. The model has been generated by superposing the structure of CCP1-4 of MCP from PDB 3O8E (Persson et al, 2010) onto the structure of the C3b-MCP (CCP1-4) complex. CCP1-2 domains of MCP, not visible in the C3b-MCP (CCP1-4) structure, are shown in grey. E. Highlight of the primary sequence differences between SPICE and VCP. The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple. The contact surface of C3b on the regulator in the C3b-SPICE (CCP1-4) complex is shown in green on the right panel.
Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode (2016)
Figure 4

Figure 4 - Structural mapping of disease-related mutations on the C3b-regulator surfaces. (A) Mapping of known disease-related mutations on C3b (Table 3). The high-resolution C3b three-dimensional structure is used as structural template, shown as white surface. The regulator contact surfaces (based on C3b-FH (CCP1-4) (Wu et al, 2009) and C3d-FH (CCP19-20) (Kajander et al, 2011; Morgan et al, 2011)) are shown as black contour. All mutations reported in Table 3 are shown in blue on the C3b surface. Disease-related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel. (B) Mapping of disease-related mutations on the FH (CCP1-4) surface using data from Table 4. Colors as in Fig. 4A. (C) Mapping of disease-related mutations on the MCP (CCP3-4) surface using data from Table 4. Colors as in Fig. 4A.
MLLT6 Maintains PD-L1 Expression and Mediates Tumor Immune Resistance (2020)
Figure 1

Figure 1. CRISPR screen identifies MLLT6 as a regulator of PD-L1.

A Schematic setup of the screen for PD-L1 regulators in the presence and absence of IFN-γ. RKO reporter cells expressing PD-L1-eGFP fusion protein (blue) were mutagenized with an sgRNA/Cas9 library. PD-L1low cells were enriched by FACS and sgRNA target genes were identified by deep sequencing. An enrichment analysis determined frequencies of different sgRNAs and their target genes.

B sgRNA frequencies (log2 of fold change) in PD-L1low cells unstimulated (left) or IFN-γ stimulated (right). Enriched sgRNAs (dashed line) are highlighted; hit genes (black, red) and controls (green, yellow or purple) are shown and labeled with target gene names.

C sgRNA frequencies (log2 of fold change) in viability screen. sgRNAs targeting controls are highlighted; essential (orange), non-essential (blue). Genes attributed to PD-L1 modulation are shown in different colors (red, yellow and purple).
MLLT6 Maintains PD-L1 Expression and Mediates Tumor Immune Resistance (2020)
Figure 2

Figure 2. MLLT6 regulates expression of PD-L1.

A PD-L1 cell surface presentation (percentage ± SD) in RKO mock control (black) or monoclonal MLLT6 knockout cells (grey) (Student's t-test; **p < 0.01; n=3 biological replicates; degrees of freedom (df)=2).

B MLLT6 BAC mediated rescue in U2OS cells (left) showing percentage ± SD of PD-L1 cell surface presentation in mock control (black), MLLT6 polyclonal knockout (light grey) and MLLT6 knockout + BAC cells (dark grey) (Student's t-test; *p < 0.05; n=3 biological replicates; df=2). Representative flow cytometry plots (right) of PD-L1 cell surface presentation after staining with anti-PD-L1 antibody (top right, bottom left, bottom right) and isotype control (top left). Percentages of cells in the PD-L1+ gate as indicated.

C Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in monoclonal RKO MLLT6 knockout, mock control or MLLT6 knockout + BAC cells visualized by immunoblotting. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.

D PD-L1 transcript levels in U2OS mock (black) and polyclonal MLLT6 knockout lines (grey) treated with and without IFN-γ normalized to expression in mock U2OS cells without IFN-γ treatment (ANOVA; **p < 0.01; n=4 biological replicates; df=12).

E Representative flow cytometry plots of PD-L1 cell surface presentation in the presence and absence of IFN-γ in mock, polyclonal MLLT6 knockout and PD-L1 knockout cells. Percentages of cells in the PD-L1+ gate after staining with anti-PD-L1 antibody or isotype control as indicated.

F Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN-γ stimulation. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.
MLLT6 Maintains PD-L1 Expression and Mediates Tumor Immune Resistance (2020)
Figure 3

Figure 3. Knockout of MLLT6 alleviates suppression of CTLs.

A Schematic of assay measuring CD8+ T cell mediated cytotoxicity. Cancer cells (grey) with or without genetic modifications were mixed with BiTE or bi-specific antibodies with or without IFN-γ and cytotoxic T lymphocytes (CTLs). Altered tumor cell numbers were determined after allowing for T cell engagement.

B Relative cell numbers (± SD; n=3; biological replicates) of U2OS mock (black) or polyclonal MLLT6 knockout (grey) cells treated with varying amounts of EpCAM-CD3 BiTE and CTLs (Student's t-test; **p < 0.01; degrees of freedom (df)=4).

C Kaplan Meier survival plot (left) showing changes in survival (percent) of mock (grey) and MLLT6 knockout (red) cells treated with CTLs and EpCAM-CD3 BiTE over a course of 9.5 hours. Representative images (right) of time-lapse fluorescence microscopy following U2OS mock (eGFP tagged, green) and MLLT6 knockout (mCherry tagged, red) cells immediately (top left and bottom left) after adding CTLs and EpCAM-CD3 BiTE and 9.5 hours later (top right and bottom right) (scale bars = 100 μm).

D Relative cell numbers (left) (± SD; n=3; biological replicates) of mock (black), PD-L1 (dark grey) or MLLT6 (light grey) knockout cells treated with BiTE or bi-specific antibodies and CTLs as indicated (Student's t-test; **p < 0.01; df=4). Representative images (right) from bright field microscopy of polyclonal U2OS mock (top), PD-L1 (middle) or MLLT6 (bottom) knockout cells treated without or with EpCAM-CD3 BiTE or bispecific antibodies.

E Relative normalized cell numbers (± SD; n=3; biological replicates) of U2OS mock (left), MLLT6 (middle) or PD-L1 (right) knockout cells treated with EpCAM-CD3 BiTE and CTLs in the presence (grey) or absence (black) of IFN-γ (Student's t-test; **p < 0.01; df=4 ).
MLLT6 Maintains PD-L1 Expression and Mediates Tumor Immune Resistance (2020)
Figure 4

Figure 4. Role of MLLT6 in gene expression.

A Scatter plots of pairwise comparisons of transcript expression in U2OS cells. Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN-γ as indicated. Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance (log10 of p-value, n=3) with candidates nominated for further validation (red).

B Correlation plots describing log2 fold change of transcripts in RNA sequencing (x axis) and Nanostring (y axis) measurements in mock cells +/- IFN-γ (left) and MLLT6 knockout cells with IFN-γ (right). Transcripts previously described to have a role in tumor immunity (red) and PD-L1 (blue) are represented on the plots as indicated.

C Changes (log2 fold) in transcript expression (left) of select immune-related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN-γ and total protein levels measured by immunoblotting (right). Numbers indicate band intensities normalized to GAPDH.
MLLT6 Maintains PD-L1 Expression and Mediates Tumor Immune Resistance (2020)
Figure 5

Figure 5. Function of MLLT6 in IFN-γ signaling.

A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN-γ. Numbers indicate band intensities normalized to GAPDH.

B Immunoblot showing total protein levels of STAT1 isoforms (α and β), IDO1, GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase (Rluc, negative control), total STAT1, STAT1α and STAT1β treated with and without IFN-γ.

C Schematic model of MLLT6 function in IFN-γ signaling.

Expanded View Figure Legends
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 1

SILAC phosphoproteomic approach for the identification of PINK1‐dependent targets

Illustration of SILAC phosphoproteomics workflow. Flp‐In T‐Rex HEK293 cells stably expressing FLAG alone were cultured in unlabelled (R0K0) medium, kinase‐inactive (KI; D384A) PINK1‐FLAG cells were “medium” (R6K4) labelled, and wild‐type (WT) PINK1‐FLAG cells were “heavy” (R10K8) labelled using SILAC media containing the respective isotopes. All conditions were treated with 10 μM CCCP for 3 h and subjected to membrane fractionation. Four biological replicates of the samples were mixed in a 1:1:1 ratio, digested and fractionated by HILIC. Phosphopeptides from these fractions were enriched by TiO2 chromatography and analysed by quantitative mass spectrometry on an Orbitrap Velos Pro mass spectrometer. Data were analysed by MaxQuant and Perseus software packages.

About 25 μg of membrane‐enriched lysate from the mass spectrometry experiments was immunoblotted with anti‐PINK1 antibody. TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm, respectively.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 3

Rab8A, Rab8B and Rab13 Ser<sup>111</sup> phosphorylations are regulated by PINK1 upon CCCP treatment

A–C

Confirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected either with HA‐Rab8A (A), HA‐Rab8B (B) or HA‐Rab13 (C) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (10 mg) were immunoprecipitated with anti‐HA agarose, resolved by SDS–PAGE and stained with colloidal Coomassie blue (second panel). Coomassie‐stained bands migrating with expected molecular mass of HA‐Rabs were excised, in‐gel digested with trypsin and subjected to high‐performance liquid chromatography with LC‐MS/MS on an LTQ‐Orbitrap mass spectrometer. Upper panel shows the extracted ion chromatogram (XIC) analysis of Ser111‐containing phosphopeptides (8A, NIEEHApSADVEK; 8B, NIEEHApSSDVER; 13, SIKENApSAGVER) with the combined signal intensity of the 2+ and 3+ forms of the peptide indicated on the y‐axis. Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1‐FLAG‐expressing cells following CCCP stimulation.

D

Characterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 4

Endogenous PINK1 regulates Rab8A, Rab8B and Rab13 Ser<sup>111</sup> phosphorylation

PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls.

Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 5

Rab8A Ser<sup>111</sup> phosphorylation is abolished in Parkinson's disease patient PINK1 fibroblasts and PINK1 knockout mouse embryonic fibroblasts (MEFs)

Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skin fibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.

Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 6

Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCP

Wild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 7

Evidence that Rab8A Ser<sup>111</sup> is not a direct substrate of PINK1

Ubiquitin, Parkin, Rab8A and Rab1A phosphosites adopt two distinct conformations. Ser65 in ubiquitin (blue) and Parkin (magenta) follows a β‐turn, before the start of the 5th β‐strand. Conversely, Ser114 in Rab1A (green) and Ser111 in Rab8A (ochre) lie after a C‐terminal helix cap just before the start of a β‐turn. Representative three‐dimensional structures are superimposed by C‐α positions for the observed phosphosites and their sequence neighbours. Side chains for observed sites are shown as sticks, and ribbons depict backbone and secondary structure. To highlight the local environment, regions more than 3 amino acids away from the phosphosites are made transparent (see Materials and Methods for PDB IDs).

In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS–PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 8

Time‐course analysis of Rab8A, Rab8B and Rab13 Ser<sup>111</sup> phosphorylation

Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.

Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1. (2015)
Figure 9

Rab8A Ser<sup>111</sup> phosphorylation impairs Rabin8‐catalysed GDP exchange <italic>in vitro</italic> and Rabin8 interaction in cells

Crystal structure of Rab8A in complex with guanine exchange factor (GEF) Rabin8 (Guo et al, 2013). Left panel: Rabin8 is shown in surface representation and Rab8A in cartoon representation. Right panel: Rabin8 is shown in cartoon representation, and Rab8A is shown in surface representation. The hydroxyl group of residue Ser111 is shown as a yellow sphere. The surfaces are colored by their positive and negative electrostatic potentials from blue to red, respectively.

Rabin8‐catalysed mant‐GDP release from mant‐GDP loaded WT, S111A and S111E mutants of Rab8A. The Rab proteins (1 μM) were incubated with 100 μM GDP in buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE), and the reaction was started by the addition of 0.5 μM Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.

Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells. Rab8A KO HeLa cells were transfected with wild‐type (WT), S111E or S111A HA‐Rab8A. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rabin8 or anti‐HA antibody. Lysates were immunoblotted with Rabin8 or anti‐HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA‐Rab8A in extracts.
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
Figure 1

Figure 1 Knockdown of Phf5a inhibits CSR.

(A) Representative FACS profiles of IgA switching in CH12F3-2A cells. The control and Phf5a siRNAs are indicated at the top. The number in each FACS profile indicates the percent (%) IgA+ cells at 24 h and 48 h after CIT stimulation.

(B) Top: Illustration of Phf5a with a central PHD domain. Bottom: Confirmation of Phf5a KD by Western blot.

(C) CSR complementation assay using siRNA-resistant Phf5a with Myc and Flag (MF) epitopes tagged at the C-terminus.

(D) Schematic of MF-tagged siRNA-resistant Phf5a (Phf5aR-MF). Asterisks (*) indicate the approximate locations of mutations in the cDNA made to generate the siRNA-resistant version. Western blot confirmed the KD efficiency of the endogenous Phf5a, and the expression of Phf5aR-MF.

(E) Quantitative RT-PCR (qRT-PCR) of μGLT, αGLT, and AID from siControl- and siPhf5a-treated samples. The values are presented as mean ± sd (n=3).

(F) Ethidium Bromide-stained gels showing the PCR detection of excision circular DNA (αCD) and Gapdh as a control in the genomic DNA samples indicated.
Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition (2021)
Figure 2

Figure 2 Knockdown of Phf5a suppresses IgH/c-Myc translocation.

(A) Schematic representation of the IgH and c-Myc chromosomal loci before and after translocation. Arrows and black bar indicate the positions of the PCR primers used to amplify the rearranged region and the probe for Southern hybridization, respectively. (B) Southern blots of the IgH/c-Myc translocation assay using genomic DNA extracted from CIT-stimulated CH12F3-2A cells treated with the siRNAs shown at the bottom of each panel. Numbers on the top indicate gel lanes.

(C) Frequency of IgH/c-Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample (Boboila et al., 2012b). The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (** p ≤ 0.01, *** p ≤ 0.001).