The Sso7d protein ofSulfolobus solfataricus: in vitro relationship among different activities

The physiological role of the nonspecific DNA-binding protein Sso7d from the crenarchaeonSulfolobus solfataricusis unknown. In vitro studies have shown that Sso7d promotes annealing of complementary DNA strands (Guagliardi et al. 1997), induces negative supercoiling (Lopez-Garcia et al. 1998), and chaperones the disassembly and renaturation of protein aggregates in an ATP hydrolysis-dependent manner (Guagliardi et al. 2000). In this study, we examined the relationships among the binding of Sso7d to double-stranded DNA, its interaction with protein aggregates, and its ATPase activity. Experiments with 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that exposed hydrophobic surfaces in Sso7d are responsible for interactions with protein aggregates and double-stranded DNA, whereas the site of ATPase activity has a non-hydrophobic character. The interactions of Sso7d with double-stranded DNA and with protein aggregates are mutually exclusive events, suggesting that the disassembly activity and the DNA-related activities of Sso7d may be competitive in vivo. In contrast, the hydrolysis of ATP by Sso7d is independent of the binding of Sso7d to double-stranded DNA or protein aggregates.

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Increased Bone Mass in Mice Lacking the Adipokine Apelin

Endocrinology ◽

10.1210/en.2012-2034 ◽

2013 ◽

Vol 154 (6) ◽

pp. 2069-2080 ◽

Cited By ~ 22

Author(s):

Lalita Wattanachanya ◽

Wei-Dar Lu ◽

Ramendra K. Kundu ◽

Liping Wang ◽

Marcia J. Abbott ◽

...

Keyword(s):

Bone Mass ◽

Bone Cells ◽

Physiological Role ◽

In Vitro Study ◽

Maximum Effect ◽

Dependent Manner ◽

Primary Mouse ◽

Skeletal Homeostasis

Abstract Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo.

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Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

Molecular Biology of the Cell ◽

10.1091/mbc.e09-12-1017 ◽

2010 ◽

Vol 21 (6) ◽

pp. 871-884 ◽

Cited By ~ 106

Author(s):

Atanas V. Koulov ◽

Paul LaPointe ◽

Bingwen Lu ◽

Abbas Razvi ◽

Judith Coppinger ◽

...

Keyword(s):

Cystic Fibrosis ◽

Atpase Activity ◽

Protein Misfolding ◽

Atp Hydrolysis ◽

Structural Basis ◽

Inherited Disease ◽

Hsp90 Chaperone ◽

Terminal Domains

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

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Ca2+-Dependent Interaction of S100A1 with F1-ATPase Leads to an Increased ATP Content in Cardiomyocytes

Molecular and Cellular Biology ◽

10.1128/mcb.02045-06 ◽

2007 ◽

Vol 27 (12) ◽

pp. 4365-4373 ◽

Cited By ~ 68

Author(s):

Melanie Boerries ◽

Patrick Most ◽

Jonathan R. Gledhill ◽

John E. Walker ◽

Hugo A. Katus ◽

...

Keyword(s):

Energy Metabolism ◽

Atpase Activity ◽

Cardiac Contractility ◽

Gel Filtration ◽

Dependent Manner ◽

Atp Content ◽

Atp Production ◽

Rat Hearts

ABSTRACT S100A1, a Ca2+-sensing protein of the EF-hand family that is expressed predominantly in cardiac muscle, plays a pivotal role in cardiac contractility in vitro and in vivo. It has recently been demonstrated that by restoring Ca2+ homeostasis, S100A1 was able to rescue contractile dysfunction in failing rat hearts. Myocardial contractility is regulated not only by Ca2+ homeostasis but also by energy metabolism, in particular the production of ATP. Here, we report a novel interaction of S100A1 with mitochondrial F1-ATPase, which affects F1-ATPase activity and cellular ATP production. In particular, cardiomyocytes that overexpress S100A1 exhibited a higher ATP content than control cells, whereas knockdown of S100A1 expression decreased ATP levels. In pull-down experiments, we identified the α- and β-chain of F1-ATPase to interact with S100A1 in a Ca2+-dependent manner. The interaction was confirmed by colocalization studies of S100A1 and F1-ATPase and the analysis of the S100A1-F1-ATPase complex by gel filtration chromatography. The functional impact of this association is highlighted by an S100A1-mediated increase of F1-ATPase activity. Consistently, ATP synthase activity is reduced in cardiomyocytes from S100A1 knockout mice. Our data indicate that S100A1 might play a key role in cardiac energy metabolism.

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AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

eLife ◽

10.7554/elife.12621 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 64

Author(s):

Steffen Preissler ◽

Cláudia Rato ◽

Ruming Chen ◽

Robin Antrobus ◽

Shujing Ding ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Atp Hydrolysis ◽

Covalent Modification ◽

Dependent Manner ◽

Gene Encoding ◽

Unfolded Protein ◽

Peptide Dissociation

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

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Mutation of the ATP-Binding Pocket of SSA1 Indicates That a Functional Interaction Between Ssa1p and Ydj1p Is Required for Post-translational Translocation Into the Yeast Endoplasmic Reticulum

Genetics ◽

10.1093/genetics/156.2.501 ◽

2000 ◽

Vol 156 (2) ◽

pp. 501-512 ◽

Cited By ~ 3

Author(s):

Amie J McClellan ◽

Jeffrey L Brodsky

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Binding Pocket ◽

Atp Binding ◽

Dominant Effect ◽

Functional Interaction ◽

Mutant Proteins

Abstract The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

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Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1820157116 ◽

2019 ◽

Vol 116 (12) ◽

pp. 5505-5513 ◽

Cited By ~ 13

Author(s):

Elda Cannavo ◽

Giordano Reginato ◽

Petr Cejka

Keyword(s):

Atp Hydrolysis ◽

Double Strand Breaks ◽

Dependent Manner ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Strand Breaks ◽

Dna Strands ◽

Dna Strand ◽

The Individual

To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 fromSaccharomyces cerevisiaeis a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA.

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The Bradyrhizobium japonicum Irr Protein Is a Transcriptional Repressor with High-Affinity DNA-Binding Activity

Journal of Bacteriology ◽

10.1128/jb.00495-08 ◽

2008 ◽

Vol 190 (15) ◽

pp. 5172-5177 ◽

Cited By ~ 20

Author(s):

Indu Sangwan ◽

Sandra K. Small ◽

Mark R. O'Brian

Keyword(s):

Dna Binding ◽

Bradyrhizobium Japonicum ◽

Iron Homeostasis ◽

Transcriptional Repressor ◽

Physiological Role ◽

Iron Limitation ◽

Dependent Manner ◽

High Affinity

ABSTRACT The Irr protein is a global regulator of iron homeostasis in Bradyrhizobium japonicum, and a subset of genes within the Irr regulon are negatively controlled under iron limitation. However, repressor function, high-affinity DNA binding in vitro, or promoter occupancy in vivo of Irr for a negatively regulated gene has not been demonstrated. Here, we show that the blr7895 and bll6680 genes are negatively regulated by Irr as determined by derepression of transcript levels in iron-limited cells of an irr mutant strain. Electrophoretic gel mobility shift analysis showed that a component in extracts of wild-type cells grown under iron limitation bound the iron control elements (ICE) within the promoters of blr7895 and bll6680 identified previously (G. Rudolph, G. Semini, F. Hauser, A. Lindemann, M. Friberg, H. Hennecke, and H. M. Fischer, J. Bacteriol. 188:733-744, 2006). Binding was not observed with extracts of cells from the parent strain grown under high iron conditions or with those from an irr mutant. Furthermore, gel mobility supershift experiments identified Irr as a component of the binding complex. Purified recombinant Irr bound to ICE DNA with high affinity in the presence of divalent metal, with K d values of 7 to 19 nM, consistent with a physiological role for Irr as a transcriptional regulator. In addition, in vitro transcription initiated from the blr7895 promoter was inhibited by Irr. Whole-cell cross-linking and immunoprecipitation experiments showed that Irr occupies the promoters of blr7895 and bll6680 in vivo in an iron-dependent manner. The findings demonstrate that Irr is a transcriptional repressor that binds DNA with high affinity.

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ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

eLife ◽

10.7554/elife.18746 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 50

Author(s):

Christopher Frederick Mugler ◽

Maria Hondele ◽

Stephanie Heinrich ◽

Ruchika Sachdev ◽

Pascal Vallotton ◽

...

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

Posttranscriptional Gene Regulation ◽

The Dead ◽

Processing Body

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

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Treatment with sodium butyrate has therapeutic benefits for Machado-Joseph disease through the induction of autophagy

10.1101/2021.04.30.442119 ◽

2021 ◽

Author(s):

Maxinne Watchon ◽

Katherine J. Robinson ◽

Luan Luu ◽

Kristy C. Yuan ◽

Albert Lee ◽

...

Keyword(s):

Sodium Butyrate ◽

Trinucleotide Repeat ◽

Swimming Performance ◽

Protein Aggregates ◽

Dependent Manner ◽

Beneficial Effects ◽

Therapeutic Benefits ◽

Machado Joseph Disease

AbstractMachado-Joseph disease (MJD) is a fatal neurodegenerative disease caused by expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of neurotoxic ataxin-3 protein aggregates, neurodegeneration and motor deficits. Here we investigated the therapeutic potential of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model MJD. MJD SH-SY5Y cells were found to contain ataxin-3 oligomeric species and protein aggregates. Interestingly, treatment with SB decreased the size of detergentinsoluble ataxin-3 aggregates in vitro. Further investigation revealed that SB treatment increased activity of the autophagy protein quality control pathway in the MJD cells and decreased presence of ataxin-3 oligomers in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo through induction of autophagy and improving swimming performance in transgenic MJD zebrafish. Co-treating the MJD zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB, suggesting that the improved swimming performance was autophagy-dependent. Furthermore, intraperitoneal injection of SB to wild type mice resulted in increased levels of neuronal LC3B levels, indicating induction of autophagy within the brain. Collectively, our findings suggest that SB can induce activity of the autophagy pathway and can produce beneficial effects in vitro and in vivo. We propose that treatment with sodium butyrate warrants further investigation for the treatment of neurodegenerative diseases underpinned by proteinopathy mechanisms, including MJD.

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Suramin and NF449 are IP5K inhibitors that disrupt inositol hexakisphosphate–mediated regulation of cullin–RING ligase and sensitize cancer cells to MLN4924/pevonedistat

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014375 ◽

2020 ◽

Vol 295 (30) ◽

pp. 10281-10292

Author(s):

Xiaozhe Zhang ◽

Shaodong Shi ◽

Yang Su ◽

Xiaoli Yang ◽

Sining He ◽

...

Keyword(s):

Atp Hydrolysis ◽

Protein S ◽

Activity Cycle ◽

Therapeutic Effects ◽

Dependent Manner ◽

Inositol Hexakisphosphate ◽

3D Genome ◽

Trial Drug

Inositol hexakisphosphate (IP6) is an abundant metabolite synthesized from inositol 1,3,4,5,6-pentakisphosphate (IP5) by the single IP5 2-kinase (IP5K). Genetic and biochemical studies have shown that IP6 usually functions as a structural cofactor in protein(s) mediating mRNA export, DNA repair, necroptosis, 3D genome organization, HIV infection, and cullin–RING ligase (CRL) deneddylation. However, it remains unknown whether pharmacological perturbation of cellular IP6 levels affects any of these processes. Here, we performed screening for small molecules that regulate human IP5K activity, revealing that the antiparasitic drug and polysulfonic compound suramin efficiently inhibits IP5K in vitro and in vivo. The results from docking experiments and biochemical validations suggested that the suramin targets IP5K in a distinct bidentate manner by concurrently binding to the ATP- and IP5-binding pockets, thereby inhibiting both IP5 phosphorylation and ATP hydrolysis. NF449, a suramin analog with additional sulfonate moieties, more potently inhibited IP5K. Both suramin and NF449 disrupted IP6-dependent sequestration of CRL by the deneddylase COP9 signalosome, thereby affecting CRL activity cycle and component dynamics in an IP5K-dependent manner. Finally, nontoxic doses of suramin, NF449, or NF110 exacerbate the loss of cell viability elicited by the neddylation inhibitor and clinical trial drug MLN4924/pevonedistat, suggesting synergistic ef-fects. Suramin and its analogs provide structural templates for designing potent and specific IP5K inhibitors, which could be used in combination therapy along with MLN4924/pevonedistat. IP5K is a potential mechanistic target of suramin, accounting for suramin's therapeutic effects.

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