Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: GAPDH is involved in the heme-maturation of myoglobin and hemoglobin

doi: 10.1096/fj.202101237RR

Figure Lengend Snippet: GAPDH associates with Hb (γ) and Hb (α) in differentiating erythroid progenitors (HiDEP-1) and silencing of GAPDH in K562 cells downregulates fetal (γ) and alpha (α) Hb heme-maturations. We performed IPs during differentiation of erythroid progenitor cells (HiDEP-1) which express Hbα and Hbγ, and found that both fetal (γ) and alpha (α) Hb binds to GAPDH. In order to further elucidate the role of GAPDH in fetal (γ) Hb heme-maturation we treated K562 cells with SA for 72 h and did simultaneous silencing of GAPDH by siRNA (80 nM). The cells were then given micromolar amounts of hemin (5 μM) for additional 3 h before harvest and cell supernatant generation. SA treated cells with GAPDH siRNA were used as controls. In other experiments the K562 cells were transfected with GAPDH siRNA or transfected with scrambled siRNA and cultured for 24–72 h and then given 50 μΜ of hemin to allow for differentiation for 24–72 h before harvest. (A, B) IPs depicting GAPDH bound to Hbγ or Hbα during HiDEP-1 differentiation and heme-stain for Hbγ/α during differentiation. (C) Densitometries comparing Hbα/γ heme-stain dimer to AHSP/Hsp90 and/or GAPDH bound to Hbα/γ as depicted in panels (A, B). (D) Representative westerns and heme-stains under indicated conditions depicting the effect of silencing GAPDH on Hbαγ heme-maturation. (E) Densitometries of Hbαγ heme-stain, and expression levels of Hbγ and GAPDH depicted in panel D. (F) Representative westerns depicting inhibition of Hbα/γ dimer formation by silencing GAPDH during K562 differentiation. Corresponding heme-stains show inhibition of heme-insertion into Hbα/γ under similar conditions. (G) Densitometries of Hbαγ heme-stain, and expression levels of Hbα/γ and GAPDH as depicted in panel F. (H) Corresponding UV-visible spectra of Hbγ/α during K562 cell differentiation ∓ GAPDH siRNA

Article Snippet: Myc-DDK tagged human hemoglobin alpha (Hbα), beta (Hbβ), human skeletal muscle myoglobin expression construct and Myc-tagged mitochondrial heme transporter cDNA construct, FLVCR1b were purchased from OriGene (Rockville, MD, USA). sGCβ1 deletion constructs (sGC-β1 Δ204–244 + Δ379–408) was a gift from Dr. Andreas Papapetropolous (Athens University, Athens, Greece).

Techniques: Transfection, Cell Culture, Staining, Expressing, Inhibition, Cell Differentiation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: GAPDH is involved in the heme-maturation of myoglobin and hemoglobin

doi: 10.1096/fj.202101237RR

Figure Lengend Snippet: Model depicting the role of GAPDH on globin heme-maturation. Cellular GAPDH acting as heme chaperon is involved in heme-maturation of Mb and alpha, beta (adult) and gamma (fetal) Hb (α, β, γ), along with specific chaperons/co-chaperons for the globins as indicated. The apo-Hbα/apo-Hbβ/γ or the apo-Mb proteins bind to specific chaperons/co-chaperons26,27 with GAPDH and hsp90 being common chaperons to both apo-Hb or apo-Mb. The Mb and Hb heme-maturation processes require an active sGCα1β1 heterodimer making cGMP and this induction maybe transcriptional. GAPDH itself delivers heme to sGCβ1 and can regulate these processes. GAPDH binds heme and may transfer its bound heme directly to the globins or transfer its heme to hsp90/AHSP to enable heme-insertion into the corresponding client globins, followed by chaperon assisted folding of these globins, resulting in heme-containing Hb dimers, tetramers and Mb monomers. GAPDH mutants H53A and K227A downregulate or inhibit Hb/Mb heme-maturation and this inhibition can be completely rescued by overexpressing GAPDH or partly by sGCβ1 overexpression. While addition of D-Ala and ferric citrate can enhance heme-insertion into Hb and Mb, a combination of overexpressed GAPDH, FLVCR1b, and supplementation of D-ALa plus ferric citrate gives the maximum heme-insertion into target Mb, suggesting that these factors act in a concerted way to enhance heme-insertion into Hb or Mb and are important for cellular heme homeostasis

Article Snippet: Myc-DDK tagged human hemoglobin alpha (Hbα), beta (Hbβ), human skeletal muscle myoglobin expression construct and Myc-tagged mitochondrial heme transporter cDNA construct, FLVCR1b were purchased from OriGene (Rockville, MD, USA). sGCβ1 deletion constructs (sGC-β1 Δ204–244 + Δ379–408) was a gift from Dr. Andreas Papapetropolous (Athens University, Athens, Greece).

Techniques: Inhibition, Over Expression

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 associates with the interphase and mitotic MT cytoskeleton. (A) Schematic representation of the Ctr TarP protein and full-length Cpn CPn0572 protein. Previously identified domains are shown: phosphorylation domain (phos), dark gray boxes; proline-rich domain (PRD), orange box; G-actin binding domain (ABD), light blue box; F-actin-binding domain (FAB), dark blue box; and vinculin-binding site (VBS), white box ( ; ). (B) Representative confocal images of HEp-2 cells expressing GFP-CPn0572 (green) for 24 h prior to fixation. Actin was visualized with Rhodamine–phalloidin staining (red) and MTs with anti-α-tubulin antibody. As secondary antibody anti-mouse-IgG conjugated to Alexa Fluor 647 was used (magenta). (C) Merged images of indicated single channels shown in (B). White boxes show enlargements. (D) Representative confocal images of HEp-2 cells transfected with plasmid expressing GFP–CPn0572 (green) for 24 h, prior to fixation. Shown is an exclusive predominant association of GFP–CPn0572 with actin. Actin was visualized with Rhodamine-phalloidin staining (red). (E) Shown is a predominant association of GFP–CPn0572 with interphase MTs visualized with anti-α-tubulin antibody (red). DAPI was used to visualize DNA (blue). (F) Quantification of HEp-2 cells transfected with a GFP–CPn0572 plasmid 24 h prior to fixation. Exclusive colocalization of GFP–CPn0572 with actin was scored in 81% (black) cells, whereas in 11% cells analyzed GFP–CPn0572 localized to both MTs and actin structures (dotted). In 7.7% cells, an exclusive GFP–CPn0572 localization to MT structures was observed (striped). Data represent a mean of three experiments, n =100 cells/experiment. (G) Diagrammatic representation of CPn0572 variants. Quantification of cells expressing GFP–CPn0572 variants and association with the MT cytoskeleton as a percentage (%) of cells counted. Association with the actin cytoskeleton is depicted as+(actin association) or−(no actin association). When no actin association was observed (four bottom CPn0572 variants), CPn0572 was either associated with MT structures (percentage given) or showed a non-specific cytoplasmic staining. * marks deletion variant with MT and actin phenotypes differing from the other protein variants (visualized in Fig. S1 , dot-like and curved-fiber phenotypes concentrated in proximity to the nucleus). (H) Schematic representation of full-length CPn0572 including the newly defined MT-binding region from aa 595–755 (purple box). (I) Confocal images of U2OS cells transfected with GFP–CPn0572 595-755 plasmid for 18 h. Mitotic metaphase is shown diagrammatically on the left. MTs were visualized by anti-α-tubulin antibody staining (red) and DNA with DAPI (blue). (J) Representative confocal image of mitotic U2OS cell expressing full length GFP-CPn0572 (green) full length for 18 h. MTs were visualized by using anti-α-tubulin antibody (magenta), actin with rhodamine-phalloidin staining (red) and DNA with DAPI. In A and G, the numbers indicate amino acid positions. For B–E, I and J images shown are representative of three or more repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Binding Assay, Expressing, Staining, Transfection, Plasmid Preparation, Variant Assay

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: Cpn TarP proteins from human and animal isolates can associate with the MT cytoskeleton. (A) Phylogenetic tree of members of the TarP protein family from different chlamydial species with the corresponding isolate indicated. Asterisks mark the TarP members selected for transfection experiment show in C. Phylogenetics were undertaken with Clustal Omega. (B) Schematic illustration of TarP family members and the C-terminus (blue) used for generation of GFP-fusion variants. (C) Representative confocal fluorescence images of HEp-2 cells expressing GFP-tagged versions of C-terminal fragments of the indicated TarP family members. Determination of colocalization with MTs is shown on the right. Cells were transfected with indicated plasmids for 18 h. MTs were visualized using anti-α-tubulin antibody (red) and DNA with DAPI (blue). White boxes show enlargements. Images shown are representative of three repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Transfection, Fluorescence, Expressing

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: Severity of alteration of MT structure correlates with time of CPn0572 595-755 expression. (A) Representative confocal fluorescence images of HEp-2 cells transfected with a plasmid encoding GFP for 18 h (top panels) or GFP–CPn0572 595-755 for the indicated times prior to fixation. MTs were visualized with anti-α-tubulin antibody (red) and DNA with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis of HEp-2 cells expressing GFP-CPn0572 595-755 for the indicated time points. Protein extracts were separated on a 10% SDS-PAGE followed by Western blot analysis. Western blot was probed with anti-GFP or anti-GAPDH antibodies. Blot shown is representative of two repeats. (C) Explanatory cartoon to show co-dependency of GFP–CPn0572 595-755 expression levels and the increase of aberrant MTs (only the 0 h and 18 h time points represent actual measurements). (D) Quantification (mean±s.e.m.) of MT phenotypes shown in A. Typical examples of MTs phenotypes are shown for (i) GFP-expressing control cells (18 h), (ii) GFP–CPn0572 595-755 -expressing cells (6 h) and (iii) GFP–CPn0572 595-755 -expressing cells (18 h). MT thickness was determined by calculating the average diameter measured at three different positions of a single MT bundle using ImageJ. Thin MTs were defined as MTs with <0.302 µm in diameter; thick MTs were defined as MTs with ≥0.302 µm in diameter. n =3 cells (30 MTs measured per condition). *** P <0.001; ** P <0.005; ns, not significant (two-tailed unpaired Student's t -test). (E) Quantification (mean±s.e.m.) of the percentage of GFP- or GFP–CPn0572 595-755 -expressing mitotic U2OS cells with spindle defects. n =3 independent experiments each representing 50 cells. *** P <0.001 (two-tailed unpaired Student's t -test). (F) Confocal images of U2OS cells transfected with a plasmid encoding GFP or GFP-CPn0572 595-755 for 18 h. Top images show a GFP-expressing mitotic cell in anaphase. Bottom images show a representative example of the main spindle defect observed in GFP–CPn0572 595-755 - expressing cells. MTs were visualized by using an α-tubulin antibody (red) and DNA was stained with DAPI (blue). Images shown are representative of three repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Expressing, Fluorescence, Transfection, Plasmid Preparation, Western Blot, SDS Page, Control, Two Tailed Test, Staining

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 associated with MTs during chlamydial infection impacts infection efficiency. (A) PCR and (B) western blot verification of Ctr expressing CPn0572–FLAG. For B cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG (pMH16) for 48 h were lysed and proteins were separated on 10% SDS-PAGE. For following western blot analysis anti-CPn0572 (generated in our lab), anti-FLAG and anti-DnaK antibodies were used. Images shown are representative of one (A) or two (B) repeats. (C) Confocal images of HEp-2 cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG for 15 min. CPn0572–FLAG was visualized with anti-FLAG antibody (green) and DNA with DAPI (blue). (D) Quantification (mean±s.e.m.) of inclusion size [µm 2 ] 24 hpi of Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG. n =2, each representing 30 cells. * P <0.05 (two-tailed unpaired Student's t -test). (E) Representative images from three independent experiments showing single focal planes of HEp-2 cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG (MOI=0.5, 48 hpi). Cells were treated with 0.5% Triton X-100 for 30 s and fixed with 0.5% glutaraldehyde. Images show a focal plane between the inclusion and the plasma membrane. (F) Quantification (mean±s.e.m.) of the diameter of MTs measured at the inclusion or distal of the inclusion in cells infected with Ctr - or CPn0572-expressing Ctr . MT diameter was measured by measuring the diameter at three different positions of one MT fiber using ImageJ. Data represent measurements of MT diameter for 10 MTs ( n =4 cells). *** P <0.001; ns, not significant (two-tailed unpaired Student's t -test). (G) Representative images from three repeats of Caco-2 cells infected with Ctr transformed with plasmid encoding CPn0572–FLAG for 48 h. For E and G, CPn0572–FLAG was visualized with anti-FLAG (green), MTs with anti-α-tubulin antibody (red) and eukaryotic and chlamydial DNA with DAPI (blue). For C, E and G, white boxes show enlargements (zoom). Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Infection, Western Blot, Expressing, Transformation Assay, Plasmid Preparation, SDS Page, Generated, Two Tailed Test, Membrane

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 595-755 protects MTs against cold-induced MT depolymerization. (A,B) Representative confocal images of HEp-2 cells transfected with plasmids encoding GFP (A) or GFP–CPn0572 595-755 (B) for 18 h. MTs were wild-type (after incubation at 37°C) or depolymerized through incubation on ice for 15 or 60 min and then visualized by anti-α-tubulin antibody (red) and DAPI to stain DNA (blue). Boxed regions correspond to the enlarged images shown on the right. Scale bars: 10 µm. (C) Confocal images of HEp-2 cells transfected with a plasmid encoding GFP or GFP–CPn0572 595-755 for 18 h. MTs were visualized with an anti-acetylated-α-tubulin antibody (red) and DNA with DAPI (blue). Images shown in A–C are representative of three or more repeats. Scale bars: 10 µm. (D) A typical example of a Western blot analysis of HEp-2 cells expressing GFP or GFP-CPn0572 595-755 for 18 h. Protein extracts were separated on a 10% SDS-PAGE followed by western blot analysis. Western blot was probed with anti-GFP, anti-acetylated-α-tubulin, anti-α-tubulin or anti-GAPDH antibodies. (E) Quantification of relative acetylated-α-tubulin levels shown in D. Band intensities from Western blots of acetylated-α-tubulin in cells expressing GFP–CPn0572 595-755 were determined and compared relative to control cells (GFP alone=1). ImageJ 1.47v was used for band intensity quantification. Error bars denote ±s.e.m., n =3 independent experiments. * P <0.05 (two-tailed unpaired Student's t -test).

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Transfection, Incubation, Staining, Plasmid Preparation, Western Blot, Expressing, SDS Page, Control, Two Tailed Test