A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in and Other Denitrifying Bacteria.

The transmembrane nitrate reductase (Nar) is the first enzyme in the dissimilatory alternate anaerobic nitrate respiratory chain in denitrifying bacteria. To date, there has been no real-time method to determine its specific activity embedded in its native membrane; here, we describe such a new method, which is useful with the inside-out membranes of and other denitrifying bacteria. This new method takes advantage of the native coupling of the endogenous NADH dehydrogenase or Complex I with the reduction of nitrate by Nar through the quinone pool of the inner membranes of .

Cross-sex hormonal replacement: Some effects over mitochondria.

Transgender is a term for people whose gender identity or expression differs from their natal sex. These individuals often seek cross-hormonal therapy to simulate the individual´s desired gender. However, the use of estrogens and testosterone has side effects such as a higher propensity to cancer, weight changes and cardiovascular diseases.

Inhibitory to non-inhibitory evolution of the subunit of the FF-ATPase of and -proteobacteria as related to mitochondrial endosymbiosis.

The subunit is a potent inhibitor of the FF-ATPase of (PdFF-ATPase) and related -proteobacteria different from the other two canonical inhibitors of bacterial () and mitochondrial (IF) FF-ATPases. mimics mitochondrial IF in its inhibitory N-terminus, blocking the PdFF-ATPase activity as a unidirectional pawl-ratchet and allowing the PdFF-ATP synthase turnover. is essential for the respiratory growth of , as we showed by a knockout.

Rotary properties of hybrid F-ATPases consisting of subunits from different species.

F-ATPase (F) is an ATP-driven rotary motor protein ubiquitously found in many species as the catalytic portion of FF-ATP synthase. Despite the highly conserved amino acid sequence of the catalytic core subunits: α and β, F shows diversity in the maximum catalytic turnover rate and the number of rotary steps per turn. To study the design principle of F, we prepared eight hybrid Fs composed of subunits from two of three genuine Fs: thermophilic PS3 (TF), bovine mitochondria (MF), and (PdF), differing in the and the number of rotary steps.

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF Subunits of the FF-ATPase as Related to the Endosymbiotic Origin of Mitochondria.

The F1FO-ATP synthase nanomotor synthesizes >90% of the cellular ATP of almost all living beings by rotating in the “forward” direction, but it can also consume the same ATP pools by rotating in “reverse.” To prevent futile F1FO-ATPase activity, several different inhibitory proteins or domains in bacteria (ε and ζ subunits), mitochondria (IF1), and chloroplasts (ε and γ disulfide) emerged to block the F1FO-ATPase activity selectively. In this study, we analyze how these F1FO-ATPase inhibitory proteins have evolved.

Putative Repurposing of Lamivudine, a Nucleoside/Nucleotide Analogue and Antiretroviral to Improve the Outcome of Cancer and COVID-19 Patients.

Lamivudine, also widely known as 3TC belongs to a family of nucleotide/nucleoside analogues of cytidine or cytosine that inhibits the Reverse Transcriptase (RT) of retroviruses such as HIV. Lamivudine is currently indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection or for chronic Hepatitis B (HBV) virus infection associated with evidence of hepatitis B viral replication and active liver inflammation. HBV reactivation in patients with HBV infections who receive anticancer chemotherapy can be a life-threatening complication during and after the completion of chemotherapy.

Regulation of bacterial ATP synthase activity: A gear-shifting or a pawl-ratchet mechanism?

The F F -ATP synthase, a widely distributed nanomotor responsible of ATP synthesis, rotates its central rotor reversibly: In the clockwise direction when viewed from the Fo (with the observer facing the positive side of the energy transducing membrane and looking down into the negative side of the membrane), it functions as ATP synthase, while in counterclockwise sense, it operates as a proton-pumping ATP hydrolase. Regulation exerted by naturally occurring inhibitory proteins of the enzyme appears to function by avoiding ATP hydrolysis while preserving ATP synthesis. The work of Liu et al.

The 3 × 120° rotary mechanism of F-ATPase is different from that of the bacterial and mitochondrial F-ATPases.

The rotation of F-ATPase (PdF) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or below , PdF showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF executes binding, hydrolysis, and probably product release at the same dwell.

Microencapsulated Pomegranate Reverts High-Density Lipoprotein (HDL)-Induced Endothelial Dysfunction and Reduces Postprandial Triglyceridemia in Women with Acute Coronary Syndrome.

(1) Background: the composition of high-density lipoproteins (HDL) becomes altered during the postprandial state, probably affecting their functionality vis-à-vis the endothelium. Since acute coronary syndrome (ACS) in women is frequently associated with endothelial dysfunction, it is likely that HDL are unable to improve artery vasodilation in these patients. Therefore, we characterized HDL from women with ACS in fasting and postprandial conditions.

Control of rotation of the FF-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF proteins.

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase.

Unidirectional regulation of the FF-ATP synthase nanomotor by the ζ pawl-ratchet inhibitor protein of Paracoccus denitrificans and related α-proteobacteria.

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF subunits, prevent the wasteful CCW FF-ATPase activity by blocking γ rotation at the α/β/γ interface of the F portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F-ATPase inhibitor named ζ.

The Biological Role of the ζ Subunit as Unidirectional Inhibitor of the FF-ATPase of Paracoccus denitrificans.

The biological roles of the three natural FF-ATPase inhibitors, ε, ζ, and IF, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF, but in the FF-ATPase of Paracoccus denitrificans (PdFF), it is a monogenic and supernumerary subunit. Here, we constructed a P.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation.

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria.

The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF1) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P.

Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution.

The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P.

Novel mutations in the transcriptional activator domain of the human TBX20 in patients with atrial septal defect.

Background: The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD) were found in the DNA-binding T-box domain, none in the transcriptional activator domain.

Methods: We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD) using the High Resolution Melting (HRM) method and DNA sequencing.

New insights on the mechanism of the K(+-) independent activity of crenarchaeota pyruvate kinases.

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position.

The ζ subunit of the F1FO-ATP synthase of α-proteobacteria controls rotation of the nanomotor with a different structure.

The ζ subunit is a novel natural inhibitor of the α-proteobacterial F1FO-ATPase described originally in Paracoccus denitrificans. To characterize the mechanism by which this subunit inhibits the F1FO nanomotor, the ζ subunit of Paracoccus denitrificans (Pd-ζ) was analyzed by the combination of kinetic, biochemical, bioinformatic, proteomic, and structural approaches. The ζ subunit causes full inhibition of the sulfite-activated PdF1-ATPase with an apparent IC50 of 270 nM by a mechanism independent of the ε subunit.

Atypical cristae morphology of human syncytiotrophoblast mitochondria: role for complex V.

Mitochondrial complexes I, III(2), and IV from human cytotrophoblast and syncytiotrophoblast associate to form supercomplexes or respirasomes, with the following stoichiometries: I(1):(III(2))(1) and I(1):(III(2))(1-2):IV(1-4). 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.

What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes.

Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140 residues and a diminished overall mean hydrophobicity when compared with their mtDNA-encoded counterparts.

Structure of dimeric F1F0-ATP synthase.

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c(10) ring and the other stator subunits at the F(0)-F(0) dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å.

A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related alpha-proteobacteria.

The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes.

A deletion in the PRKAR1A gene is associated with Carney complex.

Mutations of the PRKAR1A gene are an important cause of Carney complex (CC). The PRKAR1A gene encodes the type 1A regulatory subunit of cAMP-dependent protein kinase A. We have identified one mutation of PRKAR1A (553delG) in three members of the same family affected by CC.

Regulation of the F1F0-ATP synthase rotary nanomotor in its monomeric-bacterial and dimeric-mitochondrial forms.

The F(1)F(0)-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover.