Landscape of the mitochondrial Hsp90 metabolome in tumours.

Reprogramming of tumour cell metabolism contributes to disease progression and resistance to therapy, but how this process is regulated on the molecular level is unclear. Here we report that heat shock protein 90-directed protein folding in mitochondria controls central metabolic networks in tumour cells, including the electron transport chain, citric acid cycle, fatty acid oxidation, amino acid synthesis and cellular redox status. Specifically, mitochondrial heat shock protein 90, but not cytosolic heat shock protein 90, binds and stabilizes the electron transport chain Complex II subunit succinate dehydrogenase-B, maintaining cellular respiration under low-nutrient conditions, and contributing to hypoxia-inducible factor-1α-mediated tumorigenesis in patients carrying succinate dehydrogenase-B mutations. Thus, heat shock protein 90-directed proteostasis in mitochondria regulates tumour cell metabolism, and may provide a tractable target for cancer therapy.

PI(3)P-independent and -dependent pathways function together in a vacuolar translocation sequence to target malarial proteins to the host erythrocyte.

Malaria parasites export 'a secretome' of hundreds of proteins, including major virulence determinants, from their endoplasmic reticulum (ER), past the parasite plasma and vacuolar membranes to the host erythrocyte. The export mechanism is high affinity (nanomolar) binding of a host (cell) targeting (HT) motif RxLxE/D/Q to the lipid phosphatidylinositol 3-phosphate (PI(3)P) in the ER. Cleavage of the HT motif releases the secretory protein from the ER membrane. The HT motif is thought to be the only export signal resident in an N-terminal vacuolar translocation sequence (VTS) that quantitatively targets green fluorescent protein to the erythrocyte. We have previously shown that the R to A mutation in the HT motif, abrogates VTS binding to PI(3)P (K(d)>5 µM). We now show that remarkably, the R to A mutant is exported to the host erythrocyte, for both membrane and soluble reporters, although the efficiency of export is reduced to ~30% of that seen with a complete VTS. Mass spectrometry indicates that the R to A mutant is cleaved at sites upstream of the HT motif. Antibodies to upstream sequences confirm that aberrantly cleaved R to A protein mutant is exported to the erythrocyte. These data suggest that export mechanisms, independent of PI(3)P as well as those dependent on PI(3)P, function together in a VTS to target parasite proteins to the host erythrocyte.

Endoplasmic reticulum PI(3)P lipid binding targets malaria proteins to the host cell.

Hundreds of effector proteins of the human malaria parasite Plasmodium falciparum constitute a "secretome" carrying a host-targeting (HT) signal, which predicts their export from the intracellular pathogen into the surrounding erythrocyte. Cleavage of the HT signal by a parasite endoplasmic reticulum (ER) protease, plasmepsin V, is the proposed export mechanism. Here, we show that the HT signal facilitates export by recognition of the lipid phosphatidylinositol-3-phosphate (PI(3)P) in the ER, prior to and independent of protease action. Secretome HT signals, including those of major virulence determinants, bind PI(3)P with nanomolar affinity and amino acid specificities displayed by HT-mediated export. PI(3)P-enriched regions are detected within the parasite's ER and colocalize with endogenous HT signal on ER precursors, which also display high-affinity binding to PI(3)P. A related pathogenic oomycete's HT signal export is dependent on PI(3)P binding, without cleavage by plasmepsin V. Thus, PI(3)P in the ER functions in mechanisms of secretion and pathogenesis.

Deciphering the human platelet sheddome.

Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function.

N-terminal sequence analysis of proteins and peptides.

Automated N-terminal sequence analysis involves a series of chemical reactions that derivatize and remove one amino acid at a time from the N-terminus of purified peptides or intact proteins. At least several picomoles of a purified protein or 10 to 20 pmol of a purified peptide with an unmodified N-terminus is required to obtain useful sequence information. In recent years, the demand for N-terminal sequencing has decreased substantially as some applications for protein identification and characterization can now be more effectively performed using mass spectrometry. However, N-terminal sequencing remains the method of choice for verifying the N-terminal boundary of recombinant proteins, determining the N-terminus of protease-resistant domains, identifying proteins isolated from species where most of the genome has not yet been sequenced, and mapping modified or crosslinked sites in proteins that prove to be refractory to analysis by mass spectrometry.

Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection.

Infection of human erythrocytes by the apicomplexan malaria parasite Plasmodium falciparum results in endovacuolar uptake of 4 host proteins that reside in erythrocyte detergent-resistant membranes (DRMs). Whether this vacuolar transport reflects selective uptake of host DRM proteins remains unknown. A further complication is that DRMs of vastly different protein and cholesterol contents have been isolated from erythrocytes. Here we show that isolated DRMs containing the highest cholesterol-to-protein ratio have low protein mass. Liquid chromatography, mass spectrometry, and antibody-based studies reveal that the major DRM proteins are band 3, flotillin-1 and -2, peroxiredoxin-2, and stomatin. Band 3 and stomatin, which reflect the bulk mass of erythrocyte DRM proteins, and all tested non-DRM proteins are excluded from the vacuolar parasite. In contrast, flotillin-1 and -2 and 8 minor DRM proteins are recruited to the vacuole. These data suggest that DRM association is necessary but not sufficient for vacuolar recruitment and there is active, vacuolar uptake of a subset of host DRM proteins. Finally, the 10 internalized DRM proteins show varied lipid and peptidic anchors indicating that, contrary to the prevailing model of apicomplexan vacuole formation, DRM association, rather than lipid anchors, provides the preferred criteria for protein recruitment to the malarial vacuole.