Identification of the mitochondrial glutamate transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms

The mitochondrial carriers are a family of transport proteins in the inner membranes of mitochondria. They shuttle substrates, metabolites, and cofactors through this membrane and connect cytoplasm functions with others in the matrix. Glutamate is co-transported with H(+) (or exchanged for OH(-)), but no protein has ever been associated with this activity. Two human expressed sequence tags encode proteins of 323 and 315 amino acids with 63% identity that are related to the aspartate-glutamate carrier, a member of the carrier family. They have been overexpressed in Escherichia coli and reconstituted into phospholipid vesicles. Their transport properties demonstrate that the two proteins are isoforms of the glutamate/H(+) symporter described in the past in whole mitochondria. Isoform 1 is expressed at higher levels than isoform 2 in all the tissues except in brain, where the two isoforms are expressed at comparable levels. The differences in expression levels and kinetic parameters of the two isoforms suggest that isoform 2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and isoform 1 becomes operative to accommodate higher demands associated with specific metabolic functions such as ureogenesis.     

An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein

The prion protein (PrP^C) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrP^C proteoforms have been hampered by the lack of methods able to identify all PrP^C-derived proteoforms. Building on previous knowledge of PrP^C endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrP^C constitutive processing and the relative abundance of PrP^C proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrP^C proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrP^C-derived fragments. Endoproteolytic processing of PrP^C was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrP^C was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrP^C is not affected by PrP^C overexpression or host factors other than PrP^C but can be impacted by PrP^C primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrP^C and for a comprehensive appraisal of PrP^C proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.     

Characterization of mitochondrial dicarboxylate/tricarboxylate transporters from grape berries

Grape berries (Vitis vinifera L fruit) exhibit a double-sigmoid pattern of development that results from two successive periods of vacuolar swelling during which the nature of accumulated solutes changes significantly. Throughout the first period, called green or herbaceous stage, berries accumulate high levels of organic acids, mainly malate and tartrate. At the cellular level fruit acidity comprises both metabolism and vacuolar storage. Malic acid compartmentation is critical for optimal functioning of cytosolic enzymes. Therefore, the identification and characterization of the carriers involved in malate transport across sub-cellular compartments is of great importance. The decrease in acid content during grape berry ripening has been mainly associated to mitochondrial malate oxidation. However, no Vitis vinifera mitochondrial carrier involved in malate transport has been reported to date. Here we describe the identification of three V. vinifera mitochondrial dicarboxylate/tricarboxylate carriers (VvDTC1-3) putatively involved in mitochondrial malate, citrate and other di/tricarboxylates transport. The three VvDTCs are very similar, sharing a percentage of identical residues of at least 83 %. Expression analysis of the encoding VvDTC genes in grape berries shows that they are differentially regulated exhibiting a developmental pattern of expression. The simultaneous high expression of both VvDTC2 and VvDTC3 in grape berry mesocarp close to the onset of ripening suggests that these carriers might be involved in the transport of malate into mitochondria.     

Small Ruminant Nor98 Prions Share Biochemical Features with Human Gerstmann-Str?ussler-Scheinker Disease and Variably Protease-Sensitive Prionopathy

Prion diseases are classically characterized by the accumulation of pathological prion protein (PrP^Sc) with the protease resistant C-terminal fragment (PrP^res) of 27–30 kDa. However, in both humans and animals, prion diseases with atypical biochemical features, characterized by PK-resistant PrP internal fragments (PrP^res) cleaved at both the N and C termini, have been described. In this study we performed a detailed comparison of the biochemical features of PrP^Sc from atypical prion diseases including human Gerstmann-Sträussler-Scheinker disease (GSS) and variably protease-sensitive prionopathy (VPSPr) and in small ruminant Nor98 or atypical scrapie. The kinetics of PrP^res production and its cleavage sites after PK digestion were analyzed, along with the PrP^Sc conformational stability, using a new method able to characterize both protease-resistant and protease-sensitive PrP^Sc components. All these PrP^Sc types shared common and distinctive biochemical features compared to PrP^Sc from classical prion diseases such as sporadic Creutzfeldt-Jakob disease and scrapie. Notwithstanding, distinct biochemical signatures based on PrP^res cleavage sites and PrP^Sc conformational stability were identified in GSS A117V, GSS F198S, GSS P102L and VPSPr, which allowed their specific identification. Importantly, the biochemical properties of PrP^Sc from Nor98 and GSS P102L largely overlapped, but were distinct from the other human prions investigated. Finally, our study paves the way towards more refined comparative approaches to the characterization of prions at the animal–human interface.     

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD⁺ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD⁺ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD⁺ content, we have expressed plant and yeast mitochondrial NAD⁺ carriers in human cells and observed a profound increase in mitochondrial NAD⁺. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD⁺ content. Surprisingly, constitutive redistribution of NAD⁺ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD⁺ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD⁺ levels. These results suggest that a mitochondrial NAD⁺ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD⁺ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.     

shape Ogmogaster antarcticus Johnston, 1931 (Digenea: Notocotylidae) in shape Balaenoptera physalus (L.): first record in the Mediterranean Sea

Detailed parasitological investigations were carried out on the stomach and the intestine of a young male Balaenoptera physalus found stranded along the east coast of the northern Tyrrhenian Sea. Thousands of digeneans were found in both the caecum and the colon. Morphological and morphometric studies were carried out on these parasites, which were identified as Ogmogaster antarcticus Johnston, 1931. In contrast to the other species of the genus, O. antarcticus has a large geographical distribution and it is not specific to its definitive hosts. This is the first record of O. antarcticus from B. physalus in the Mediterranean Sea, a finding which helps confirm the world-wide distribution of this parasite.     

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.     

A single amino acid residue in bank vole prion protein drives permissiveness to Nor98/atypical scrapie and the emergence of multiple strain variants

Prions are infectious agents that replicate through the autocatalytic misfolding of the cellular prion protein (PrP^C) into infectious aggregates (PrP^Sc) causing fatal neurodegenerative diseases in humans and animals. Prions exist as strains, which are encoded by conformational variants of PrP^Sc. The transmissibility of prions depends on the PrP^C sequence of the recipient host and on the incoming prion strain, so that some animal prion strains are more contagious than others or are transmissible to new species, including humans. Nor98/atypical scrapie (AS) is a prion disease of sheep and goats reported in several countries worldwide. At variance with classical scrapie (CS), AS is considered poorly contagious and is supposed to be spontaneous in origin. The zoonotic potential of AS, its strain variability and the relationships with the more contagious CS strains remain largely unknown. We characterized AS isolates from sheep and goats by transmission in ovinised transgenic mice (tg338) and in two genetic lines of bank voles, carrying either methionine (BvM) or isoleucine (BvI) at PrP residue 109. All AS isolates induced the same pathological phenotype in tg338 mice, thus proving that they encoded the same strain, irrespective of their geographical origin or source species. In bank voles, we found that the M109I polymorphism dictates the susceptibility to AS. BvI were susceptible and faithfully reproduced the AS strain, while the transmission in BvM was highly inefficient and was characterized by a conformational change towards a CS-like prion strain. Sub-passaging experiments revealed that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible animals. These findings add new clues for a better comprehension of strain selection dynamics in prion infections and have wider implications for understanding the origin of contagious prion strains, such as CS.