Fig 1: Proteomic analysis of Pf-derived EV subpopulationsTotal proteins were extracted from the two EV fractions (F3 and F4) and subjected to LC–MS/MS analysis. GO annotation analysis of the two EV fractions.The figure presents the most intense proteins in the EV repertoire (not including the HBB and HBA dominant proteins). The rectangular areas are proportional to the median intensity of the protein in either F3 or F4 EVs.Subcellular localization of identified proteins by GO annotation. Other—mitochondria, early and late endosome, lysosome, cytoskeleton, secreted, and so on. Quantification and averaging of four independent biological replicates. Reference list: Homo sapiens or Plasmodium falciparum (all genes in the database).Western blot analysis of uRBCs or iRBCs and Pf-derived EVs. Anti-PSMA1, anti-PSMB2 (proteasome subunits) and anti-C3 (complement system protein) antibodies were used. As a positive control, antibodies against the EV markers HSP90 and Annexin 7 were used. C3b subunit is detected within Pf-derived EVs. As a negative control, antibodies against the RBC membrane marker Ankyrin-1 were used. Source data are available online for this figure.
Fig 2: A de novo-designed C-PLoop protein binds the 20S proteasome.a To examine whether C-PLoop can protect a-synuclein (a-syn) from the 20S-mediated proteolysis, we incubated a mixture of a-syn and 20S proteasomes from archaea, yeast, and rat with C-PLoop to perform time-dependent degradation assays. Regardless of the 20S proteasome species, the addition of C-PLoop rescued the degradation of a-syn. Quantification of a-syn levels from three independent experiments is displayed on the bottom as mean intensities; error bars represent SD. Free b archaeal and c rat 20S proteasomes and 20S complexes pre-incubated with C-PLoop, were examined by native MS. For each sample, the most intense charge state obtained in the MS spectrum was subjected to MS/MS analysis (inset in (b) and (c) shows the MS spectrum of the free 20S proteasomes; the 65+ charge state highlighted in red was subjected to MS/MS analysis). Comparison of the free 20S spectrum (top panels b, c) with a mixture of the 20S proteasome and C-PLoop (lower panels b, c) revealed additional peaks that corresponded in mass to C-PLoop. By extrapolation, we can therefore conclude that prior to MS/MS analysis, C-PLoop binds to the 20S proteasome. Blue circles correspond to a-subunits of the archaea and rat 20S proteasomes; orange circles represent C-PLoop. d–g For cellular experiments, HA-tagged C-PLoop was overexpressed in HEK293T cells, stably expressing the FLAG-tagged PSMB2 subunit of the 20S proteasome. Lysates were subjected to IP using either d anti-FLAG-affinity gel, e anti-HA, or f anti-PSMD1 antibodies, or g uncoupled protein G beads as a control. Total starting lysate (L), unbound proteins (UB), and IP samples were analyzed by Western blot using anti-PSMA1, anti-FLAG, anti-HA, or anti-PSMD1 antibodies. h Bands corresponding to HA (i.e., C-PLoop) in the FLAG (20S) IP and FLAG (20S) in the HA IP were quantified and compared with the protein G control. Quantifications demonstrate the average of three independent experiments. Measurements were subjected to a one-tailed Student t-test analysis. Error bars represent SEM. * for IP:FLAG (20S) and IP:C-PLoop-HA represents p-values = 0.0350 and 0.0380, respectively. Source data are provided with this paper.
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