scholarly journals Mitochondrial DNA metabolism is coupled with 20S proteasome function via regulation of deoxyribonucleotide homeostasis in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Xin Jie Chen ◽  
Xiaowen Wang ◽  
Arnav Rana ◽  
Liam P. Coyne ◽  
Daniel M. Loh ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
20S Proteasome ◽  
Dependent Manner ◽  
Quiescent Cells ◽  
Subtle Effect ◽  
Metabolic States ◽  
Mtdna Replication ◽  
Deoxyribonucleoside Triphosphate ◽  
A Minor ◽  
Respiratory Conditions

The synthesis of mitochondrial DNA (mtDNA) is not coupled with cell cycle. Previous studies have shown that the size of deoxyribonucleoside triphosphate (dNTP) pools plays an important role in regulating mtDNA replication and amplification. In yeast, dNTPs are synthesized by the cytosolic ribonucleotide reductase (RNR). It is currently poorly understood as to how RNR activity is regulated in non-dividing or quiescent cells to finely tune mtDNA metabolism to cope with different metabolic states. Here, we show that defect in the 20S proteasome drastically destabilizes mtDNA. The mtDNA instability phenotype in 20S proteasome mutants is suppressed by overexpression of RNR3 or by the deletion of SML1, encoding a minor catalytic subunit and an intrinsic inhibitor of RNR respectively. We found that Sml1 is stabilized in the 20S proteasomal mutants, suggesting that 20S affects mtDNA stability by stabilizing Sml1. Interestingly, defect in the regulatory 19S proteasomal function has only subtle effect on mtDNA stability, supporting a role of the 20S proteasome in dNTP homeostasis independent of 19S. Finally, we found that when cells are transitioned from glycolytic to oxidative growth, Sml1 level is reduced in a 20S-dependent manner. In summary, our study establishes a link between cellular proteostasis and mtDNA metabolism through the regulation of dNTP homeostasis. We propose that increased degradation of Sml1 by the 20S proteasome under respiratory conditions provides a mechanism to stimulate dNTP synthesis and promote mtDNA amplification.

scholarly journals Tissue-specific distribution of cross-linked somatostatin receptor proteins in the rat

1992 ◽  
Vol 282 (2) ◽  
pp. 339-344 ◽  
Author(s):  
C B Srikant ◽  
K K Murthy ◽  
Y C Patel
Keyword(s):  
Exocrine Pancreas ◽  
Receptor Protein ◽  
Cross Linking ◽  
Molecular Heterogeneity ◽  
Dependent Manner ◽  
Tissue Specific ◽  
Receptor Proteins ◽  
Specific Distribution ◽  
A Minor ◽  
The Brain

Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and exhibits SS-14-and SS-28-selective subtypes. Whether such subtypes arise from molecular heterogeneity of the receptor protein has not been definitively established. Previous reports characterizing the molecular properties of the SS receptor by the cross-linking approach have yielded divergent size estimates ranging from 27 kDa to 200 kDa. In order to resolve this discrepancy, as well as to determine whether SS-14 and SS-28 interact with specific receptor proteins, we have cross-linked radioiodinated derivatives of [125I-Tyr11]SS-14 (T*-SS-14) and [Leu8,D-Trp22,125I-Tyr25]SS-28 (LTT*-SS-28) to membrane SS receptors in rat brain, pituitary, exocrine pancreas and adrenal cortex using a number of chemical and photoaffinity cross-linking agents. The labelled cross-linked receptor proteins were analysed by SDS/PAGE under reducing conditions followed by autoradiography. Our findings indicate that the pattern of specifically labelled cross-linked SS receptor proteins is sensitive to the concentration of chemical cross-linking agents such as disuccinimidyl suberate and dithiobis-(succinimidyl propionate). Labelled high-molecular-mass complexes of cross-linked receptor-ligand proteins were observed only when high concentrations of these cross-linkers were employed. Using optimized low concentrations of cross-linkers, however, two major labelled bands of 58 +/- 3 kDa and 27 +/- 2 kDa were detected. These two bands were identified as specifically labelled SS receptor proteins subsequent to cross-linking with a number of photoaffinity cross-linking agents as well. We demonstrate here that the 58 kDa protein is the major SS receptor protein in the rat pituitary, adrenal and exocrine pancreas, whereas the 27 kDa moiety represents the principal form in the brain. Additionally, the presence of a minor specifically labelled band of 32 kDa was detected uniquely in the brain, and a minor labelled protein of 42 kDa was observed in the pancreas. The labelling pattern obtained with LTT*-SS-28 was identical to that observed with T*-SS-14. Labelling of the 27 kDa band by either ligand was inhibited by SS-14 and SS-28 in a dose-dependent manner. Densitometric quantification showed that SS-14 exhibited greater than 2-fold greater potency than SS-28 for inhibiting the labelling of the 27 kDa species. These findings emphasize the need for careful interpretation of cross-linking data obtained for SS receptors, and provide evidence for molecular heterogeneity and for a tissue-specific distribution of the two principal SS receptor proteins.

Phase I Trial of the Proteasome Inhibitor PS-341 in Patients With Refractory Hematologic Malignancies

2002 ◽  
Vol 20 (22) ◽  
pp. 4420-4427 ◽  
Author(s):  
Robert Z. Orlowski ◽  
Thomas E. Stinchcombe ◽  
Beverly S. Mitchell ◽  
Thomas C. Shea ◽  
Albert S. Baldwin ◽  
...  
Keyword(s):  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Complete Response ◽  
Hematologic Malignancies ◽  
Maximum Tolerated Dose ◽  
20S Proteasome ◽  
Dependent Manner ◽  
Mantle Cell ◽  
Heavily Pretreated ◽  
Systemic Hypersensitivity

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacodynamics (PD) of the proteasome inhibitor bortezomib (previously known as PS-341) in patients with refractory hematologic malignancies.PATIENTS AND METHODS: Patients received PS-341 twice weekly for 4 weeks at either 0.40, 1.04, 1.20, or 1.38 mg/m2, followed by a 2-week rest. The PD of PS-341 was evaluated by measurement of whole blood 20S proteasome activity.RESULTS: Twenty-seven patients received 293 doses of PS-341, including 24 complete cycles. DLTs at doses above the 1.04-mg/m2MTD attributed to PS-341 included thrombocytopenia, hyponatremia, hypokalemia, fatigue, and malaise. In three of 10 patients receiving additional therapy, serious reversible adverse events appeared during cycle 2, including one episode of postural hypotension, one systemic hypersensitivity reaction, and grade 4 transaminitis in a patient with hepatitis C and a substantial acetaminophen ingestion. PD studies revealed PS-341 induced 20S proteasome inhibition in a time-dependent manner, and this inhibition was also related to both the dose in milligrams per meter squared, and the absolute dose of PS-341. Among nine fully assessable patients with heavily pretreated plasma cell dyscrasias completing one cycle of therapy, there was one complete response and a reduction in paraprotein levels and/or marrow plasmacytosis in eight others. In addition, one patient with mantle cell lymphoma and another with follicular lymphoma had shrinkage of nodal disease.CONCLUSION: PS-341 was well tolerated at 1.04 mg/m2on this dose-intensive schedule, although patients need to be monitored for electrolyte abnormalities and late toxicities. Additional studies are indicated to determine whether incorporation of dose/body surface area yields a superior PD model to dosing without normalization. PS-341 showed activity against refractory multiple myeloma and possibly non-Hodgkin’s lymphoma in this study, and merits further investigation in these populations.

Endoplasmic reticulum membrane isolated from small-intestinal epithelial cells: enzyme and protein components

1981 ◽  
Vol 52 (1) ◽  
pp. 215-222
Author(s):  
M. Fujita ◽  
H. Ohta ◽  
T. Uezato
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cells ◽  
Cytochrome C ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Cytochrome C Reductase ◽  
Basolateral Membranes ◽  
Nadh Cytochrome ◽  
Protein Components ◽  
A Minor

Endoplasmic reticulum membrane-rich fraction was obtained by subfractionation of the light microsomes from mouse jejunal mucosal epithelial cells. It was marked by high glucose-6-phosphatase, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase activities and low Na+,K+-ATPase activity. The enrichment of Na+,K+-ATPase was 180-fold higher in the basolateral membranes than in the endoplasmic reticulum membrane-rich fraction relative to glucose-6-phosphatase. The protein peak that was phosphorylated in a Na-dependent manner was prominent in the basolateral membranes while it was a minor peak in the endoplasmic reticulum membrane-rich fraction. Under the electron microscope the fraction was seen to be composed of homogeneous small vesicles with thin smooth membranes.

Expression of senescence in Neurospora intermedia

1986 ◽  
Vol 28 (3) ◽  
pp. 459-467 ◽  
Author(s):  
A. J. F. Griffiths ◽  
S. Kraus ◽  
H. Bertrand
Keyword(s):  
Mitochondrial Dna ◽  
Genetic Factors ◽  
Growth Arrest ◽  
Growth Front ◽  
Continuous Growth ◽  
Quiescent Cells ◽  
Neurospora Intermedia ◽  
Aerial Hyphae ◽  
Similar Basis ◽  
Transfer Interval

Kalilo cytoplasms of Neurospora intermedia have been shown to express senescence in two ways. First, by the previously reported way of death in a subculture series, and second, as reported here, by growth cessation in a 50-cm race tube. Only those cultures that are sufficiently far advanced in the development of senescence will stop growth in the length of a race tube. Resumption of growth occurs in most cases of growth arrest in race tubes. Although in subculture series growth resumption is rare, there is probably a similar basis: mitochondrial DNA (mtDNA) studies on one such case showed that growth resumption is associated with a resurgence of normal mtDNA and a decline of abnormal genomes. When senescent cultures in race tubes were sampled by removing mycelial cores, longer grown cultures were shown to be able to support less growth than younger cultures of the same genotype, and the growth front was generally able to support less growth than other regions. Therefore senescence in both transfer series and in race tubes involves the accumulation of genetic factors unable to suppport continuous growth. The expression of senescence is considered to be more efficient in subculture series than in race tubes. In such series, conidia or aerial hyphae work equally well as transfer inocula, but 1-mm hyphal tips cut from growing mycelium do not promote senescence when used as inocula. Furthermore, varying the transfer interval does not affect senescence. It is concluded that there is some feature of the development or germination of quiescent cells that enhances the expression of senescence in Neurospora.Key words: Neurospora, senescence, mitochondria, growth.

scholarly journals Mitochondrial fission is required for proper nucleoid distribution within mitochondrial networks

2021 ◽  
Author(s):  
Hema Saranya Ilamathi ◽  
Mathieu Ouellet ◽  
Rasha Sabouny ◽  
Justine Desrochers-Goyette ◽  
Matthew A Lines ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Network Structure ◽  
Mitochondrial Fission ◽  
Healthy Population ◽  
Primary Cells ◽  
Mitochondrial Homeostasis ◽  
Mtdna Replication ◽  
Mitochondrial Networks ◽  
Regular Spacing

Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.

scholarly journals Mitochondrial DNA segregation and replication restrict the transmission of detrimental mutation

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Zhe Chen ◽  
Zong-Heng Wang ◽  
Guofeng Zhang ◽  
Christopher K.E. Bleck ◽  
Dillon J. Chung ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Stress Test ◽  
Mitochondrial Fission ◽  
Minor Role ◽  
Deleterious Mutations ◽  
Mtdna Mutations ◽  
Electron Transport Chains ◽  
Repair Mechanisms ◽  
A Minor ◽  
Pole Plasm

Although mitochondrial DNA (mtDNA) is prone to accumulate mutations and lacks conventional DNA repair mechanisms, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations is restricted through the maternal lineage is debated. Here, we demonstrate that mitochondrial fission, together with the lack of mtDNA replication, segregate mtDNA into individual organelles in the Drosophila early germarium. After mtDNA segregation, mtDNA transcription begins, which activates respiration. Mitochondria harboring wild-type genomes have functional electron transport chains and propagate more vigorously than mitochondria containing deleterious mutations in hetreoplasmic cells. Therefore, mtDNA expression acts as a stress test for the integrity of mitochondrial genomes and sets the stage for replication competition. Our observations support selective inheritance at the organelle level through a series of developmentally orchestrated mitochondrial processes. We also show that the Balbiani body has a minor role in mtDNA selective inheritance by supplying healthy mitochondria to the pole plasm. These two mechanisms may act synergistically to secure the transmission of functional mtDNA through Drosophila oogenesis.

scholarly journals Specificity and Prevalence of Natural Bovine Antimannan Antibodies

1999 ◽  
Vol 6 (6) ◽  
pp. 946-952 ◽  
Author(s):  
Abhay Srinivasan ◽  
Yawei Ni ◽  
Ian Tizard
Keyword(s):  
Age Groups ◽  
Inhibitory Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Serum Samples ◽  
Adult Cattle ◽  
Calcium Dependent ◽  
High Concentrations ◽  
Carbohydrate Components ◽  
A Minor

ABSTRACT Immune responses to the carbohydrate components of microorganisms, mediated both by antibodies and by lectins, are an important part of host defense. In the present experiments, the specificity and presence of natural bovine antibodies against mannan, a common fungal antigen, were examined by enzyme-linked immunosorbent assay (ELISA), usingSaccharomyces cerevisiae mannan as an antigen. The results showed that all serum samples from animals of three age groups (newborn, calf, and adult) tested contained antimannan antibodies, and the titer of these antibodies increased significantly in adults. However, titers among individual adult cattle differed widely. Inhibition assays showed that yeast mannan was the strongest inhibitor.d-Mannose exhibited only a minor inhibitory effect at high concentrations. This suggests that most of these antibodies recognize an oligosaccharide-based epitope(s) different from those recognized by lectins. Cattle possess three serum C-type lectins (collectins) capable of recognizing mannan in a calcium-dependent manner. Addition of EDTA to the reaction did not reduce antibody binding, suggesting that the binding of these antibodies to mannan was not affected by the presence of collectin. The antibodies purified from either calf or adult serum by mannan-Sepharose affinity chromatography consisted of mainly immunoglobulin G (IgG) and a smaller amount of IgM. IgG1 was shown to be the dominant antimannan IgG isotype by isotype-specific ELISA. Together, these results demonstrate the production of natural antimannan antibodies in cattle in an age-dependent manner. These antibodies might be involved in defending the host against mannan-containing pathogens as a specific line of defense in conjunction with the innate response by lectins.

scholarly journals DRP1 regulates endoplasmic reticulum sheets to control mitochondrial DNA replication and segregation

2021 ◽  
Author(s):  
Hema Saranya Ilamathi ◽  
Sara Benhammouda ◽  
Justine Desrochers-Goyette ◽  
Matthew A Lines ◽  
Marc Germain
Keyword(s):  
Endoplasmic Reticulum ◽  
Mitochondrial Dna ◽  
Protein Complexes ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Mitochondrial Diseases ◽  
Contact Sites ◽  
Transport Chain ◽  
Essential Components ◽  
Mtdna Replication

Mitochondria are multi-faceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) codes for several essential components of the electron transport chain, and mtDNA maintenance defects lead to mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is regulated by mitochondrial dynamics. Specifically, mitochondrial fusion is essential for mtDNA maintenance. In contrast, while loss of mitochondrial fission causes the aggregation of nucleoids (mtDNA-protein complexes), its role in nucleoid distribution remains unclear. Here, we show that the mitochondrial fission protein DRP1 regulates nucleoid segregation by altering ER sheets, the ER structure associated with protein synthesis. Specifically, DRP1 loss or mutation leads to altered ER sheets that physically interact with mitobulbs, mitochondrial structures containing aggregated nucleoids. Importantly, nucleoid distribution and mtDNA replication were rescued by expressing the ER sheet protein CLIMP63. Thus, our work identifies a novel mechanism by which DRP1 regulates mtDNA replication and distribution.

Abstract 201: Enhancing Pgc-1α Activity Improves Heart Function Through Activating Mitochondrial Biogenesis in Chagas Disease

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Xianxiu Wan ◽  
Jianjun Wen ◽  
Koo Sue-jie
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Biogenesis ◽  
Heart Function ◽  
Mrna Levels ◽  
Beneficial Effects ◽  
Expression Of Genes ◽  
Improve Heart Function ◽  
Mtdna Replication ◽  
Substantial Decline ◽  
Nuclear Levels

Chronic chagasic cardiomyopathy (CCM) is presented with ventricular hypertrophy and contractile dysfunction that can lead to heart failure. I have found that a substantial decline in mitochondrial biogenesis and SIRT1/PGC-1α activity ensue in chronic chagasic mice. It was evidenced by the decline in mitochondrial DNA content as well as mRNA levels of mitochondrial encoded genes and mtDNA replication machinery. Further, the activity of SIRT1 (required for PGC-1α activation) was decreased and associated with decreased nuclear levels of PGC-1-regulated NRF1 transcription factor in chagasic hearts. The mitochondrial size and number were also reduced in chagasic heart, determined by electron microscopy. Therefore, we hypothesized that enhancing the SIRT1/PGC-1α activity by SIRT1 agonist would improve heart function through activating mitochondrial biogenesis in Chagasic disease. Mice were infected with T. cruzi, and beginning at day 90 post-infection (pi), treated with resveratrol (SIRT1 agonist) or metformin (AMPK agonist, can enhance SIRT1 activity) for 21 days; and then heart function was monitored at 150 days pi. We found that treatment with resveratrol partially attenuated the heart dysfunction (stroke volume, cardiac output, ejection fraction, heart rate) and cardiac hypertrophy in chagasic mice. These benefits were associated with improved expression of the mitochondrial DNA encoded genes and mtDNA content though the expression of genes involved in mtDNA replication was not improved. Treatment with metformin was not significantly beneficial in improving the CCM outcomes. The partial beneficial effects of resveratrol could be due to inefficient activation of SIRT1 or delayed start of the treatment. We plan to treat mice with SIRT1 agonist SIRT1720 (10 fold more active than resveratrol) during the indeterminate phase of T. cruzi infection in next set of experiments. This study will improve our understanding of the molecular and immune mechanisms of chagasic heart disease and will provide a novel treatment for chronically-infected chagasic patients.

scholarly journals Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired

2017 ◽  
Vol 114 (47) ◽  
pp. 12466-12471 ◽  
Author(s):  
Paulina H. Wanrooij ◽  
Martin K. M. Engqvist ◽  
Josefin M. E. Forslund ◽  
Clara Navarrete ◽  
Anna Karin Nilsson ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Dna ◽  
Excision Repair ◽  
Genome Replication ◽  
Dntp Pool ◽  
Frequent Exchange ◽  
Repair Mechanisms ◽  
Ribonucleoside Triphosphate ◽  
Deoxyribonucleoside Triphosphate ◽  
Mitochondrial Dna Polymerase

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.

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