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.

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.

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 Å resolution reveals a globular structure with a subtilisin-like fold, a Ser 475 -Glu 272 -Asp 360 catalytic triad, and an octahedrally coordinated Ca 2؉ -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 Asp 276 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.

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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.