[PDF] Characterizing Mitochondrial Function And Structure eBook

Author : H. Tedeschi
Publisher : Springer Science & Business Media
Page : 291 pages
File Size : 22,56 MB
Release : 2012-12-06
Category : Medical
ISBN : 3709184126

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In the past few years, the body of experimental work on the structure, function and assembly processes of mitochondria has expanded rapidly. No one person can believe himself or herself completely in control of the burgeoning literature without possessing serious omissions or blind spots. In the present monograph I have attempted a critical evaluation of the literature. I believe that the common thread of single authorship outweighs the shortcomings of one person presenting many disparate viewpo,ints. It is my hope that the end product represents a comprehensive and coordinated review of the subject matter to the present date. Although the bulk of this monograph was completed by October 1974, I have made some attempts to update several of the sections at later times. Albany, N. Y. , November 1975 H. TEDESCHI Contents A. Structure of Mitochondria 1. General Organization . a) Conventional Thin Section Electron Microscopy b) Negative Staining Techniques . 3 c) Freeze-Cleavage . . . . . . . 4 2. Special Organization and Inclusions 10 a) Prismatic or Atypical Cristae 10 b) Filaments and Tubules . . 11 c) Amorphous Inclusions 12 d) Intramitochondrial Granules 12 e) DNA Containing Fibers 14 3. Topography of the Mitochondrial Transducing Systems 15 B. The Assembly of Mitochondria 18 1. Mitochondrial DNA and Its Genetic Role 19 2. Transcription of the Mitochondrial DNA . 28 a) mRNA . . . . . . . . . . . . . . 30 b) The Machinery for Transcription and Translation 32 c) Turnover of Mitochondrial RNA 36 3. Protein Synthesis . . . . . . . . 37 a) Mitochondrial Protein Synthesis and Its Characteristics 37 b) Role of Mitochondrial Protein Synthesis . .

Author : S. Meuer
Publisher : Springer Science & Business Media
Page : 390 pages
File Size : 29,40 MB
Release : 2012-12-06
Category : Science
ISBN : 3642595243

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The first comprehensive treatise on Rapid Cycle Real-Time PCR. With amplification times of 15-30 minutes of on-line detection and analysis, nucleic acid quantification of mutation analysis finally becomes a routine, powerful and rapid method. Focusing primarily on the LightCycler, an instrument that combines Rapid Cycle PCR with fluorescent monitoring, this technology provides convenient analysis by melting temperatures. PCR products can be identified by product Tm, and single base mismatches can easily be genotyped by probe Tm. Methods chapters detail the theory behind quantification of mutation analysis; the design of synthesis of fluorescent hybridization probes of the preparation of template DNA. Application chapters apply nucleid acid quantification to infectious organisms of intracellular messengers and mutation detection to somatic of acquired mutations.

Author : Olga Zurita Rendón
Publisher :
Page : pages
File Size : 11,13 MB
Release : 2015
Category :
ISBN :

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"The main function of mitochondria is the generation of cellular energy through the oxidative phosphorylation (OXPHOS) pathway. Calcium homeostasis, apoptotic signaling, the citric acid and urea cycles, iron sulfur cluster, steroid and heme synthesis are other biosynthetic pathways that take place within mitochondria, making this organelle indispensable for the proper function of the cell. Mitochondrial disorders have an incidence of at least 1 in 5000 live births and they range from neonatal fatalities to late-onset neurodegeneration. Mammalian mitochondria have a circular genome reminiscent of their prokaryotic origins. It is organized into protein/DNA complexes called nucleoids, which are necessary for its stability, expression and segregation. Mitochondrial DNA (mtDNA) encodes 13 subunits of the OXPHOS complexes I, III, IV and V, 22 tRNAs and two rRNAs.Complex I (NADH ubiquinone oxidoreductase) is the first complex of the OXPHOS pathway. It is responsible for the oxidization of NADH and for the pumping of protons from the mitochondrial matrix to the intermembrane space, in this way contributing to the formation of a proton gradient that is ultimately used to synthesize ATP. Complex I deficiency is the most common cause of mitochondrial disease in infants. Mutational analyses have identified defects in several of the structural components of the complex, however these mutations explain only 50% of the cases, implicating nuclear-encoded chaperones or assembly factors as an important cause of disease. In this study we demonstrate that complex I biogenesis is nucleated by an early subcomplex of 315 kDa containing at least the nuclear-encoded subunit NDUFS2 and the mtDNA-encoded subunit ND1. The assembly factors NDUFAF3, NDUFAF4, NDUFAF7, C8orf38 and, C20orf7 are necessary for the assembly and stabilization of the 315 kDa intermediate. By using RNAi technology to knock-down the expression of NDUFAF2, NDUFAF3, NDUFAF4, C8orf38, C20orf7 and, NDUFAF7 we demonstrate that early complex I assembly defects result in the proteolytic degradation of the ND1 subunit by the inner membrane protease m-AAA AFG3L2, in this manner regulating the latter steps of the assembly pathway. We performed an in depth functional characterization of the NDUFAF7 assembly factor and demonstrated that it is responsible for the symmetric dimethylation of Arg85 of the NDUFS2 subunit after it assembles into complex I, stabilizing an early assembly intermediate.We showed that the AAA+ LONP1 protease, which is part of the protein quality control system of the mitochondrial matrix, plays an essential role in the maintenance and expression of the mitochondrial genome. LONP1 depletion selectively impairs the degradation and processing of the mitochondrial targeting sequence of the nucleoid components, SSBP1 and MTERFD3, the RNA granule protein, FASTKD2 and, the matrix protease, CLPX. Likewise, LONP1 knock-down caused the accumulation of protein aggregates primarily containing soluble DNA/RNA Associated Proteins (DRAPs) and mitochondrial ribosomal structural subunits, which ultimately triggered mitophagy.This work contributes to our understanding of mitochondrial biogenesis by describing in detail the chaperone regulatory system involved in the early assembly steps of complex I and by demonstrating that the LONP1 protease is required in the degradation and processing of some of the key players involved in the expression of the mitochondrial genome." --

Author : Dejana Mokranjac
Publisher : Humana Press
Page : 409 pages
File Size : 12,16 MB
Release : 2018-07-20
Category : Science
ISBN : 9781493983094

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This volume compiles a broad range of step-by-step protocols, complementary to the ones published in the first edition of this book, to study various aspects of mitochondrial structure and function in different model organisms, both in vitro and in vivo. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Mitochondria: Practical Protocols, Second Edition aims to be useful for beginners as well as for experienced researchers in the field.

Author : Jonathan Michael Schmitz
Publisher :
Page : 0 pages
File Size : 34,88 MB
Release : 2023
Category :
ISBN :

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As central hubs of metabolism, mitochondria are the nexus of multiple interconnected biochemical pathways critical for proper cellular function. Mitochondrial dysfunction interferes with these metabolic pathways, resulting in numerous human diseases-many of which are incurable. Identifying the genetic source of mitochondrial dysfunction is made challenging by the fact that nearly a quarter of the mitochondrial proteome is uncharacterized. Motivated by the connection of these uncharacterized mitochondrial proteins to human disease, the field of mitochondrial biochemistry has been a hotbed for the functional annotation of these proteins. In this dissertation, I give an overview of an experimental framework named "systems biochemistry" used to study uncharacterized proteins and my efforts to characterize two pairs of uncharacterized mitochondrial proteins in Saccharomyces cerevisiae. In the first part of chapter 1, I give a general overview of uncharacterized proteins and systems biochemistry, a multidisciplinary framework of study which combines the breadth of systems biology with the depth of biochemical follow-up. In the second part of chapter 1, I give a broad overview of flavonoid biosynthesis in plants, introduce chalcone isomerases, and highlight two uncharacterized mitochondrial proteins in S. cerevisiae that are homologous to plant chalcone isomerases. In chapter 2, I detail how systems biochemistry was employed to characterize the uncharacterized mitochondrial proteins Cqd1p and Cqd2p. Using a forward-genetic screen on yeast sensitized to oxidative stress, Cqd1p and Cqd2p were identified as proteins involved in the distribution of the redox-active mitochondrial lipid coenzyme Q (CoQ). We achieved this by developing and applying a method that isolates inner mitochondrial membrane lipid nanoparticles to demonstrate that CoQ levels are altered in S. cerevisiae that lack Cqd1p and/or Cqd2p-supporting the hypothesis that these proteins are involved in CoQ distribution. In chapter 3, I present the initial functional characterization of the uncharacterized mitochondrial proteins Aim18p and Aim46p. I demonstrate how Aim18p and Aim46p are mitochondrial proteins with sequence and structural homology to chalcone isomerase-like proteins in plants and how they lack the canonical functions of this protein family. Instead, these proteins are hemoproteins, and I show how this heme-binding function is widely distributed across homologs of Aim18p and Aim46p in multiple phyla of fungi. In chapter 4, I summarize the major findings of chapters 2 and 3; I then detail the next steps of studying Cqd1p and Cqd2p in the context of CoQ distribution and highlight what work remains to characterize Aim18p and Aim46p as hemoproteins. Taken together, the work presented in this dissertation is another example of how systems biochemistry has the power to advance our knowledge of the uncharacterized mitochondrial proteome.