Journal: Molecular Microbiology

Article Title: Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

doi: 10.1111/j.1365-2958.2009.06639.x

Figure Lengend Snippet: Analysis of Pf_calpain expression using C-terminally tagged chimeras. A. Strategy used to create C-terminally tagged calpains by integration at the 3′ end of the endogenous locus. We generated a set of plasmids containing 1.3 kb of sequence from the 3′ end of the Pf_calpain ORF. Downstream of this sequence we placed different tags in frame: 6-his, GFP, 2×-myc and 2×-flag. A human dihydrofolate reductase (hDHFR) cassette was used for positive drug selection. The relative positions of NsiI and SphI restriction sites, used to detect integration, as well as of the probe are indicated. B. Southern blots of restricted genomic DNA. Calpain-6-his, calpain-GFP, calpain-2×-myc and calpain-2×-flag integrant clones gave the bands expected of a single-crossover recombination (white arrows) and plasmid (grey arrows). The asterisks indicate extra bands probably due to concatamerization with plasmid rearrangement, a frequent event in P. falciparum . The 3D7 parental strain shows a single band (black arrows) of the correct size, which is absent in the clones. The difference in size of the smallest DNA fragment reflects the difference in size between GFP and the smaller tags. C. Immunoprecipitated calpain was detected by Western blot analysis. We pulled down calpain using anti-calpain antiserum #19, including as control its pre-immune serum (right panel). For each immunoprecipitation, we used 5 × 10 8 asynchronous parasites of the parental strain 3D7 (WT) or the calpain-GFP clone (C3). BiP content of each lysate is shown in the panel below. Calpain-GFP was detected by the use of a third antibody, mouse anti-GFP. Pf_calpain is an extremely low-abundance protein that has an apparent molecular weight over 250 kDa (black arrow).

Article Snippet: Other antibodies: rabbit anti-GFP 6556 (Abcam) diluted for microscopy (Mic) 1:200 and for WBs 1:5000; goat anti-GFP 5450 (Abcam) Mic 1:25; monoclonal anti-GFP (JL8, BD) WB 1:1000; rabbit anti-calpain #17 WB 1:250; monoclonal Anti-flag M2 Mic 1:100; monoclonal anti-His Mic 1:100; rat and rabbit anti-Bip (MR4) Mic 1:1500 and WB 1:10 000; monoclonal anti-hNop1 (Abcam) Mic 1:12.5; polyclonal anti-Pf_Nop1 ( Figueiredo et al ., 2005 ); Mic 1:25; anti-calpain #35 Mic 1:50 (whole cell) and 1:400 (purified nuclei); monoclonal anti-PMV , WB 1/400.

Techniques: Expressing, Generated, Sequencing, Selection, Clone Assay, Plasmid Preparation, Immunoprecipitation, Western Blot, Molecular Weight

Journal: Molecular Microbiology

Article Title: Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

doi: 10.1111/j.1365-2958.2009.06639.x

Figure Lengend Snippet: Immunofluorescence localization of calpain. A. Immunofluorescence images of clones expressing calpain-6-his, calpain-GFP and calpain-2×-flag are presented. The images are representative of the overall population and were obtained using tag-specific primary antibodies and a proper secondary antibody conjugated to Alexa-Fluor (AF) 488. The immunofluorescence distribution in all the clones highlights a region closely associated with the nucleus but not completely merging with the DAPI signal. B. Colocalization of fluorescence signal distributions from both anti-flag antibody and anti-calpain antibody with that of a C-terminal peptide of calpain (#35, ). Images a and b show the calpain-2×-flag clone stained with monoclonal anti-flag antibody/goat AF488-conjugated anti-mouse antibody and either rabbit anti-calpain #35/goat AF555-conjugated anti-rabbit or pre-immune serum of the same rabbit/goat AF555-conjugated anti-rabbit. The 3D7 parental strain, stained as in a, is positive with anti-calpain #35, but not with the anti-flag antibody. C. The relative distributions of calpain-2×-flag (green channel) and BiP (red channel) signals were analysed by confocal microscopy. Here two representative images show the signal detected in a perinuclear plane (a) and in a nuclear plane (b). The right-most panels present the enlarged merge images. The nucleus in (a) was detected acquiring the Topro3-signal with increased pin-hole that rendered a non-confocal image. Bars, 5 μm.

Article Snippet: Other antibodies: rabbit anti-GFP 6556 (Abcam) diluted for microscopy (Mic) 1:200 and for WBs 1:5000; goat anti-GFP 5450 (Abcam) Mic 1:25; monoclonal anti-GFP (JL8, BD) WB 1:1000; rabbit anti-calpain #17 WB 1:250; monoclonal Anti-flag M2 Mic 1:100; monoclonal anti-His Mic 1:100; rat and rabbit anti-Bip (MR4) Mic 1:1500 and WB 1:10 000; monoclonal anti-hNop1 (Abcam) Mic 1:12.5; polyclonal anti-Pf_Nop1 ( Figueiredo et al ., 2005 ); Mic 1:25; anti-calpain #35 Mic 1:50 (whole cell) and 1:400 (purified nuclei); monoclonal anti-PMV , WB 1/400.

Techniques: Immunofluorescence, Clone Assay, Expressing, Fluorescence, Staining, Confocal Microscopy

Journal: Molecular Microbiology

Article Title: Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

doi: 10.1111/j.1365-2958.2009.06639.x

Figure Lengend Snippet: Immunofluorescence and EM analyses reveal that Pf_calpain is nucleolar. A. Colocalization of calpain and Nop1, a nucleolar marker. Using specific antibodies for GFP or for flag epitope in combination with proper AF488-conjugated secondary antibodies, we detected the cellular distribution of calpain in the clones expressing calpain-GFP and calpain-2×-flag in relation to the nucleolar compartment labelling obtained with an anti-Pf_Nop1 serum (red channel). The nuclei are stained with DAPI. Bar, 5 μm. Enlargements of the nuclei are provided in . B. Immuno-EM was done using pre-embedding labelling with anti-flag and anti-Pf_Nop1 after tetanolysin treatment to permeabilize erythrocytes. The image presents the nuclear section of a representative early trophozoite. At the upper left corner are visible the red blood cell (R) and a cytostome (C). The nucleus (N) appears thoroughly circumscribed by its double membrane closely associated with ER (E). Inside the nucleus, labelling due to the immunodetection of calpain-flag (gold particles 12 nm – open arrows in the enlarged panel) and Pf_Nop1 (gold particles 18 nm – solid arrows in the enlarged panel) reveals staining of an electron-dense nuclear subcompartment. Some particles are also found in the perinuclear ER (asterisk). Bars, 0.5 μm. C. Confocal analysis of purified nuclei from 3D7 using anti-calpain antiserum #35. We generally detected one or two spots of approximate diameter of 0.5 μm. Colocalizazion of Nop1 and calpain in an isolated nucleus by confocal analysis is provided in .

Article Snippet: Other antibodies: rabbit anti-GFP 6556 (Abcam) diluted for microscopy (Mic) 1:200 and for WBs 1:5000; goat anti-GFP 5450 (Abcam) Mic 1:25; monoclonal anti-GFP (JL8, BD) WB 1:1000; rabbit anti-calpain #17 WB 1:250; monoclonal Anti-flag M2 Mic 1:100; monoclonal anti-His Mic 1:100; rat and rabbit anti-Bip (MR4) Mic 1:1500 and WB 1:10 000; monoclonal anti-hNop1 (Abcam) Mic 1:12.5; polyclonal anti-Pf_Nop1 ( Figueiredo et al ., 2005 ); Mic 1:25; anti-calpain #35 Mic 1:50 (whole cell) and 1:400 (purified nuclei); monoclonal anti-PMV , WB 1/400.

Techniques: Immunofluorescence, Marker, FLAG-tag, Clone Assay, Expressing, Staining, Immunodetection, Purification, Isolation

Journal: Molecular Microbiology

Article Title: Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

doi: 10.1111/j.1365-2958.2009.06639.x

Figure Lengend Snippet: Localization of YFP-HC3 in transfected parasites and mammalian cells. A. Single YFP chimera localization. Left, P. falciparum (bars, 5 μm); right, human 293T cells (bars, 1 μm). Between the panels are schematics of the constructs expressed. Green circle, HC3 subdomain. B. Enlargement of nuclei of fixed P. falciparum cells expressing YFP-NLS, showing discrete subnuclear spots. Bar, 1 μm. C. Colocalization of P. falciparum YFP-HC3 fusion (YFP) with anti-Pf_Nop1. DAPI staining is also shown. Merges are to the right. D. Multi-YFP chimera localization. Cells and schematics as in (A). Enlargements of the nuclei are provided in . E and F. Expression pattern of the YFP chimeras used in mammalian cells (E) and P. falciparum (F) by Western blot using anti-GFP antibody. G. Tabulation of construct sizes, putative nuclear entry mode and significant localization. C, cytoplasmic; N, nuclear; No, nucleolar. H. Live P. falciparum colocalization of Td-Tomato (a), Td-Tomato-HC3 chimera (b) with sytoRNA green, a dye to detect the nucleolus in live cells. Bar, 5 μm. Enlargements of the nuclei are provided in .

Article Snippet: Other antibodies: rabbit anti-GFP 6556 (Abcam) diluted for microscopy (Mic) 1:200 and for WBs 1:5000; goat anti-GFP 5450 (Abcam) Mic 1:25; monoclonal anti-GFP (JL8, BD) WB 1:1000; rabbit anti-calpain #17 WB 1:250; monoclonal Anti-flag M2 Mic 1:100; monoclonal anti-His Mic 1:100; rat and rabbit anti-Bip (MR4) Mic 1:1500 and WB 1:10 000; monoclonal anti-hNop1 (Abcam) Mic 1:12.5; polyclonal anti-Pf_Nop1 ( Figueiredo et al ., 2005 ); Mic 1:25; anti-calpain #35 Mic 1:50 (whole cell) and 1:400 (purified nuclei); monoclonal anti-PMV , WB 1/400.

Techniques: Transfection, Construct, Expressing, Staining, Western Blot

Journal: Molecular Microbiology

Article Title: Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

doi: 10.1111/j.1365-2958.2009.06639.x

Figure Lengend Snippet: HC1 is membrane anchored as a consequence of acylation. A. The sequence of HC1. The amino acids predicted to be modified by myristate (Myr) and palmitate (Pal) are indicated by outlined letters. The bar indicates the peptide used to generate anti-calpain antiserum #17. Amino acids included in the longer version of the calpain N-terminal HC1 chimeras (NtL) and excluded from the shorter version (Nt) are encircled. Asterisk, potential cleavage site. B. Analysis of constructs in P. falciparum . Stable lines of transfected parasites were generated, using plasmids that drive the episomal expression of chimeras carrying wild-type HC1 or mutants in amino acids 2 and 3. Immunoprecipitated chimeras were analysed by Western blot using rabbit anti-GFP antibody (a) and, after stripping of the membrane, rabbit-anti-calpain antiserum #17, which recognizes only the unmyristoylated constructs (b). C and D. Analysis of constructs in mammalian cells. 293T cells were transfected with plasmids encoding each of the chimeras as in (B). The cells were grown in normal (C), or labelling media (D). Cell lysates were analysed by Western blotting (C) using anti-GFP (a) to monitor expression levels or anti-calpain antiserum #17 (b). Alternatively, cells were labelled with 3 H-myristate (a) or 3 H-palmitate (b), lysates were immunoprecipitated with anti-GFP antibody and fluorography was performed (D). 3 H-palmitate but not 3 H-myristate label was sensitive to hydroxylamine (not shown). E. Fluorescence of live transfected P. falciparum (left; bars, 5 μm) and fixed human 293T cells (right; bar, 1 μm). Schematics of the constructs are shown. Light green oval, HC1 subdomain. Below each schematic is the mutation constructed. Images are labelled as in . NtL chimeras are shown in .

Article Snippet: Other antibodies: rabbit anti-GFP 6556 (Abcam) diluted for microscopy (Mic) 1:200 and for WBs 1:5000; goat anti-GFP 5450 (Abcam) Mic 1:25; monoclonal anti-GFP (JL8, BD) WB 1:1000; rabbit anti-calpain #17 WB 1:250; monoclonal Anti-flag M2 Mic 1:100; monoclonal anti-His Mic 1:100; rat and rabbit anti-Bip (MR4) Mic 1:1500 and WB 1:10 000; monoclonal anti-hNop1 (Abcam) Mic 1:12.5; polyclonal anti-Pf_Nop1 ( Figueiredo et al ., 2005 ); Mic 1:25; anti-calpain #35 Mic 1:50 (whole cell) and 1:400 (purified nuclei); monoclonal anti-PMV , WB 1/400.

Techniques: Sequencing, Modification, Construct, Transfection, Generated, Expressing, Immunoprecipitation, Western Blot, Stripping Membranes, Fluorescence, Mutagenesis

Journal: Molecular Microbiology

Article Title: Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

doi: 10.1111/j.1365-2958.2009.06639.x

Figure Lengend Snippet: Interaction of HC1 and HC3 regions in reporter targeting. A and B. Fluorescence of live P. falciparum (left; bars, 5 μm) and fixed human 293T cells (right; bar, 1 μm). As in and , schematics of the constructs and mutations are shown and images are labelled at the top. Chimeras shown have the short HC1 version (Nt) (A) or long one (NtL) (B). C. Analysis of expressed constructs in P. falciparum . As in , chimeras, immunoprecipitated from stably transfected parasites, were analysed by Western blot using rabbit anti-GFP antibody (top) and, after stripping of the membrane, rabbit-anti-calpain antiserum #17 (bottom). D and E. Analysis of constructs in mammalian cells. 293T cells were transfected with plasmids encoding each of the chimeras as in (C). The cells were grown in normal (D), or labelling media (E). In (D), cell lysates were analysed by Western blotting using anti-GFP (top) to monitor expression levels or anti-calpain antiserum #17 (bottom). E. Alternatively, lysates were immunoprecipitated with anti-GFP antibody and fluorography was performed. F. Cellular fractionation analysis of Ntl-YFP-NLS and its two HC1 mutants, G2A and C3A. The distribution of the small chimeras (panel a) and Plasmepsin V (PMV), a membrane marker (panel b) in the total lysate (1/2 vol.), soluble (1 vol.) and membrane fractions (1 vol.) is shown.

Article Snippet: Other antibodies: rabbit anti-GFP 6556 (Abcam) diluted for microscopy (Mic) 1:200 and for WBs 1:5000; goat anti-GFP 5450 (Abcam) Mic 1:25; monoclonal anti-GFP (JL8, BD) WB 1:1000; rabbit anti-calpain #17 WB 1:250; monoclonal Anti-flag M2 Mic 1:100; monoclonal anti-His Mic 1:100; rat and rabbit anti-Bip (MR4) Mic 1:1500 and WB 1:10 000; monoclonal anti-hNop1 (Abcam) Mic 1:12.5; polyclonal anti-Pf_Nop1 ( Figueiredo et al ., 2005 ); Mic 1:25; anti-calpain #35 Mic 1:50 (whole cell) and 1:400 (purified nuclei); monoclonal anti-PMV , WB 1/400.

Techniques: Fluorescence, Construct, Immunoprecipitation, Stable Transfection, Transfection, Western Blot, Stripping Membranes, Expressing, Cell Fractionation, Marker

Journal: Molecular Microbiology

Article Title: Fatty acid acylation regulates trafficking of the unusual Plasmodium falciparum calpain to the nucleolus

doi: 10.1111/j.1365-2958.2009.06639.x

Figure Lengend Snippet: Localization of wild-type HC1-YFP-HC3 and full-length calpain-GFP in parasites treated with palmitoylation inhibitors. Live fluorescence of stably transfected P. falciparum episomally expressing NtL-YFP-NLS (A) and integration clone C3 expressing calpain-GFP (B). Images are labelled at the top for phase contrast, YFP, nuclear stain and merged channels. No drug controls; 2.5 μg ml −1 cerulenin; 10 μg ml −1 2-Bromo-palmitate are shown. Incubations were carried out for 20 h. Bars, 5 μm. (C) Cellular fractionation analysis of palmitoylation inhibitor-treated NtL-YFP-NLS. The distribution of the small chimera (panel a) and Plasmepsin V (panel b) in the total lysates (Tot, 1/2 vol.), soluble (Sol, 1 vol.) and membrane fractions (Membr, 1 vol.) is shown.

Article Snippet: Other antibodies: rabbit anti-GFP 6556 (Abcam) diluted for microscopy (Mic) 1:200 and for WBs 1:5000; goat anti-GFP 5450 (Abcam) Mic 1:25; monoclonal anti-GFP (JL8, BD) WB 1:1000; rabbit anti-calpain #17 WB 1:250; monoclonal Anti-flag M2 Mic 1:100; monoclonal anti-His Mic 1:100; rat and rabbit anti-Bip (MR4) Mic 1:1500 and WB 1:10 000; monoclonal anti-hNop1 (Abcam) Mic 1:12.5; polyclonal anti-Pf_Nop1 ( Figueiredo et al ., 2005 ); Mic 1:25; anti-calpain #35 Mic 1:50 (whole cell) and 1:400 (purified nuclei); monoclonal anti-PMV , WB 1/400.

Techniques: Fluorescence, Stable Transfection, Transfection, Expressing, Staining, Cell Fractionation

Journal: The American Journal of Pathology

Article Title: Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

doi: 10.1016/j.ajpath.2014.11.026

Figure Lengend Snippet: Cathepsin S inhibitor RO5444101 decreases plaque size, macrophage accumulation, and growth differentiation factor-15 (GDF15) expression in the atherosclerotic arteries of Apoe−/− mice with chronic renal disease (CRD). A and D: Quantification of atherosclerotic plaque size at the lesser curvature of the aortic arch using hematoxylin and eosin (H&E) staining. B and E: Mac3 staining in the aortic arch (B) shows macrophage accumulation on representative images; quantitative analysis of Mac3 staining (E). C and F: Immunohistochemical staining for a proinflammatory cytokine, GDF15, in the aortic arch (C) and its quantification (F). Data represent means ± SD. ∗P < 0.05, ∗∗∗P < 0.001.

Article Snippet: Immunohistochemical analysis for macrophages (anti-mouse mac3; BD Biosciences, San Jose, CA), cathepsin S (anti-mouse cathepsin S; Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (goat anti-mouse polyclonal antibody; Serotech, Dusseldorf, Germany), and growth differentiation factor-15 (GDF15) (rabbit anti–Gdf-15 polyclonal antibody; Bioss Antibodies, Woburn, MA) was performed using the avidin-biotin peroxidase method.

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: The American Journal of Pathology

Article Title: Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

doi: 10.1016/j.ajpath.2014.11.026

Figure Lengend Snippet: Cathepsin S (CatS)–specific inhibition by RO5444101 attenuates interferon γ (IFN-γ)–induced expression of growth differentiation factor-15 (GDF15) and monocyte chemotactic protein-1 (MCP-1)/CCL2 in human and mouse macrophages. Cultured human primary macrophages (MΦ) differentiated from peripheral blood monocytes and mouse macrophage-like RAW264.7 cells were pretreated with cathepsin S inhibitor RO5444101 and then were incubated with IFN-γ. RO5444101 or siRNA against cathepsin S reduced IFN-γ–induced GDF15 (A) and MCP-1/CCL2 (B) expression in both human and mouse macrophages. N = 4 for all experiments. Data represent means ± SD. ∗P < 0.05 between comparisons. Ctrl, control; hIFN-γ, human IFN-γ; mIFN-γ, mouse IFN-γ.

Article Snippet: Immunohistochemical analysis for macrophages (anti-mouse mac3; BD Biosciences, San Jose, CA), cathepsin S (anti-mouse cathepsin S; Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (goat anti-mouse polyclonal antibody; Serotech, Dusseldorf, Germany), and growth differentiation factor-15 (GDF15) (rabbit anti–Gdf-15 polyclonal antibody; Bioss Antibodies, Woburn, MA) was performed using the avidin-biotin peroxidase method.

Techniques: Inhibition, Expressing, Cell Culture, Incubation

Journal: The American Journal of Pathology

Article Title: Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

doi: 10.1016/j.ajpath.2014.11.026

Figure Lengend Snippet: Cathepsin S inhibition in atherosclerotic plaques in chronic renal disease. Inhibition of cathepsin S may reduce lesion size via decreasing growth differentiation factor-15 (GDF15), a modulator of macrophage chemotaxis. Cathepsin S inhibition also may decrease osteogenic stimuli, such as osteopontin and osteocalcin, by suppressing elastin degradation. CCR2, C-C chemokine receptor type 2.

Article Snippet: Immunohistochemical analysis for macrophages (anti-mouse mac3; BD Biosciences, San Jose, CA), cathepsin S (anti-mouse cathepsin S; Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (goat anti-mouse polyclonal antibody; Serotech, Dusseldorf, Germany), and growth differentiation factor-15 (GDF15) (rabbit anti–Gdf-15 polyclonal antibody; Bioss Antibodies, Woburn, MA) was performed using the avidin-biotin peroxidase method.

Techniques: Inhibition, Chemotaxis Assay