Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: α-Spectrin is mainly degraded in the RBCs after P. falciparum invasion. ( A ) Western blots of α-spectrin and β-spectrin in P. falciparum -iRBCs at different erythrocytic stages. R, T, and S indicate P. falciparum at the ring, trophozoite, and schizont stages, respectively. ( B and C ) Relative quantification of α-spectrin and β-spectrin protein levels obtained in panel A. ( D and E ) α-Spectrin and β-spectrin in iRBCs were detected via immunofluorescence staining. ( F ) The bubble chart represents the enrichment of Plasmodium proteins in KEGG pathways over 8-, 16-, 24-, 32-, 40-, and 48-h post-parasite invasion. The size of the circles indicates the number of proteins enriched in the signaling pathway, and the color of each circle indicates the enrichment score. The enrichment score was calculated by comparing the proteins’ enrichment in iRBCs to that in the RBCs, with blue representing the lowest score and orange the highest score. ( G ) The heatmap represents the abundance of α-spectrin ubiquitinated sites in iRBCs over 8-, 16-, 24-, 32-, 40-, and 48-h post-parasite invasion. Only the lysine 2321 in α-spectrin was ubiquitinated in uninfected RBC, nine more lysine residues were ubiquitinated after parasite invasion. ( H ) The distribution of ubiquitinated lysine residues in α-spectrin of P. falciparum -iRBCs is graphically represented.

Article Snippet: Following incubation, agarose beads were washed five times with 1 mL of co-IP lysis buffer and eluted with 15 µL of 5× SDS sample loading buffer (Beyotime, cat#P0015L) boiled for 10 min. Proteins were released, separated on 6% SDS-PAGE, immunoblotted using primary antibodies with mouse anti-α-spectrin (Abnova, cat#MAB2515, 1:1,000) and rabbit anti-ubiquitin (PTM Biolabs, cat#PTM1106, 1:1,000), and then incubated with the differentially labeled species-specific secondary antibodies, HRP-conjugated goat anti-rabbit IgG (BBI Life Sciences Corporation, cat#D110058, 1:10,000) and HRP-conjugated goat anti-mouse IgG (BBI Life Sciences Corporation, cat#D110087, 1:10,000).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: PfPI3K regulates P. falciparum ubiquitin-proteasome system. ( A ) Western blots of α-spectrin and β-spectrin in the ring-stage P. falciparum -iRBCs with or without the treatment of the PfPI3K inhibitor LY294002 (50 µM). ( B and C ) Relative quantification of α-spectrin and β-spectrin protein levels shown in panel A. ( D ) Immunofluorescence staining of α-spectrin with a protein-specific antibody in cells with or without LY294002 (50 µM) treatment. ( E ) Functional categorization of proteins identified in iRBC of ring-stage parasites by proteomic analysis after treatment with LY294002. ( F ) Categorization of identified proteins based on structural domains in proteins identified in iRBC of ring-stage parasites after treatment with LY294002. Proteins associated with ubiquination and proteasome are highlighted in blue color. ( G ) Downregulated proteins were identified in iRBC of ring-stage parasites by proteomic analysis after treatment with LY294002. Ubiquitin-conjugating enzymes E2 K (UBE2K) were the proteins predominantly affected by the inhibitor. ( H ) Western blots of UBE2K in ring-stage P. falciparum -iRBCs with or without the treatment of the PfPI3K inhibitor LY294002 (50 µM). ( I ) Relative quantification of UBE2K protein level shown in panel H. Inhibition of PfPI3K activity significantly downregulated the UBE2K.

Article Snippet: Following incubation, agarose beads were washed five times with 1 mL of co-IP lysis buffer and eluted with 15 µL of 5× SDS sample loading buffer (Beyotime, cat#P0015L) boiled for 10 min. Proteins were released, separated on 6% SDS-PAGE, immunoblotted using primary antibodies with mouse anti-α-spectrin (Abnova, cat#MAB2515, 1:1,000) and rabbit anti-ubiquitin (PTM Biolabs, cat#PTM1106, 1:1,000), and then incubated with the differentially labeled species-specific secondary antibodies, HRP-conjugated goat anti-rabbit IgG (BBI Life Sciences Corporation, cat#D110058, 1:10,000) and HRP-conjugated goat anti-mouse IgG (BBI Life Sciences Corporation, cat#D110087, 1:10,000).

Techniques: Western Blot, Immunofluorescence, Staining, Functional Assay, Inhibition, Activity Assay

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: Host NEDD4L is necessary for the ubiquitination of α-spectrin in the blood stage of Plasmodium parasites. ( A ) Of the 18 E3 ubiquitin ligases potentially involved in the interaction with α-spectrin, 3 were identified in the proteomic analysis, and none were involved in β-spectrin ubiquitination. The black and blue circles represent the predicted E3 ubiquitin ligase to α-spectrin and β-spectrin, respectively ( http://ubibrowser.bio-it.cn/ ); The green circle represents the E3 ubiquitin ligase identified by LC-MS/MS in iRBCs. ( B ) NEDD4L was detected via immunofluorescence staining in RBCs and iRBCs with an anti-NEDD4L antibody. ( C ) Western blots of NEDD4L in whole-cell extracts from RBCs, iRBCs, and purified P. falciparum 3D7 with an anti-NEDD4L antibody. NEDD4L was detected in both RBCs and iRBCs but not in the purified P. falciparum . ( D ) Western blots of NEDD4L and P-NEDD4L with anti-NEDD4L and anti-P-NEDD4L antibodies in the ring stage of P. falciparum -iRBCs after treatment with the PfPI3K and E3 ubiquitin ligase inhibitors LY294002 (50 µM) and heclin (5 µM), respectively, using e-BLOT touch imager. ( E and F ) Relative quantification of the NEDD4L and P-NEDD4L protein levels shown in panel D. ( G and H ) The ubiquitinated α-spectrin could not be degraded in RBCs. However, it was completely degraded in iRBCs with ring-stage parasites. The PfPI3K inhibitor LY294002 (50 µM) partially inhibited ubiquitination and degradation of α-spectrin, but the effect of NEDD4L inhibitor heclin (5 µM) was more significant. ( I ) RBC lysates were subjected to co-immunoprecipitation (IP) using anti-α-spectrin antibodies, and α-spectrin-ubiquitin complexes were detected with anti-ubiquitin antibody immunoblotting. Ubiquitination was observed on α-spectrin in RBC lysates (input). The α-spectrin-ubiquitin complex was not detected in the IP with an irrelevant IgG control. ( J ) Heclin showed significant inhibition of parasite proliferation in a dose-dependent manner, compared to the control medium. ( K and L ) Heclin treatment significantly reduced parasitemia in P. berghei ANKA-infected mice. The control mice were the same as that illustrated in . ( M ) Statistical analysis of the luminescence showed that heclin treatment significantly reduced parasitemia in P. berghei ANKA-infected mice.

Article Snippet: Following incubation, agarose beads were washed five times with 1 mL of co-IP lysis buffer and eluted with 15 µL of 5× SDS sample loading buffer (Beyotime, cat#P0015L) boiled for 10 min. Proteins were released, separated on 6% SDS-PAGE, immunoblotted using primary antibodies with mouse anti-α-spectrin (Abnova, cat#MAB2515, 1:1,000) and rabbit anti-ubiquitin (PTM Biolabs, cat#PTM1106, 1:1,000), and then incubated with the differentially labeled species-specific secondary antibodies, HRP-conjugated goat anti-rabbit IgG (BBI Life Sciences Corporation, cat#D110058, 1:10,000) and HRP-conjugated goat anti-mouse IgG (BBI Life Sciences Corporation, cat#D110087, 1:10,000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Staining, Western Blot, Purification, Immunoprecipitation, Control, Inhibition, Infection

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: The 26S proteasome was involved in α-spectrin degradation at the early erythrocytic stage P. falciparum . ( A and B ) The heatmaps represent the abundance of the 20S ( A ) and 26S ( B ) proteasomes in the iRBCs over 8-, 16-, 24-, 32-, 40-, and 48-h post-parasite invasion. ( C and D ) Treatment with the PfPI3K and 26S proteasome inhibitors, LY294002 and MG132, did not alter PSMA1 (20S proteasome) and PSMD1 (26S proteasome) transcription in ring-stage P. falciparum . ( E ) Western blots show that treatment with the respective PfPI3K and 26S proteasome inhibitors, LY294002 (50 µM) and MG132 (100 nM), did not affect PSMA1 and PSMD1 expression. ( F and G ) Relative quantification of PSMA1 and PSMD1 protein levels shown in panel E. ( H and I ) The ubiquitinated α-spectrin completely degraded iRBCs with ring-stage parasites (R). PfPI3K inhibitor LY294002 (50 µM) partially inhibited the ubiquitination and degradation of α-spectrin, but the 26S proteasome inhibitor MG132 (100 nM) only inhibited the degradation of α-spectrin in iRBCs. ( J ) MG132 significantly inhibited P. falciparum proliferation in vitro in a dose-dependent manner, compared to the medium control. ( K and L ) MG132 treatment significantly reduced parasitemia in P. berghei ANKA-infected mice. The experiment was carried out continuously with the same group of mice injected with a saline solution ( , control). ( M ) Statistical analysis of the luminescence showed that MG132 treatment significantly reduced parasitemia in P. berghei ANKA-infected mice.

Article Snippet: Following incubation, agarose beads were washed five times with 1 mL of co-IP lysis buffer and eluted with 15 µL of 5× SDS sample loading buffer (Beyotime, cat#P0015L) boiled for 10 min. Proteins were released, separated on 6% SDS-PAGE, immunoblotted using primary antibodies with mouse anti-α-spectrin (Abnova, cat#MAB2515, 1:1,000) and rabbit anti-ubiquitin (PTM Biolabs, cat#PTM1106, 1:1,000), and then incubated with the differentially labeled species-specific secondary antibodies, HRP-conjugated goat anti-rabbit IgG (BBI Life Sciences Corporation, cat#D110058, 1:10,000) and HRP-conjugated goat anti-mouse IgG (BBI Life Sciences Corporation, cat#D110087, 1:10,000).

Techniques: Western Blot, Expressing, In Vitro, Control, Infection, Injection, Saline

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: A schematic illustration of P. falciparum PI3K signaling in the regulation of selective α-spectrin processing. PfPI3K phosphorylates and activates P. falciparum ubiquitin-protein ligase, which promotes α-spectrin ubiquitination and degradation during the early stage of P. falciparum development.

Article Snippet: Following incubation, agarose beads were washed five times with 1 mL of co-IP lysis buffer and eluted with 15 µL of 5× SDS sample loading buffer (Beyotime, cat#P0015L) boiled for 10 min. Proteins were released, separated on 6% SDS-PAGE, immunoblotted using primary antibodies with mouse anti-α-spectrin (Abnova, cat#MAB2515, 1:1,000) and rabbit anti-ubiquitin (PTM Biolabs, cat#PTM1106, 1:1,000), and then incubated with the differentially labeled species-specific secondary antibodies, HRP-conjugated goat anti-rabbit IgG (BBI Life Sciences Corporation, cat#D110058, 1:10,000) and HRP-conjugated goat anti-mouse IgG (BBI Life Sciences Corporation, cat#D110087, 1:10,000).

Techniques:

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: α-Spectrin is mainly degraded in the RBCs after P. falciparum invasion. ( A ) Western blots of α-spectrin and β-spectrin in P. falciparum -iRBCs at different erythrocytic stages. R, T, and S indicate P. falciparum at the ring, trophozoite, and schizont stages, respectively. ( B and C ) Relative quantification of α-spectrin and β-spectrin protein levels obtained in panel A. ( D and E ) α-Spectrin and β-spectrin in iRBCs were detected via immunofluorescence staining. ( F ) The bubble chart represents the enrichment of Plasmodium proteins in KEGG pathways over 8-, 16-, 24-, 32-, 40-, and 48-h post-parasite invasion. The size of the circles indicates the number of proteins enriched in the signaling pathway, and the color of each circle indicates the enrichment score. The enrichment score was calculated by comparing the proteins’ enrichment in iRBCs to that in the RBCs, with blue representing the lowest score and orange the highest score. ( G ) The heatmap represents the abundance of α-spectrin ubiquitinated sites in iRBCs over 8-, 16-, 24-, 32-, 40-, and 48-h post-parasite invasion. Only the lysine 2321 in α-spectrin was ubiquitinated in uninfected RBC, nine more lysine residues were ubiquitinated after parasite invasion. ( H ) The distribution of ubiquitinated lysine residues in α-spectrin of P. falciparum -iRBCs is graphically represented.

Article Snippet: The cell protein (500 μg) was incubated with mouse anti-α-spectrin monoclonal antibody (1 μg, Abnova, cat#MAB2515) overnight rotating at 4°C, and mouse IgG (1 μg, Solarbio, cat#SP031) was used as a control.

Techniques: Western Blot, Quantitative Proteomics, Immunofluorescence, Staining

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: PfPI3K regulates P. falciparum ubiquitin-proteasome system. ( A ) Western blots of α-spectrin and β-spectrin in the ring-stage P. falciparum -iRBCs with or without the treatment of the PfPI3K inhibitor LY294002 (50 µM). ( B and C ) Relative quantification of α-spectrin and β-spectrin protein levels shown in panel A. ( D ) Immunofluorescence staining of α-spectrin with a protein-specific antibody in cells with or without LY294002 (50 µM) treatment. ( E ) Functional categorization of proteins identified in iRBC of ring-stage parasites by proteomic analysis after treatment with LY294002. ( F ) Categorization of identified proteins based on structural domains in proteins identified in iRBC of ring-stage parasites after treatment with LY294002. Proteins associated with ubiquination and proteasome are highlighted in blue color. ( G ) Downregulated proteins were identified in iRBC of ring-stage parasites by proteomic analysis after treatment with LY294002. Ubiquitin-conjugating enzymes E2 K (UBE2K) were the proteins predominantly affected by the inhibitor. ( H ) Western blots of UBE2K in ring-stage P. falciparum -iRBCs with or without the treatment of the PfPI3K inhibitor LY294002 (50 µM). ( I ) Relative quantification of UBE2K protein level shown in panel H. Inhibition of PfPI3K activity significantly downregulated the UBE2K.

Article Snippet: The cell protein (500 μg) was incubated with mouse anti-α-spectrin monoclonal antibody (1 μg, Abnova, cat#MAB2515) overnight rotating at 4°C, and mouse IgG (1 μg, Solarbio, cat#SP031) was used as a control.

Techniques: Ubiquitin Proteomics, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining, Functional Assay, Inhibition, Activity Assay

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: Host NEDD4L is necessary for the ubiquitination of α-spectrin in the blood stage of Plasmodium parasites. ( A ) Of the 18 E3 ubiquitin ligases potentially involved in the interaction with α-spectrin, 3 were identified in the proteomic analysis, and none were involved in β-spectrin ubiquitination. The black and blue circles represent the predicted E3 ubiquitin ligase to α-spectrin and β-spectrin, respectively ( http://ubibrowser.bio-it.cn/ ); The green circle represents the E3 ubiquitin ligase identified by LC-MS/MS in iRBCs. ( B ) NEDD4L was detected via immunofluorescence staining in RBCs and iRBCs with an anti-NEDD4L antibody. ( C ) Western blots of NEDD4L in whole-cell extracts from RBCs, iRBCs, and purified P. falciparum 3D7 with an anti-NEDD4L antibody. NEDD4L was detected in both RBCs and iRBCs but not in the purified P. falciparum . ( D ) Western blots of NEDD4L and P-NEDD4L with anti-NEDD4L and anti-P-NEDD4L antibodies in the ring stage of P. falciparum -iRBCs after treatment with the PfPI3K and E3 ubiquitin ligase inhibitors LY294002 (50 µM) and heclin (5 µM), respectively, using e-BLOT touch imager. ( E and F ) Relative quantification of the NEDD4L and P-NEDD4L protein levels shown in panel D. ( G and H ) The ubiquitinated α-spectrin could not be degraded in RBCs. However, it was completely degraded in iRBCs with ring-stage parasites. The PfPI3K inhibitor LY294002 (50 µM) partially inhibited ubiquitination and degradation of α-spectrin, but the effect of NEDD4L inhibitor heclin (5 µM) was more significant. ( I ) RBC lysates were subjected to co-immunoprecipitation (IP) using anti-α-spectrin antibodies, and α-spectrin-ubiquitin complexes were detected with anti-ubiquitin antibody immunoblotting. Ubiquitination was observed on α-spectrin in RBC lysates (input). The α-spectrin-ubiquitin complex was not detected in the IP with an irrelevant IgG control. ( J ) Heclin showed significant inhibition of parasite proliferation in a dose-dependent manner, compared to the control medium. ( K and L ) Heclin treatment significantly reduced parasitemia in P. berghei ANKA-infected mice. The control mice were the same as that illustrated in . ( M ) Statistical analysis of the luminescence showed that heclin treatment significantly reduced parasitemia in P. berghei ANKA-infected mice.

Article Snippet: The cell protein (500 μg) was incubated with mouse anti-α-spectrin monoclonal antibody (1 μg, Abnova, cat#MAB2515) overnight rotating at 4°C, and mouse IgG (1 μg, Solarbio, cat#SP031) was used as a control.

Techniques: Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Staining, Western Blot, Purification, Quantitative Proteomics, Immunoprecipitation, Control, Inhibition, Infection

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: The 26S proteasome was involved in α-spectrin degradation at the early erythrocytic stage P. falciparum . ( A and B ) The heatmaps represent the abundance of the 20S ( A ) and 26S ( B ) proteasomes in the iRBCs over 8-, 16-, 24-, 32-, 40-, and 48-h post-parasite invasion. ( C and D ) Treatment with the PfPI3K and 26S proteasome inhibitors, LY294002 and MG132, did not alter PSMA1 (20S proteasome) and PSMD1 (26S proteasome) transcription in ring-stage P. falciparum . ( E ) Western blots show that treatment with the respective PfPI3K and 26S proteasome inhibitors, LY294002 (50 µM) and MG132 (100 nM), did not affect PSMA1 and PSMD1 expression. ( F and G ) Relative quantification of PSMA1 and PSMD1 protein levels shown in panel E. ( H and I ) The ubiquitinated α-spectrin completely degraded iRBCs with ring-stage parasites (R). PfPI3K inhibitor LY294002 (50 µM) partially inhibited the ubiquitination and degradation of α-spectrin, but the 26S proteasome inhibitor MG132 (100 nM) only inhibited the degradation of α-spectrin in iRBCs. ( J ) MG132 significantly inhibited P. falciparum proliferation in vitro in a dose-dependent manner, compared to the medium control. ( K and L ) MG132 treatment significantly reduced parasitemia in P. berghei ANKA-infected mice. The experiment was carried out continuously with the same group of mice injected with a saline solution ( , control). ( M ) Statistical analysis of the luminescence showed that MG132 treatment significantly reduced parasitemia in P. berghei ANKA-infected mice.

Article Snippet: The cell protein (500 μg) was incubated with mouse anti-α-spectrin monoclonal antibody (1 μg, Abnova, cat#MAB2515) overnight rotating at 4°C, and mouse IgG (1 μg, Solarbio, cat#SP031) was used as a control.

Techniques: Western Blot, Expressing, Quantitative Proteomics, Ubiquitin Proteomics, In Vitro, Control, Infection, Injection, Saline

Journal: mBio

Article Title: Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion

doi: 10.1128/mbio.03510-23

Figure Lengend Snippet: A schematic illustration of P. falciparum PI3K signaling in the regulation of selective α-spectrin processing. PfPI3K phosphorylates and activates P. falciparum ubiquitin-protein ligase, which promotes α-spectrin ubiquitination and degradation during the early stage of P. falciparum development.

Article Snippet: The cell protein (500 μg) was incubated with mouse anti-α-spectrin monoclonal antibody (1 μg, Abnova, cat#MAB2515) overnight rotating at 4°C, and mouse IgG (1 μg, Solarbio, cat#SP031) was used as a control.

Techniques: Ubiquitin Proteomics