Federico Martínez

Atypical Cristae Morphology of Human Syncytiotrophoblast Mitochondria: ROLE FOR COMPLEX V

Mitochondrial complexes I, III2, and IV from human cytotrophoblast and syncytiotrophoblast associate to form supercomplexes or respirasomes, with the following stoichiometries: I1:(III2)1 and I1:(III2)1–2:IV1–4. The content of respirasomes was similar in both cell types after isolating mitochondria. However, syncytiotrophoblast mitochondria possess low levels of dimeric complex V and do not have orthodox cristae morphology. In contrast, cytotrophoblast mitochondria show normal cristae morphology and a higher content of ATP synthase dimer. Consistent with the dimerizing role of the ATPase inhibitory protein (IF1) (García, J. J., Morales-Ríos, E., Cortés-Hernandez, P., and Rodríguez-Zavala, J. S. (2006) Biochemistry 45, 12695–12703), higher relative amounts of IF1 were observed in cytotrophoblast when compared with syncytiotrophoblast mitochondria. Therefore, there is a correlation between dimerization of complex V, IF1 expression, and the morphology of mitochondrial cristae in human placental mitochondria. The possible relationship between cristae architecture and the physiological function of the syncytiotrophoblast mitochondria is discussed.

The effect of osmolarity on human placental mitochondria function

Human placental explants survive large changes in osmolarity, but the mechanism for this property is unknown. The goal of this work was to examine the effect of osmolarity on human placental mitochondria. Mitochondria from human term placenta were isolated by differential centrifugation, and incubated in the presence of different concentrations of sucrose or KCl, to modify the osmolarity of the media. Rat liver mitochondria were used as control. The parameters studied were: respiration rate, adenine nucleotide hydrolysis, calcium transport, membrane potential, and mitochondrial morphology. Stimulation of the mitochondrial respiration rate and an increase in Ca2+ transport was observed in the presence of K+. With sucrose, Ca2+ transport showed a complex kinetic behavior, whereas the respiratory control was slightly diminished. Although the ATPase activity was enhanced in the absence of a respiratory substrate, no change in ATP hydrolysis due to osmolarity was observed. ADP hydrolysis was inhibited by a high K+ concentration, but not by sucrose. The membrane potential was not modified by osmolarity, even in the absence of sucrose or K+ in the medium. Mitochondria isolated with KCl showed aggregation, whereas dispersed mitochondria were observed with sucrose. This study showed that sucrose-induced changes in osmolarity, does not modify metabolic and transport properties of human placental mitochondria, whereas KCl-induced osmolarity changes does affect these functions.

DNA moves sequentially towards the nuclear matrix during DNA replication in vivo

BackgroundIn the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM). There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops.ResultsIn the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved.ConclusionsLooped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

Progesterone synthesis by human placental mitochondria is sensitive to PKA inhibition by H89

The transfer of cholesterol to mitochondria, which might involve the phosphorylation of proteins, is the rate-limiting step in human placental steroidogenesis. Protein kinase A (PKA) activity and its role in progesterone synthesis by human placental mitochondria were assessed in this study. The results showed that PKA and phosphotyrosine phosphatase D1 are associated with syncytiotrophoblast mitochondrial membrane by an anchoring kinase cAMP protein-121. The ³²P-labeled of four major proteins was analyzed. The specific inhibitor of PKA, H89, decreased progesterone synthesis in mitochondria while in mitochondrial steroidogenic contact sites protein-phosphorylation was diminished, suggesting that PKA plays a role in placental hormone synthesis. In isolated mitochondria, PKA activity was unaffected by the addition of cAMP suggesting a constant activity of this kinase in the syncytiotrophoblast. The presence of PKA and phosphotyrosine phosphatase D1 anchored to mitochondria by an anchoring kinase cAMP protein-121 indicated that syncytiotrophoblast mitochondria contain a full phosphorylation/dephosphorylation system.

Regulation of mitochondrial cholesterol metabolism.

Cholesterol is required for the growth and replication of mammalian cells. As an integral component of membranes, particularly the plasmalemma, cholesterol facilitates membrane curvature and increases resistance to physical damage. The insertion of cholesterol into membranes increases fluidity (Chapman, 1975), a property closely correlated with the cholesterol/phospholipid (C/PL) ratio. Cholesterol is also the substrate for the production of bile acids and steroid hormones. Because of cholesterol’s importance to cellular function, intracellular cholesterol concentrations are highly regulated (Yeagle, 1985; Lange and Ramos, 1983). Indeed, increases or decreases in this sterol can produce major alterations in cell physiology.

Decreased exchange of adenine nucleotides in human placental mitochondria

The purpose of this work was to study the exchange of adenine nucleotides in mitochondria isolated from human placenta tissue. The results indicate that ADP and ATP are translocated at a lower rate than those reported for rat liver mitochondria. It is proposed that the limited transport is due to the particular lipid composition of placental mitochondria membrane, which induces an arrest in membrane fluidity with the consequent restriction in adenine nucleotide translocase mobility.

The alternative NADH dehydrogenase is present in mitochondria of some animal taxa

The distribution of the alternative NADH dehydrogenase (NDH-2) in the living world was explored. The enzyme, although present in representatives of all living kingdoms, does not have a universal distribution. With the exception of ε-proteobacteria, the enzyme was found in all eubacterial groups. In contrast with the known presence of the NDH-2 in Archaea, the alternative oxidase (AOX) is absent in this group. With regard to the Eukarya domain, the NDH-2 was found in representatives of Protista, Fungi, Plantae, and Animalia. In the latter, however, the presence of the enzyme was restricted to some primitive Metazoa (Placozoa and Cnidaria), and two members of the Deuterostomate lineage of the Bilateria (Echinodermata and Urochordata). No evidence for the presence of the NDH-2 was found in any representative of the Protostomate branch of the Bilateria, contrasting with the existence of the AOX in this same group. It is worth mentioning that those animal species containing the NDH-2 also have an AOX. The actual distribution of the NDH-2 in the various living kingdoms is discussed within the framework of the endosymbiotic theory; in addition, a hypothesis is proposed to explain the disappearance of the alternative NDH-2 and AOX from the majority of the animals.

Modulation of matrix Ca2+ content by the ADP/ATP carrier in brown adipose tissue mitochondria. Influence of membrane lipid composition

The role of the adenine nucleotide translocase on Ca2+ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 μM Ca2+, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca2+ release, even though it inhibits the ADP exchange reaction. However, when the Ca2+ concentration was increased to 150 μM, carboxyatractyloside did induce Ca2+ release, and ADP favored Ca2+ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It is suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.

Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast

BACKGROUND STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. METHODS Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. RESULTS STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. CONCLUSION Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. GENERAL SIGNIFICANCE Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.

Multiple functions of syncytiotrophoblast mitochondria.

The human placenta plays a central role in pregnancy, and the syncytiotrophoblast cells are the main components of the placenta that support the relationship between the mother and fetus, in apart through the production of progesterone. In this review, the metabolic processes performed by syncytiotrophoblast mitochondria associated with placental steroidogenesis are described. The metabolism of cholesterol, specifically how this steroid hormone precursor reaches the mitochondria, and its transformation into progesterone are reviewed. The role of nucleotides in steroidogenesis, as well as the mechanisms associated with signal transduction through protein phosphorylation and dephosphorylation of proteins is discussed. Finally, topics that require further research are identified, including the need for new techniques to study the syncytiotrophoblast in situ using non-invasive methods.