Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate

Quantification of von Willebrand factor and ADAMTS-13 after traumatic injury: a pilot study

Background Von Willebrand factor (VWF) is an acute phase reactant synthesized in the megakaryocytes and endothelial cells. VWF forms ultra-large multimers (ULVWF) which are cleaved by the metalloprotease ADAMTS-13, preventing spontaneous VWF–platelet interaction. After trauma, ULVWF is released into circulation as part of the acute phase reaction. We hypothesized that trauma patients would have increased levels of VWF and decreased levels of ADAMTS-13 and that these patients would have accelerated thrombin generation.Methods We assessed plasma concentrations of VWF antigen and ADAMTS-13 antigen, the Rapid Enzyme Assays for Autoimmune Diseases (REAADS) activity of VWF, which measure exposure of the platelet-binding A1 domain, and thrombin generation kinetics in 50 samples from 30 trauma patients and an additional 21 samples from volunteers. Samples were analyzed at 0 to 2 hours and at 6 hours from the time of injury. Data are presented as median (IQR) and Kruskal-Wallis test was performed between trauma patients and volunteers at both time points.Results REAADS activity was greater in trauma patients than volunteers both at 0 to 2 hours (190.0 (132.0–264.0) vs. 92.0 (71.0–114.0), p<0.002) and at 6 hours (167.5 (108.0–312.5.0) vs. 92.0 (71.0–114.0), p<0.001). ADAMTS-13 antigen levels were also decreased in trauma patients both at 0 to 2 hours (0.84 (0.51–0.94) vs. 1.00 (0.89–1.09), p=0.010) and at 6 hours (0.653 (0.531–0.821) vs. 1.00 (0.89–1.09), p<0.001). Trauma patients had accelerated thrombin generation kinetics, with greater peak height and shorter time to peak than healthy volunteers at both time points.Discussion Trauma patients have increased exposure of the VWF A1 domain and decreased levels of ADAMTS-13 compared with healthy volunteers. This suggests that the VWF burst after trauma may exceed the proteolytic capacity of ADAMTS-13, allowing circulating ULVWF multimers to bind platelets, potentially contributing to trauma-induced coagulopathy.Level of evidence Prospective case cohort study

Immunofluorescence microscopy analysis of TbIP₃R.

<p>(A) TbIP₃R co-localized with TbVP1 in acidocalcisomes of PCF trypanosomes (Pearson's correlation coefficient of 0.8399). <i>Yellow</i> in merge images indicates co-localization. <i>Scale bars</i> = 10 µm. (B) Western blot analysis of TbIP₃R expressed in PCF trypanosomes using polyclonal anti-TbIP₃R antibody. Lysate containing 30 µg of protein from PCF trypanosomes was subjected to SDS/PAGE on 4–15% polyacrylamide gel, and transferred to a nitrocellulose membrane. Molecular weight markers at <i>left</i> and <i>arrow</i> shows the band corresponding to TbIP₃R.</p

Immunofluorescence microscopy and western blot analysis of V-H⁺-ATPase subunit <i>a</i> in PCF trypanosomes.

<p>Epitope-tagged V-H⁺-ATPase subunit <i>a</i> co-localizes with TbVP1 to the acidocalcisomes (A), with TbGRASP to the Golgi complex (B) with TbCATL (C) and with p67 (D) and to lysosomes (Pearson's correlation coefficients of 0.631, 0.539, 0.804, and 0.754, respectively). <i>Yellow</i> in merge images indicate co-localization (also shown with <i>arrows</i> in (B–D)). <i>Scale bars</i> for (A–D) = 10 µm. (E) Confirmation of tagging by western blot analyses with monoclonal anti-HA in PCF trypanosomes. HRP-conjugated goat anti-mouse was used as a secondary antibody. Magic Mark XP (Invitrogen) was used as a molecular weight marker and <i>arrow</i> shows band corresponding to TbVA<i>a</i>. Tubulin (Tub) was used as a loading control (<i>bottom panel</i>).</p

Distribution on iodixanol gradients of organellar markers from PCF trypanosomes.

<p>(A) Photograph showing bands obtained after the second iodixanol gradient centrifugation. Fraction 5 corresponds to the purified acidocalcisomes. (B) Protein distribution. (C) TbVP1 activity (measured as the AMDP-sensitive P_i release) is concentrated in fractions 3 and 5. (D) Mitochondrial marker distribution, succinate cytochrome c reductase. (E) Glycosomal marker distribution, hexokinase. (F) Lysosomal marker distribution, α-mannosidase. In (B–F) the <i>y-</i>axis indicates relative distribution; the <i>x-</i>axis indicates fraction number; <i>bars</i> show means ± SD (as a percentage of the total recovered activity) from two or three independent experiments. (G) SDS-PAGE of Fraction 5 from a representative acidocalcisome (ACCS) fractionation stained with Coomassie brilliant blue. The relative intensities of the bands were obtained from a bitmap file of the gel image and is shown on the right. Background was subtracted. The approximate localization of the acidocalcisome proteins identified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004555#ppat-1004555-t002" target="_blank">Table 2</a> is shown. BenchMark protein markers are shown at the left.</p

Immunofluorescence microscopy and western blot analysis of proteins involved in phosphorus metabolism.

<p>TbVtc1 (A), TbVtc4 (B), TbPho91 (C), and TbAP (D) were 3×HA epitope-tagged in situ and co-localized with TbVP1 in acidocalcisomes of PCF trypanosomes (Pearson's correlation coefficients of 0.873, 0.734, 0.728, and 0.680, respectively). <i>Yellow</i> in merge images indicate co-localization. <i>Scale bars</i> for (A–D) = 10 µm. Western blot analyses with monoclonal anti-HA showing labeling of TbVtc1 (E), TbVtc4 (F), TbPho91 (G), and TbAP (H) in PCF trypanosomes. Molecular weight markers at <i>left</i>, and <i>arrows</i> show the corresponding bands identified. Tubulin (<i>Tub</i>) was used as a loading control.</p

Schematic representation of the acidocalcisome of <i>T. brucei</i>.

<p>The identified acidocalcisome proteins corresponding to the pumps, exchangers, transporters or protein complexes in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004555#ppat-1004555-t002" target="_blank">Table 2</a> are shown in this model. The newly identified acidocalcisome proteins in this study are marked in blue.</p

Identification of acidocalcisome protein candidates in <i>T. brucei</i>, showing localization and essentiality in BSF or PCF.

<p>MW, molecular weight; TMD, transmembrane domains; Ac, acidocalcisome; IP₃, inositol 1,4,5-trisphosphate; PPase, pyrophosphatase; -, not tested.</p><p>*Proteins for which peptides were not found in the acidocalcisome proteome.</p><p>Identification of acidocalcisome protein candidates in <i>T. brucei</i>, showing localization and essentiality in BSF or PCF.</p