N. Yadava - Academia.edu (original) (raw)

Papers by N. Yadava

Research paper thumbnail of Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins

Molecular and biochemical parasitology, 1997

A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the ... more A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophores...

Research paper thumbnail of Real-time monitoring of mitochondrial and cellular bioenergetics in cultured neurons in a microplate

Research paper thumbnail of Development and Characterization of a Conditional Mitochondrial Complex I Assembly System

Journal of Biological Chemistry, 2004

We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line,... more We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line, CCL16-B2, that does not express the NDUFA1 gene (encoding the MWFE protein). In this mutant, a hemagglutinin (HA) epitope-tagged MWFE protein was expressed from a doxycycline-inducible promoter. The expression of the protein was absolutely dependent on the presence of doxycycline, and the gene could be turned off completely by removal of doxycycline. These experiments demonstrated a key role of MWFE in the pathway of complex I assembly. Upon induction the MWFE.HA protein reached steady-state levels within 24 h, but the appearance of fully active complex I was delayed by another approximately 24 h. The MWFE appeared in a precomplex that probably includes one or more subunits encoded by mtDNA. The fate of MWFE and the stability of complex I were themselves very tightly linked to the activity of mitochondrial protein synthesis and to the assembly of subunits encoded by mtDNA (ND1-6 and ND4L). This novel conditional system can shed light not only on the mechanism of complex I assembly but emphasizes the role of subunits previously thought of as "accessory." It promises to have broader applications in the study of cellular energy metabolism and production of reactive oxygen species and related processes.

Research paper thumbnail of Species-specific and Mutant MWFE Proteins. THEIR EFFECT ON THE ASSEMBLY OF A FUNCTIONAL MAMMALIAN MITOCHONDRIAL COMPLEX I

Journal of Biological Chemistry, 2002

The MWFE protein (70 amino acids) is highly conserved in evolution, but the human protein (80% id... more The MWFE protein (70 amino acids) is highly conserved in evolution, but the human protein (80% identical to hamster) does not complement a null mutation in Chinese hamster cells. We have identified a small protein segment where significant differences exist between rodents and primates, illustrating very specifically the need for compatibility of the nuclear and mitochondrial genomes in the assembly of complex I. The segment between amino acids 39 and 46 appears to be critical for species-specific compatibility. Amino acid substitutions in this region were tested that caused a reduction of activity of the hamster protein or converted the inactive human protein into a partially active one. Such mutations could be useful in making mice with partial complex I activity as models for mitochondrial diseases. Their potential as dominant negative mutants was explored. More deleterious mutations in the NDUFA1 gene were also characterized. A conservative substitution, R50K, or a short C-terminal deletion makes the protein completely inactive. In the absence of MWFE, no high molecular weight complex was detectable by Blue Native-gel electrophoresis. The MWFE protein itself is unstable in the absence of assembled mitochondrially encoded integral membrane proteins of complex I.

Research paper thumbnail of Mitochondrial Dysfunction Impairs Tumor Suppressor p53 Expression/Function

Journal of Biological Chemistry, 2011

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying m... more Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.

Research paper thumbnail of The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Journal of Biological Chemistry, 2014

Background: At the redox potential of NADH/NAD ϩ , at least four mitochondrial sites produce supe... more Background: At the redox potential of NADH/NAD ϩ , at least four mitochondrial sites produce superoxide/H 2 O 2 . Results: We compare their capacities in situ in isolated mitochondria. Conclusion: Maximum capacities of complexes were 2-oxoglutarate dehydrogenase Ͼ pyruvate dehydrogenase Ͼ branchedchain 2-oxoacid dehydrogenase Ͼ complex I. Significance: H 2 O 2 production from 2-oxoacid dehydrogenases can be considerable but may previously have been misattributed to complex I.

Research paper thumbnail of Episomal Expression of Specific Sense and Antisense mRNAs in Leishmania amazonensis: Modulation of gp63 Level in Promastigotes and Their Infection of Macrophages In Vitro

Infection and Immunity, 2000

The major surface glycoprotein (gp63) of Leishmania amazonensis is a metalloprotease implicated i... more The major surface glycoprotein (gp63) of Leishmania amazonensis is a metalloprotease implicated in the infection of mammalian macrophages. The expression of gp63 and its participation in this infection were further examined by modulating the level of this molecule in a virulent gp63-abundant wild-type clone. Promastigotes were transfected with gp63 genes cloned into a Leishmania-specific vector in two different orientations, leading to the expression of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sense transcripts and suppressed to a very low level in those with antisense transcripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfectants differed significantly only in the level of gp63, thereby allowing specific evaluation of this molecule in leishmanial infection of macrophages in vitro. Kinetic studies of infection in vitro indicate that gp63 plays a role not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.

Research paper thumbnail of Disruption of Mitochondrial Function during Apoptosis Is Mediated by Caspase Cleavage of the p75 Subunit of Complex I of the Electron Transport Chain

Cell, 2004

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation... more Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.

Research paper thumbnail of Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins

Molecular and biochemical parasitology, 1997

A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the ... more A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophores...

Research paper thumbnail of Real-time monitoring of mitochondrial and cellular bioenergetics in cultured neurons in a microplate

Research paper thumbnail of Development and Characterization of a Conditional Mitochondrial Complex I Assembly System

Journal of Biological Chemistry, 2004

We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line,... more We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line, CCL16-B2, that does not express the NDUFA1 gene (encoding the MWFE protein). In this mutant, a hemagglutinin (HA) epitope-tagged MWFE protein was expressed from a doxycycline-inducible promoter. The expression of the protein was absolutely dependent on the presence of doxycycline, and the gene could be turned off completely by removal of doxycycline. These experiments demonstrated a key role of MWFE in the pathway of complex I assembly. Upon induction the MWFE.HA protein reached steady-state levels within 24 h, but the appearance of fully active complex I was delayed by another approximately 24 h. The MWFE appeared in a precomplex that probably includes one or more subunits encoded by mtDNA. The fate of MWFE and the stability of complex I were themselves very tightly linked to the activity of mitochondrial protein synthesis and to the assembly of subunits encoded by mtDNA (ND1-6 and ND4L). This novel conditional system can shed light not only on the mechanism of complex I assembly but emphasizes the role of subunits previously thought of as "accessory." It promises to have broader applications in the study of cellular energy metabolism and production of reactive oxygen species and related processes.

Research paper thumbnail of Species-specific and Mutant MWFE Proteins. THEIR EFFECT ON THE ASSEMBLY OF A FUNCTIONAL MAMMALIAN MITOCHONDRIAL COMPLEX I

Journal of Biological Chemistry, 2002

The MWFE protein (70 amino acids) is highly conserved in evolution, but the human protein (80% id... more The MWFE protein (70 amino acids) is highly conserved in evolution, but the human protein (80% identical to hamster) does not complement a null mutation in Chinese hamster cells. We have identified a small protein segment where significant differences exist between rodents and primates, illustrating very specifically the need for compatibility of the nuclear and mitochondrial genomes in the assembly of complex I. The segment between amino acids 39 and 46 appears to be critical for species-specific compatibility. Amino acid substitutions in this region were tested that caused a reduction of activity of the hamster protein or converted the inactive human protein into a partially active one. Such mutations could be useful in making mice with partial complex I activity as models for mitochondrial diseases. Their potential as dominant negative mutants was explored. More deleterious mutations in the NDUFA1 gene were also characterized. A conservative substitution, R50K, or a short C-terminal deletion makes the protein completely inactive. In the absence of MWFE, no high molecular weight complex was detectable by Blue Native-gel electrophoresis. The MWFE protein itself is unstable in the absence of assembled mitochondrially encoded integral membrane proteins of complex I.

Research paper thumbnail of Mitochondrial Dysfunction Impairs Tumor Suppressor p53 Expression/Function

Journal of Biological Chemistry, 2011

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying m... more Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.

Research paper thumbnail of The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Journal of Biological Chemistry, 2014

Background: At the redox potential of NADH/NAD ϩ , at least four mitochondrial sites produce supe... more Background: At the redox potential of NADH/NAD ϩ , at least four mitochondrial sites produce superoxide/H 2 O 2 . Results: We compare their capacities in situ in isolated mitochondria. Conclusion: Maximum capacities of complexes were 2-oxoglutarate dehydrogenase Ͼ pyruvate dehydrogenase Ͼ branchedchain 2-oxoacid dehydrogenase Ͼ complex I. Significance: H 2 O 2 production from 2-oxoacid dehydrogenases can be considerable but may previously have been misattributed to complex I.

Research paper thumbnail of Episomal Expression of Specific Sense and Antisense mRNAs in Leishmania amazonensis: Modulation of gp63 Level in Promastigotes and Their Infection of Macrophages In Vitro

Infection and Immunity, 2000

The major surface glycoprotein (gp63) of Leishmania amazonensis is a metalloprotease implicated i... more The major surface glycoprotein (gp63) of Leishmania amazonensis is a metalloprotease implicated in the infection of mammalian macrophages. The expression of gp63 and its participation in this infection were further examined by modulating the level of this molecule in a virulent gp63-abundant wild-type clone. Promastigotes were transfected with gp63 genes cloned into a Leishmania-specific vector in two different orientations, leading to the expression of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sense transcripts and suppressed to a very low level in those with antisense transcripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfectants differed significantly only in the level of gp63, thereby allowing specific evaluation of this molecule in leishmanial infection of macrophages in vitro. Kinetic studies of infection in vitro indicate that gp63 plays a role not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.

Research paper thumbnail of Disruption of Mitochondrial Function during Apoptosis Is Mediated by Caspase Cleavage of the p75 Subunit of Complex I of the Electron Transport Chain

Cell, 2004

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation... more Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.