scholarly journals A Single Conserved Residue Mediates Binding of the Ribonucleotide Reductase Catalytic Subunit RRM1 to RRM2 and Is Essential for Mouse Development

2015 ◽  
Vol 35 (17) ◽  
pp. 2910-2917 ◽  
Author(s):  
Julia Specks ◽  
Emilio Lecona ◽  
Andrés J. Lopez-Contreras ◽  
Oscar Fernandez-Capetillo
Keyword(s):  
Ribonucleotide Reductase ◽  
Mammalian Cells ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Mouse Development ◽  
Genetic Studies ◽  
Content Type ◽  
The Impact ◽  
Rate Limiting

The ribonucleotide reductase (RNR) complex, composed of a catalytic subunit (RRM1) and a regulatory subunit (RRM2), is thought to be a rate-limiting enzymatic complex for the production of nucleotides. In humans, theRrm1gene lies at 11p15.5, a tumor suppressor region, and RRM1 expression in cancer has been shown to predict responses to chemotherapy. Nevertheless, whether RRM1 is essential in mammalian cells and what the effects of its haploinsufficiency are remain unknown. To model RNR function in mice we used a mutation previously described inSaccharomyces cerevisiae(Rnr1-W688G) which, despite being viable, leads to increased interaction of the RNR complex with its allosteric inhibitor Sml1. In contrast to yeast, homozygous mutant mice carrying theRrm1mutation (Rrm1WG/WG) are not viable, even at the earliest embryonic stages. Proteomic analyses failed to identify proteins that specifically bind to the mutant RRM1 but revealed that, in mammals, the mutation prevents RRM1 binding to RRM2. Despite the impact of the mutation,Rrm1WG/+mice and cells presented no obvious phenotype, suggesting that the RRM1 protein exists in excess. Our work reveals that binding of RRM1 to RRM2 is essential for mammalian cells and provides the first loss-of-function model of the RNR complex for genetic studies.

scholarly journals Analysis of heat-induced protein aggregation in human mitochondria

2018 ◽  
Author(s):  
Anne Wilkening ◽  
Cornelia Rüb ◽  
Marc Sylvester ◽  
Wolfgang Voos
Keyword(s):  
Heat Stress ◽  
Mammalian Cells ◽  
Elevated Temperatures ◽  
Protein Biosynthesis ◽  
High Sensitivity ◽  
Small Subset ◽  
Loss Of Function ◽  
Mitochondrial Translation ◽  
The Impact ◽  
Induced Aggregation

AbstractAs proteins in mammalian cells exhibit optimal stability at natural temperatures, small temperature variations may cause unfolding and subsequent non-specific aggregation. As this process leads to a loss of function of the affected polypeptides as well as to further cytotoxic stress, aggregate formation has been recognized as a major pathogenic factor in human diseases. In this study we determined the impact of physiological heat stress on mammalian mitochondria on a proteomic level. The overall solubility of endogenous mitochondrial proteins was only marginally affected by a treatment at elevated temperatures. However, we identified a small subset of polypeptides that exhibited an exceptionally high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat stress conditions. In parallel, mitochondrial translation as well as the import of cytosolic proteins was defective in heat stressed mitochondria. Both types of nascent polypeptides, derived from translation as well as from import exhibited a strong heat-induced aggregation tendency. We propose a model that a quick and specific inactivation of elongation factors may prevent an accumulation of misfolded nascent polypeptides and thereby attenuate proteotoxicity under stress.

scholarly journals Tornadic Shear Stress Induces a Transient, Calcineurin-Dependent Hypervirulent Phenotype in Mucorales Molds

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Sebastian Wurster ◽  
Alexander M. Tatara ◽  
Nathaniel D. Albert ◽  
Ashraf S. Ibrahim ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Shear Stress ◽  
Mechanical Stress ◽  
Fungal Growth ◽  
High Energy ◽  
Infection Model ◽  
High Morbidity ◽  
Sequencing Analysis ◽  
Loss Of Function ◽  
Content Type ◽  
The Impact

ABSTRACT Trauma-related necrotizing myocutaneous mucormycosis (NMM) has a high morbidity and mortality in victims of combat-related injuries, geometeorological disasters, and severe burns. Inspired by the observation that several recent clusters of NMM have been associated with extreme mechanical forces (e.g., during tornados), we studied the impact of mechanical stress on Mucoralean biology and virulence in a Drosophila melanogaster infection model. In contrast to other experimental procedures to exert mechanical stress, tornadic shear challenge (TSC) by magnetic stirring induced a hypervirulent phenotype in several clinically relevant Mucorales species but not in Aspergillus or Fusarium. Whereas fungal growth rates, morphogenesis, and susceptibility to noxious environments or phagocytes were not altered by TSC, soluble factors released in the supernatant of shear-challenged R. arrhizus spores rendered static spores hypervirulent. Consistent with a rapid decay of TSC-induced hypervirulence, minimal transcriptional changes were revealed by comparative RNA sequencing analysis of static and shear-challenged Rhizopus arrhizus. However, inhibition of the calcineurin/heat shock protein 90 (hsp90) stress response circuitry by cyclosporine and tanespimycin abrogated the increased pathogenicity of R. arrhizus spores following TSC. Similarly, calcineurin loss-of-function mutants of Mucor circinelloides displayed no increased virulence capacity in flies after undergoing TSC. Collectively, these results establish that TSC induces hypervirulence specifically in Mucorales and point out the calcineurin/hsp90 pathway as a key orchestrator of this phenotype. Our findings invite future studies of topical calcineurin inhibitor treatment of wounds as an adjunct mitigation strategy for NMM following high-energy trauma. IMPORTANCE Given the limited efficacy of current medical treatments in trauma-related necrotizing mucormycosis, there is a dire need to better understand the Mucoralean pathophysiology in order to develop novel strategies to counteract fungal tissue invasion following severe trauma. Here, we describe that tornadic shear stress challenge transiently induces a hypervirulent phenotype in various pathogenic Mucorales species but not in other molds known to cause wound infections. Pharmacological and genetic inhibition of calcineurin signaling abrogated hypervirulence in shear stress-challenged Mucorales, encouraging further evaluation of (topical) calcineurin inhibitors to improve therapeutic outcomes of NMM after combat-related blast injuries or violent storms.

scholarly journals Theapt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus

2016 ◽  
Vol 82 (10) ◽  
pp. 3070-3081 ◽  
Author(s):  
Changyi Zhang ◽  
Qunxin She ◽  
Hongkai Bi ◽  
Rachel J. Whitaker
Keyword(s):  
Evolutionary Ecology ◽  
Population Genomics ◽  
Genetic Manipulation ◽  
Model Organisms ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Genetic Studies ◽  
Content Type ◽  
Sulfolobus Islandicus ◽  
Mutation Assay

ABSTRACTSulfolobus islandicusserves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker inS. islandicusto expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) inS. islandicusM.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded byM164_0158(apt). The application of theaptgene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations inS. islandicusM.16.4 at theaptlocus. Moreover, the general conservation ofaptgenes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that theaptlocus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeonS. islandicus.IMPORTANCECurrently, thepyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since mostSulfolobusmutants constructed by the research community were derived from genetic hosts lacking thepyrEFgenes, thepyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of theapt/6-MP counterselection system for theSulfolobusmodel renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with apyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies amongS. islandicusstrains.

Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase

1994 ◽  
Vol 14 (11) ◽  
pp. 7265-7275
Author(s):  
M Matsuoka ◽  
J Y Kato ◽  
R P Fisher ◽  
D O Morgan ◽  
C J Sherr
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Regulatory Protein ◽  
Cyclin A ◽  
Catalytic Subunit ◽  
Regulatory Control ◽  
Cyclin D ◽  
Enzymatic Activation ◽  
Rate Limiting

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.

The DNA damage-inducible gene DIN1 of Saccharomyces cerevisiae encodes a regulatory subunit of ribonucleotide reductase and is identical to RNR3

1990 ◽  
Vol 10 (10) ◽  
pp. 5553-5557
Author(s):  
K Yagle ◽  
K McEntee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Regulatory Elements ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Subunit Gene ◽  
Gene Encoding ◽  
Sequence Elements ◽  
Inducible Gene

The sequence of the DIN1 gene of Saccharomyces cerevisiae is identical to RNR3, a gene encoding a DNA damage-inducible regulatory subunit of ribonucleotide reductase. Two sequence elements located upstream of DIN1 (RNR3) are homologous to putative DNA damage regulatory elements in the promoter of the reductase catalytic subunit gene, RNR2. The transcript start sites for DIN1(RNR3) have been localized, and induction by different agents has been compared with other DNA damage-regulated genes.

scholarly journals The DNA damage-inducible gene DIN1 of Saccharomyces cerevisiae encodes a regulatory subunit of ribonucleotide reductase and is identical to RNR3.

1990 ◽  
Vol 10 (10) ◽  
pp. 5553-5557 ◽  
Author(s):  
K Yagle ◽  
K McEntee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Regulatory Elements ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Subunit Gene ◽  
Gene Encoding ◽  
Sequence Elements ◽  
Inducible Gene

The sequence of the DIN1 gene of Saccharomyces cerevisiae is identical to RNR3, a gene encoding a DNA damage-inducible regulatory subunit of ribonucleotide reductase. Two sequence elements located upstream of DIN1 (RNR3) are homologous to putative DNA damage regulatory elements in the promoter of the reductase catalytic subunit gene, RNR2. The transcript start sites for DIN1(RNR3) have been localized, and induction by different agents has been compared with other DNA damage-regulated genes.

Russia builds gold reserves to hedge against risk

2018 ◽  
Keyword(s):  
Exchange Rate ◽  
Central Bank ◽  
Financial Support ◽  
Mining Industry ◽  
International Reserves ◽  
Real Economy ◽  
Economic Resilience ◽  
Content Type ◽  
The Impact ◽  
Rate Limiting

Subject Russia's strategy of building up gold reserves. Significance The Central Bank of Russia (CBR) surpassed its Chinese equivalent in January to become the world's fifth-largest holder of gold reserves. Gold purchases have accelerated, as the CBR diversifies its holdings away from assets such as dollars that are more volatile or whose liquidity is vulnerable to US sanctions. This is part of a broader objective of increasing Russia's international reserves in the interests of economic resilience. Impacts CBR gold purchases do not affect the ruble's exchange rate, limiting the impact of reserve building on the real economy. Higher gold reserves would facilitate possible future non-dollar-denominated trading arrangements. Increased CBR purchases provide financial support for the domestic mining industry. Reserves are a non-productive use of assets that could have otherwise been invested to stimulate the economy.

scholarly journals Activation of cyclin-dependent kinase 4 (cdk4) by mouse MO15-associated kinase.

1994 ◽  
Vol 14 (11) ◽  
pp. 7265-7275 ◽  
Author(s):  
M Matsuoka ◽  
J Y Kato ◽  
R P Fisher ◽  
D O Morgan ◽  
C J Sherr
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Regulatory Protein ◽  
Cyclin A ◽  
Catalytic Subunit ◽  
Regulatory Control ◽  
Cyclin D ◽  
Enzymatic Activation ◽  
Rate Limiting

The assembly of functional holoenzymes composed of regulatory D-type cyclins and cyclin-dependent kinases (cdks) is rate limiting for progression through the G1 phase of the mammalian somatic cell cycle. Complexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphorylation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrate that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39MO15) can assemble with a regulatory protein present in either insect or mammalian cells to generate a CAK activity capable of phosphorylating and enzymatically activating both cdk2 and cdk4 in complexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78:713-724, 1994]) can assemble with p39MO15 to activate both cyclin A-cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally reminiscent of cyclin-cdk complexes themselves. Antisera produced to the p39MO15 subunit can completely deplete mammalian cell lysates of CAK activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detected both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of cyclin-cdk complexes, CAK appears to be neither rate limiting for the emergence of cells from quiescence nor subject to upstream regulatory control by stimulatory mitogens.

scholarly journals The spindle assembly function of Caenorhabditis elegans katanin does not require microtubule-severing activity

2011 ◽  
Vol 22 (9) ◽  
pp. 1550-1560 ◽  
Author(s):  
Karen Perry McNally ◽  
Francis J. McNally
Keyword(s):  
Caenorhabditis Elegans ◽  
Spindle Pole ◽  
Catalytic Subunit ◽  
Meiotic Spindle ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Microtubule Binding ◽  
Microtubule Severing ◽  
Terminal Domain ◽  
Meiotic Spindles

Katanin is a heterodimeric microtubule-severing protein that is conserved among eukaryotes. Loss-of-function mutations in the Caenorhabditis elegans katanin catalytic subunit, MEI-1, cause specific defects in female meiotic spindles. To determine the relationship between katanin’s microtubule-severing activity and its role in meiotic spindle formation, we analyzed the MEI-1(A338S) mutant. Unlike wild-type MEI-1, which mediated disassembly of microtubule arrays in Xenopus fibroblasts, MEI-1(A338S) had no effect on fibroblast microtubules, indicating a lack of microtubule-severing activity. In C. elegans, MEI-1(A338S) mediated assembly of extremely long bipolar meiotic spindles. In contrast, a nonsense mutation in MEI-1 caused assembly of meiotic spindles without any poles as assayed by localization of the spindle-pole protein, ASPM-1. These results indicated that katanin protein, but not katanin’s microtubule-severing activity, is required for assembly of acentriolar meiotic spindle poles. To understand the nonsevering activities of katanin, we characterized the N-terminal domain of the katanin catalytic subunit. The N-terminal domain was necessary and sufficient for binding to the katanin regulatory subunit. The katanin regulatory subunit in turn caused a dramatic change in the microtubule-binding properties of the N-terminal domain of the catalytic subunit. This unique bipartite microtubule-binding structure may mediate the spindle-pole assembly activity of katanin during female meiosis.

scholarly journals Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

1998 ◽  
Vol 18 (3) ◽  
pp. 1379-1387 ◽  
Author(s):  
Jinghua Yu ◽  
Yitao Zhang ◽  
James McIlroy ◽  
Tamara Rordorf-Nikolic ◽  
George A. Orr ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Thermal Inactivation ◽  
Insect Cells ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Phosphatidylinositol 3

ABSTRACT We propose a novel model for the regulation of the p85/p110α phosphatidylinositol 3′-kinase. In insect cells, the p110α catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110α is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110α/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110α. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110α dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110α or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110α, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110α in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110α. Thus, we have experimentally distinguished two effects of p85 on p110α: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110α is both stabilized and inhibited by dimerization with p85.

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