Mechanism of platelet α-granule biogenesis: study of cargo transport and the VPS33B-VPS16B complex in a model system.

Platelet α-granules play important roles in platelet function. They contain hundreds of proteins that are synthesized by the megakaryocyte or taken up by endocytosis. The trafficking pathways that mediate platelet α-granule biogenesis are incompletely understood, especially with regard to cargo synthesized by the megakaryocyte. Vacuolar-protein sorting 33B (VPS33B) and VPS16B are essential proteins for α-granule biogenesis, but they are largely uncharacterized. Here, we adapted a powerful method to directly map the pathway followed by newly synthesized cargo proteins to reach α-granules. Using this method, we revealed the recycling endosome as a key intermediate compartment in α-granule biogenesis. We then used CRISPR/Cas9 gene editing to knock out VPS33B in pluripotent stem cell-derived immortalized megakaryocyte cells (imMKCLs). Consistent with the observations in platelets from patients with VPS33B mutation, VPS33B-knockout (KO) imMKCLs have drastically reduced levels of α-granule proteins platelet factor 4, von Willebrand factor, and P-selectin. VPS33B and VPS16B form a distinct and small complex in imMKCLs with the same hydrodynamic radius as the recombinant VPS33B-VPS16B heterodimer purified from bacteria. Mechanistically, the VPS33B-VPS16B complex ensures the correct trafficking of α-granule proteins. VPS33B deficiency results in α-granule cargo degradation in lysosomes. VPS16B steady-state levels are significantly lower in VPS33B-KO imMKCLs, suggesting that VPS16B is destabilized in the absence of its partner. Exogenous expression of green fluorescent protein-VPS33B in VPS33B-KO imMKCLs reconstitutes the complex, which localizes to the recycling endosome, further defining this compartment as a key intermediate in α-granule biogenesis. These results advance our understanding of platelet α-granule biogenesis and open new avenues for the study of these organelles.

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

Identification and quantification of host proteins in the vesicular fluid of porcine Taenia solium cysticerci.

The host-parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pig's sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3-8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3-8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11-13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.

A novel monoclonal antibody against the C-terminus of ß-tubulin recognizes endocytic organelles in Trypanosoma cruzi.

Microtubule cytoskeleton is a dynamic structure involved in the maintenance of eukaryote cell shape, motion of cilia and flagellum, and intracellular movement of vesicles and organelles. Many antibodies against tubulins have been described, most of them against the C-terminal portion, which is exposed at the outside of the microtubules. By generating a novel set of monoclonal antibodies against the cytoskeleton of Trypanosoma cruzi, a flagellate protozoan that causes Chagas' disease, we selected a clone (mAb 3G4) that recognizes ß-tubulin. The epitope for mAb 3G4 was mapped by pepscan to a highly conserved sequence motif found between α-helices 11 and 12 of the C-terminus of ß-tubulin in eukaryotes. It labels vesicular structures in both T. cruzi and mammalian cells, colocalizing respectively with a major cysteine protease (Cruzipain) and lysosome associated protein (LAMP2) respectively, but it does not label regular microtubules on these cellular models. We propose that the epitope recognized by mAb 3G4 is exposed only in a form of tubulin associated with endosomes.

Chitosomes loaded with cranberry proanthocyanidins attenuate the bacterial lipopolysaccharide-induced expression of iNOS and COX-2 in raw 264.7 macrophages.

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its positively charged amino group. This interaction allows stable covered vesicles (chitosomes) to be developed as a suitable targeted carrier and controlled release system. This study investigated the effect of chitosomes on the activation of cranberry proanthocyanidins (PAC) in Raw 264.7 macrophages. Chitosomes were characterized according to size, zeta potential, PAC-loading, and release properties. Results showed an increase in the net positive charge and size of the liposomes as the concentration of chitosan was increased, suggesting an effective covering of the vesicles by means of electrostatic interactions, as shown by transmission electron microscopy and fluorescence microscopy. About 85% of the PAC that was loaded remained in the chitosomes after release studies for 4 hours in phosphate-buffered saline. Cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are associated with inflammation. Activated RAW 264.7 macrophages increase the expression of COX-2 and iNOS in response to bacterial infection and inflammation; we, therefore, tested the ability of the PAC-loaded chitosomes to attenuate COX-2 and iNOS expression in LPS (lipopolysaccharide)-stimulated macrophages. Increasing the amount of PAC loaded into the chitosomes caused a dose-dependent attenuation of iNOS and COX-2 expression in LPS-stimulated macrophages. A 2% v/v PAC-loaded chitosomes formulation almost completely attenuated the LPS-induced expression of iNOS and COX-2. PAC-loaded chitosomes were more active than PAC alone, suggesting that the macrophage response to LPS occurs after endocytosis of the PAC-loaded chitosomes.

Peritrophic membrane protein in the larval stingless bee Melipona quadrifasciata anthidioides: immunolocalization of secretory sites.

The origin of the peritrophic membrane (PM) in bees is still a matter of debate. It is either of type I (synthesized by the entire midgut epithelium) or of type II (released from the anterior midgut end). The present study identified secretory sites of peritrophin-55 kDa, a PM protein in larvae of Melipona quadrifasciata anthidioides. Peritrophin-55 was isolated from PMs and was used for the production of a polyclonal antibody. Our study demonstrates the presence of peritrophin-55 in vesicles and on microvilli of digestive cells and in the PM. It suggests that the PM is of type-I, being specific for the larval phase of this stingless bee.