The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD ؉ glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca 2؉ to isolated rat heart mitochondria resulted in a profound decrease in their NAD ؉ content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD ؉ depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD ؉ available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD ؉ content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD ؉ , which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 M CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD ؉ contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD ؉ , which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart. Depending on the duration and severity of myocardial ischemia, reperfusion can result in either recovery of contractile function or rapid transition toward tissue necrosis (for review see Refs. 1-3). Paradoxically, both events require coupled mitochondrial respiration (4). Indeed, cyanide (5) or 2,4-dinitrophenol (6) largely reduce the release of intracellular enzymes, the marker of cell death induced by postischemic reperfusion. However, after more than 25 years, the specific mechanisms underlying these phenomenological observations have yet to be elucidated.A large body of experimental evidence suggests that a suboptimal mitochondrial function could produce low levels of ATP, which in the presence of even a modest rise in [Ca 2ϩ ] i might cause hypercontracture in isolated cardiomyocytes (7) and sarcolemma rupture in intact hearts (8,9). Such a sequence of events could be set in motion by the opening of the mitochondrial PTP, 1 a high conductance channel located in the inner mitochondrial membrane (10). The open probability of this channel is regulated by several factors including mitochondrial membrane potential difference (⌬ m ), Ca 2ϩ , matrix pH, and CsA, a high affinity inhibitor (1...

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2 (AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct. Immunogold electron microscopy and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 (associated with early endosomes); Rab7 (late endosomes); and Rab11 and Rab25 (recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, vesicle-associated membrane protein 2, and vesicle-associated membrane protein 3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network, components of the exocyst complex, and several motor proteins including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB. Beyond this, identification of multiple endoplasmic reticulum-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum. These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, trans-Golgi network, and rough endoplasmic reticulum. Molecular & Cellular Proteomics 4:1095-1106, 2005. Aquaporin-2 (AQP2)1 is the vasopressin-regulated molecular water channel of the renal collecting duct where it constitutes the major route of water movement across the apical plasma membrane. Vasopressin rapidly increases the water permeability of the collecting duct epithelium by binding to V 2 receptors in the basolateral plasma membrane and inducing the cAMP-dependent trafficking of AQP2-containing vesicles from throughout the cytoplasm to the apical region of collecting duct principal cells followed by fusion of these vesicles with the apical membrane of collecting duct cells (1). Although this fundamental mechanism is well established, there is little information about the specific intracellular protein trafficking pathways involved and the nature of the intracellular compartments in which AQP2 resides.Until now, studies to identify the intracellular localization of AQP2 in collecting duct cells have depended largely on two fundamental approaches, namely immunoelectron microscopy and immunofluorescence immunocytochemistry with confocal microscopy. Immunoelectron microscopy (1, 2) has demonstrated ...

We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of (at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1⌬ mutant strain was constructed. Coupled mitochondria isolated from flx1⌬ mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1⌬ mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial role in both bioenergetics and cellular regulation (for reviews see Refs. 1 and 2).As far as mammalian mitochondria are concerned, we have demonstrated that in rat liver the main source of intramitochondrial flavin cofactors is riboflavin (Rf) 1 taken up from the cytosol. FAD synthesis occurs inside the organelle from imported Rf and mitochondrial ATP, consistent with the presence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2) (3, 4). Newly synthesized FAD can be either efficiently incorporated into newly imported apo-flavoproteins (5, 6) or can be exported into the outer mitochondrial compartments, where it is reconverted to Rf by FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) in a recycling pathway, i.e. the Rf-FAD cycle (4, 7). This novel mitochondrial pathway is assumed to play a central role in cellular Rf homeostasis and in flavoprotein biogenesis (5,8).The origin of flavin cofactors in yeast mitochondria is still controversially discussed. It has been reported that yeast mitochondria do not contain their own FAD synthetase activity an...

Recent studies elucidated how riboflavin transporters and FAD forming enzymes work in humans and create a coordinated flavin network ensuring the maintenance of cellular flavoproteome. Alteration of this network may be causative of severe metabolic disorders such as multiple acyl-CoA dehydrogenase deficiency (MADD) or Brown-Vialetto-van Laere syndrome. A crucial step in the maintenance of FAD homeostasis is riboflavin uptake by plasma and mitochondrial membranes. Therefore, studies on recently identified human plasma membrane riboflavin transporters are presented, together with those in which still unidentified mitochondrial riboflavin transporter(s) have been described. A main goal of future research is to fill the gaps still existing as for some transcriptional, functional and structural details of human FAD synthases (FADS) encoded by FLAD1 gene, a novel "redox sensing" enzyme. In the frame of the hypothesis that FADS, acting as a "FAD chaperone", could play a crucial role in the biogenesis of mitochondrial flavo-proteome, several basic functional aspects of flavin cofactor delivery to cognate apo-flavoenzyme are also briefly dealt with. The establishment of model organisms performing altered FAD homeostasis will improve the molecular description of human pathologies. The molecular and functional studies of transporters and enzymes herereported, provide guidelines for improving therapies which may have beneficial effects on the altered metabolism.

Summary Medical 3D printing is emerging as a clinically relevant imaging tool in directing preoperative and intraoperative planning in many surgical specialties and will therefore likely lead to interdisciplinary collaboration between engineers, radiologists, and surgeons. Data from standard imaging modalities such as CT, MRI, echocardiography and rotational angiography can be used to fabricate life-sized models of human anatomy and pathology, as well as patient-specific implants and surgical guides. Cardiovascular 3D printed models can improve diagnosis and allow for advanced pre-operative planning. The majority of applications reported involve congenital heart diseases, valvular and great vessels pathologies. Printed models are suitable for planning both surgical and minimally invasive procedures. Added value has been reported toward improving outcomes, minimizing peri-operative risk, and developing new procedures such as transcatheter mitral valve replacements. Similarly, thoracic surgeons are using 3D printing to assess invasion of vital structures by tumors and to assist in diagnosis and treatment of upper and lower airway diseases. Anatomic models enable surgeons to assimilate information more quickly than image review, choose the optimal surgical approach, and achieve surgery in a shorter time. Patient-specific 3D-printed implants are beginning to appear and may have significant impact on cosmetic and life-saving procedures in the future. In summary, cardiothoracic 3D printing is rapidly evolving and may be a potential game-changer for surgeons. The imager who is equipped with the tools to apply this new imaging science to cardiothoracic care is thus ideally positioned to innovate in this new emerging imaging modality.

A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein.The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo ⁄ holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin K m and V max values were 4.4^1.3 mm and 35^5 pmol´min 21´( mg protein) 21 , respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent K m and apparent V max values for FMN were 5.4^0.9 mm and 22.9^1.4 pmol´min 21´( mg matrix protein) 21 , respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.Keywords: FAD synthetase; flavin cofactors; mitochondria; riboflavin; transport.Although the biochemistry of mammalian mitochondria has been investigated in depth, the mechanisms of uptake and/or processing of vitamins and/or vitamin-derived cofactors, often necessary for assembly with their apoenzymes already imported from the cytosol, remain to be fully elucidated.The uptake of certain vitamins by mitochondria has already been reported to occur via different mechanisms, including diffusion followed by binding to intramitochondrial protein(s) [1], simple diffusion [2±4] and carrier-mediated transport [5,6]. Mitochondria are able to synthesize certain cofactors from taken up precursors, namely adenosylcobalamin [7] and CoA [8]. In contrast, mitochondria can take up cytosolically synthesized cofactors. Diffusion followed by binding to a specific high-affinity intramitochondrial protein has been proposed for pyridoxal 5 H -phosphate uptake [2], whereas thiamine diphosphate (TPP) and CoA can enter the mitochondria via a carrier-mediated process [9±11]. NAD synthesis from externally added nicotinamide mononucleotide has been reported in rat liver mitochondria (RLM) [12], while NAD permeation in mitochondria has also been shown in human cells [13].The above-described state of the art requires investigation regarding the existence of mitochondrial translocators and enzymes involved in vitamin and/or vitamin cofactor transport and metabolism in mitochondria. In particular, because RLM contain a huge amount of FAD and FMN, elucidation of the processes by which mitochondria provide and re...