scholarly journals The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation

2013 ◽  
Vol 24 (12) ◽  
pp. 1830-1841 ◽  
Author(s):  
Marta A. Uzarska ◽  
Rafal Dutkiewicz ◽  
Sven-Andreas Freibert ◽  
Roland Lill ◽  
Ulrich Mühlenhoff
Keyword(s):  
Complex Formation ◽  
Synthesis Reaction ◽  
The Core ◽  
Iron Sulfur ◽  
Cluster Transfer ◽  
Hsp70 Chaperone ◽  
Mitochondrial Hsp70 ◽  
S Proteins

The mitochondrial Hsp70 chaperone Ssq1 plays a dedicated role in the maturation of iron–sulfur (Fe/S) proteins, an essential process of mitochondria. Similar to its bacterial orthologue HscA, Ssq1 binds to the scaffold protein Isu1, thereby facilitating dissociation of the newly synthesized Fe/S cluster on Isu1 and its transfer to target apoproteins. Here we use in vivo and in vitro approaches to show that Ssq1 also interacts with the monothiol glutaredoxin 5 (Grx5) at a binding site different from that of Isu1. Grx5 binding does not stimulate the ATPase activity of Ssq1 and is most pronounced for the ADP-bound form of Ssq1, which interacts with Isu1 most tightly. The vicinity of Isu1 and Grx5 on the Hsp70 chaperone facilitates rapid Fe/S cluster transfer from Isu1 to Grx5. Grx5 and its bound Fe/S cluster are required for maturation of all cellular Fe/S proteins, regardless of the type of bound Fe/S cofactor and subcellular localization. Hence Grx5 functions as a late-acting component of the core Fe/S cluster (ISC) assembly machinery linking the Fe/S cluster synthesis reaction on Isu1 with late assembly steps involving Fe/S cluster targeting to dedicated apoproteins.

scholarly journals IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

2013 ◽  
Vol 288 (23) ◽  
pp. 16680-16689 ◽  
Author(s):  
Mineaki Seki ◽  
Yukiko Takeda ◽  
Kazuhiro Iwai ◽  
Kiyoji Tanaka
Keyword(s):  
Cluster Assembly ◽  
Core Complex ◽  
Core Component ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
The Core ◽  
Iron Sulfur ◽  
External Component

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.

scholarly journals Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1–ISCA2 by electron flow from ferredoxin FDX2

2020 ◽  
Vol 117 (34) ◽  
pp. 20555-20565 ◽  
Author(s):  
Benjamin Dennis Weiler ◽  
Marie-Christin Brück ◽  
Isabell Kothe ◽  
Eckhard Bill ◽  
Roland Lill ◽  
...  
Keyword(s):  
Electron Flow ◽  
Protein Assembly ◽  
In Vivo Studies ◽  
Cluster Assembly ◽  
Third Phase ◽  
Iron Sulfur ◽  
Cluster Transfer ◽  
In Vitro Assembly

The essential process of iron-sulfur (Fe/S) cluster assembly (ISC) in mitochondria occurs in three major phases. First, [2Fe-2S] clusters are synthesized on the scaffold protein ISCU2; second, these clusters are transferred to the monothiol glutaredoxin GLRX5 by an Hsp70 system followed by insertion into [2Fe-2S] apoproteins; third, [4Fe-4S] clusters are formed involving the ISC proteins ISCA1–ISCA2–IBA57 followed by target-specific apoprotein insertion. The third phase is poorly characterized biochemically, because previous in vitro assembly reactions involved artificial reductants and lacked at least one of the in vivo-identified ISC components. Here, we reconstituted the maturation of mitochondrial [4Fe-4S] aconitase without artificial reductants and verified the [2Fe-2S]-containing GLRX5 as cluster donor. The process required all components known from in vivo studies (i.e., ISCA1–ISCA2–IBA57), yet surprisingly also depended on mitochondrial ferredoxin FDX2 and its NADPH-coupled reductase FDXR. Electrons from FDX2 catalyze the reductive [2Fe-2S] cluster fusion on ISCA1–ISCA2 in an IBA57-dependent fashion. This previously unidentified electron transfer was occluded during previous in vivo studies due to the earlier FDX2 requirement for [2Fe-2S] cluster synthesis on ISCU2. The FDX2 function is specific, because neither FDX1, a mitochondrial ferredoxin involved in steroid production, nor other cellular reducing systems, supported maturation. In contrast to ISC factor-assisted [4Fe-4S] protein assembly, [2Fe-2S] cluster transfer from GLRX5 to [2Fe-2S] apoproteins occurred spontaneously within seconds, clearly distinguishing the mechanisms of [2Fe-2S] and [4Fe-4S] protein maturation. Our study defines the physiologically relevant mechanistic action of late-acting ISC factors in mitochondrial [4Fe-4S] cluster synthesis, trafficking, and apoprotein insertion.

BAF is required for emerin assembly into the reforming nuclear envelope

2001 ◽  
Vol 114 (24) ◽  
pp. 4575-4585 ◽  
Author(s):  
Tokuko Haraguchi ◽  
Takako Koujin ◽  
Miriam Segura-Totten ◽  
Kenneth K. Lee ◽  
Yosuke Matsuoka ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Core Region ◽  
Lamin B ◽  
The Core ◽  
Double Stranded Dna ◽  
Recessive Form ◽  
Nuclear Envelope Assembly

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

Participation of the upstream region of the fibroin gene in the formation of transcription complex in vitro

1986 ◽  
Vol 6 (11) ◽  
pp. 3928-3933
Author(s):  
M Tsuda ◽  
S Hirose ◽  
Y Suzuki
Keyword(s):  
Complex Formation ◽  
Specific Interaction ◽  
Inhibitory Effect ◽  
Silk Gland ◽  
Upstream Region ◽  
Fibroin Gene ◽  
Transcription Complexes ◽  
Posterior Silk Gland

The addition of exogenous histones has an inhibitory effect on fibroin gene transcription in posterior silk gland extracts. The histones probably disturb a process in complex formation, because when transcription complexes were constructed by preincubation of the templates with the extracts, the inhibitory effect of histones was greatly reduced. Transcription of a fibroin gene construct, pFb5' delta-238, having the upstream region beyond the TATA box was relatively less inhibited than that of pFb5' delta-44 lacking the upstream region. This tendency toward differential inhibition was observed in the silk gland extracts but not in a HeLa cell extract and persisted even after complex formation in the silk gland extracts, suggesting a specific interaction of the upstream region with some factors in the extracts. The complexes formed on pFb5' delta-44 are probably more susceptible to the inhibitory effect of histones. On the basis of these results we propose a participation of the upstream region of the fibroin gene in the formation of stable transcription complexes at the promoter through an interaction with specific factors in the silk gland. Since the transcription-enhancing effect via the upstream region is augmented at a high histone/DNA ratio, it may mimic the in vivo situation in which the fibroin gene can be transcribed in the posterior silk gland even in the presence of excess suppressive materials.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

Domains of the rat rDNA promoter must be aligned stereospecifically

1992 ◽  
Vol 12 (3) ◽  
pp. 1266-1275
Author(s):  
W Q Xie ◽  
L I Rothblum
Keyword(s):  
Protein Complexes ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Promoter Elements ◽  
Repeat Distance ◽  
Deletion Mutations ◽  
The Core ◽  
In Vivo Experiments

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G----A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant -7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60

2005 ◽  
Vol 391 (2) ◽  
pp. 185-190 ◽  
Author(s):  
Renu Wadhwa ◽  
Syuichi Takano ◽  
Kamaljit Kaur ◽  
Satoshi Aida ◽  
Tomoko Yaguchi ◽  
...  
Keyword(s):  
Heat Shock ◽  
Molecular Interactions ◽  
Heat Shock Protein 60 ◽  
Hsp60 Expression ◽  
Mitochondrial Hsp70 ◽  
Shock Proteins ◽  
First Time

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

scholarly journals FORMULATION AND IN VITRO–IN VIVO PHARMACOKINETIC EVALUATION OF CARDIOVASCULAR DRUG-LOADED PULSATILE DRUG DELIVERY SYSTEMS

2021 ◽  
pp. 144-151
Author(s):  
KUMAR BABU PASUPULETI ◽  
VENKATACHALAM A. ◽  
BHASKAR REDDY KESAVAN
Keyword(s):  
Drug Release ◽  
Ethyl Cellulose ◽  
Release Kinetic ◽  
In Vitro Drug Release ◽  
The Core ◽  
Coated Tablet ◽  
Tablet Formulations ◽  
Core Tablet

Objective: This study is to formulate Nebivolol into a Pulsatile liquid, solid composite compression coated tablet, which will delay the release of the drug in early morning hypertension conditions. Methods: The liquid, solid composite tablet was formulated and compressed with the ethylcellulose coating polymer. The percent in vitro drug release of the liquid solid composite compressed tablet was tested. Based on disintegration time and wetting time, the LCS2, LCS3, LSC6, LCS7 and LCS12 formulations were found to be the optimized solid-liquid compacts fast-dissolving core tablet formulations, which may be excellent candidates for further coating with polymer to transfer into press coated pulsatile tablet formulations. Coating the core tablet with varying ethyl cellulose concentrations resulted in five different formulations of the pulsatile press-coated tablet (CT1, CT2, CT3, CT4, CT5). In vitro drug release, in vitro release, kinetic studies, in vivo pharmacokinetic and stability tests were all performed for the prepared pulsatile press coated tablet. Results: CT3 tablets are coated with ethyl cellulose polymer, which shows maximum controlled drug release from the core tablet i.e. 96.34±1.2% at 8th h. It shows there was an efficient delay in drug release form core tablet i.e. up to 3 h, followed by the maximum amount of drug release of 96.34±2.4 at 8h. Which shows the core drug will be more efficiently protected from the gastric acid environment 1.2 pH, duodenal environment 4.0 pH and release drug only in the small intestine. Conclusion: According to the findings, CT3 Pulsatile press-coated tablet increased the bioavailability of Nebivolol by 3.11 percent.

scholarly journals A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

2005 ◽  
Vol 187 (10) ◽  
pp. 3374-3383 ◽  
Author(s):  
Christopher Stead ◽  
An Tran ◽  
Donald Ferguson ◽  
Sara McGrath ◽  
Robert Cotter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Lipid A ◽  
In Vitro Assay ◽  
Spectrometric Analysis ◽  
Vitro Assay ◽  
The Core ◽  
H Pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

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