Metabolomic Profiling of Blood-Derived Microvesicles in Breast Cancer Patients.

Malignant cells differ from benign ones in their metabolome and it is largely unknown whether this difference is reflected in the metabolic profile of their microvesicles (MV), which are secreted into the blood of cancer patients. Here, they are present together with MV from the various blood and endothelial cells. Harvesting MV from 78 breast cancer patients (BC) and 30 controls, we characterized the whole blood MV metabolome using targeted and untargeted mass spectrometry. Especially (lyso)-phosphatidylcholines and sphingomyelins were detected in a relevant abundance. Eight metabolites showed a significant discriminatory power between BC and controls. High concentrations of lysoPCaC26:0 and PCaaC38:5 were associated with shorter overall survival. Comparing BC subtype-specific metabolome profiles, 24 metabolites were differentially expressed between luminal A and luminal B. Pathway analysis revealed alterations in the glycerophospholipid metabolism for the whole cancer cohort and in the ether lipid metabolism for the molecular subtype luminal B. Although this mixture of blood-derived MV contains only a minor number of tumor MV, a combination of metabolites was identified that distinguished between BC and controls as well as between molecular subtypes, and was predictive for overall survival. This suggests that these metabolites represent promising biomarkers and, moreover, that they may be functionally relevant for tumor progression.

Folate receptor alpha defect causes cerebral folate transport deficiency: a treatable neurodegenerative disorder associated with disturbed myelin metabolism.

Sufficient folate supplementation is essential for a multitude of biological processes and diverse organ systems. At least five distinct inherited disorders of folate transport and metabolism are presently known, all of which cause systemic folate deficiency. We identified an inherited brain-specific folate transport defect that is caused by mutations in the folate receptor 1 (FOLR1) gene coding for folate receptor alpha (FRalpha). Three patients carrying FOLR1 mutations developed progressive movement disturbance, psychomotor decline, and epilepsy and showed severely reduced folate concentrations in the cerebrospinal fluid (CSF). Brain magnetic resonance imaging (MRI) demonstrated profound hypomyelination, and MR-based in vivo metabolite analysis indicated a combined depletion of white-matter choline and inositol. Retroviral transfection of patient cells with either FRalpha or FRbeta could rescue folate binding. Furthermore, CSF folate concentrations, as well as glial choline and inositol depletion, were restored by folinic acid therapy and preceded clinical improvements. Our studies not only characterize a previously unknown and treatable disorder of early childhood, but also provide new insights into the folate metabolic pathways involved in postnatal myelination and brain development.

tRNAArg-Derived Fragments Can Serve as Arginine Donors for Protein Arginylation.

Arginyltransferase ATE1 mediates posttranslational arginylation and plays key roles in multiple physiological processes. ATE1 utilizes arginyl (Arg)-tRNAArg as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we address the question of ATE1- Arg-tRNAArg specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and Ate1 knockout models, we find that, in addition to full-length tRNA, ATE1 is also able to utilize short tRNAArg fragments that bear structural resemblance to tRNA-derived fragments (tRF), a recently discovered class of small regulatory non-coding RNAs with global emerging biological role. Ate1 knockout cells show a decrease in tRFArg generation and a significant increase in the ratio of tRNAArg:tRFArg compared with wild type, suggesting a functional link between tRFArg and arginylation. We propose that generation of physiologically important tRFs can serve as a switch between translation and protein arginylation.

Gallophosphonates Containing Alkali Metal Ions. 2.(1) Synthesis and Structure of Gallophosphonates Incorporating Na(+) and K(+) Ions.

The reactions between t-BuP(O)(OH)(2) and equimolar quantities of MGaMe(4) (M = Na, K) yield ionic and alkali metal containing molecular gallophosphonates [Na(4)(&mgr;(2)-OH(2))(2)(THF)(2)][(Me(2)GaO(3)PBu-t)(2)](2).2THF (2) and [K(THF)(6)][K(5)(THF)(2){(Me(2)GaO(3)PBu-t)(2)}(3)] (3), respectively. Compounds 2 and 3are soluble in common organic solvents and have been characterized by means of analytical and spectroscopic techniques, as well as by single-crystal X-ray diffraction studies. These compounds represent the rare examples of molecular ionic phosphonate cages which contain coordinated Na(+) or K(+) ions. Compound 2 is constructed from two eight-membered Ga(2)O(4)P(2) gallium phosphonate rings which sandwich a central Na(4)(H(2)O)(2) unit. In the case of 3, three eight-membered Ga(2)O(4)P(2) gallium phosphonate units envelope an aggregated K(5) core which exists in the form of a trigonal-bipyramidal polyhedron. The Na(+) and K(+) ions in 2 and 3 are also coordinated by the endocyclic oxygen atoms of the eight-membered gallophosphonate crowns, apart from the regular exocyclic P-O coordination. Unlike the lithium gallophosphonate [Li(4)(THF)(4)][{(MeGaO(3)PBu-t)(3)(&mgr;(3)-O(2))}(2)] (1), compounds 2 and 3 do not undergo any clean cage conversion reaction in the presence of 15-crown-5 and 18-crown-6, respectively.

Structure of tripeptidyl-peptidase I provides insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 angstroms resolution reveals a globular structure with a subtilisin-like fold, a Ser475-Glu272-Asp360 catalytic triad, and an octahedrally coordinated Ca2+-binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

Cathepsin D deficiency is associated with a human neurodegenerative disorder.

Cathepsin D is a ubiquitously expressed lysosomal protease that is involved in proteolytic degradation, cell invasion, and apoptosis. In mice and sheep, cathepsin D deficiency is known to cause a fatal neurodegenerative disease. Here, we report a novel disorder in a child with early blindness and progressive psychomotor disability. Two missense mutations in the CTSD gene, F229I and W383C, were identified and were found to cause markedly reduced proteolytic activity and a diminished amount of cathepsin D in patient fibroblasts. Expression of cathepsin D mutants in cathepsin D(-/-) mouse fibroblasts revealed disturbed posttranslational processing and intracellular targeting for W383C and diminished maximal enzyme velocity for F229I. The structural effects of cathepsin D mutants were estimated by computer modeling, which suggested larger structural alterations for W383C than for F229I. Our studies broaden the group of human neurodegenerative disorders and add new insight into the cellular functions of human cathepsin D.