Regulation Of ATPase Activity Research Articles

The middle domain of Hsp104 can ensure substrates are functional after processing.

Molecular chaperones play a central role in protein disaggregation. However, the molecular determinants that regulate this process are poorly understood. Hsp104 is an AAA+ ATPase that disassembles stress granules and amyloids in yeast through collaboration with Hsp70 and Hsp40. In vitro studies show that Hsp104 processes different types of protein aggregates by partially translocating or threading polypeptides through the central pore of the hexamer. However, it is unclear how Hsp104 processing influences client protein function in vivo. The middle domain (MD) of Hsp104 regulates ATPase activity and interactions with Hsp70. Here, we tested how MD variants, Hsp104A503S and Hsp104A503V, process different protein aggregates. We establish that engineered MD variants fail to resolve stress granules but retain prion fragmentation activity required for prion propagation. Using the Sup35 prion protein, our in vitro and in vivo data indicate that the MD variants can disassemble Sup35 aggregates, but the disaggregated protein has reduced GTPase and translation termination activity. These results suggest that the middle domain can play a role in sensing certain substrates and plays an essential role in ensuring the processed protein is functional.

The regulation of ATPase activity by K+ ions in gut isolated E. coli strains and its role in propionic acid sensing

The regulation of ATPase activity by K+ ions in gut isolated E. coli strains and its role in propionic acid sensing

RNA-Seq and metabolomic analysis reveal dynamic response mechanism to hypoxia in the pearl oyster Pinctada fucata martensii

Hypoxia is becoming a stressor in aquatic environments due to human activities and climate change. To reveal the dynamic response mechanisms of pearl oyster, this study investigated transcriptomic and metabolomic changes in pearl oyster (Pinctada fucata martensii) exposed to hypoxia for 2 and 25 days. In total, 488 differentially expressed genes (DEGs) were detected between 2 and 25 days of hypoxia stress. The identified DEGs were primarily associated with Gene Ontology (GO) terms such as "histone H4 acetylation", "DNA methylation", "histone (H3-K9/H4-K20/H3-K27/H3-K4) trimethylation", "positive regulation of ATPase activity", "myosin complex", “microtubule-based processes", "actin cytoskeleton", and "glutathione peroxidase activity", as well as KEGG pathways such as "neuroactive ligand-receptor interactions", "ECM-receptor interactions", and "apoptosis". We also identified 77 significantly differential metabolites (SDMs), which were associated with 32 metabolic pathways, including arginine and proline metabolism, glycerophospholipid metabolism, aminoacyl-tRNA biosynthesis, and sphingolipid metabolism. Integrated DEGs and SDMs analyses showed that pearl oysters were found to suffer from reduced biomineralization, hindered cytoskeletal reorganization, decreased neuronal excitability, apoptosis, immune inhibition, and oxidative stress after 25 days of hypoxic stress. Overall, these results help to elucidate the complex responses of pearl oysters to hypoxic conditions.

Inhibition of Hsp90 K284 Acetylation Aalleviates Cardiac Injury After Ischemia-Reperfusion Injury.

Our objective was to determine the role of acetyl-Hsp90 and its relationship with the NF-κB p65 signaling pathway in CVDs. We investigated the effect of acetyl-Hsp90 on cardiac inflammation and apoptosis after ischemia-reperfusion injury (I/RI). The results showed that the induction of acetyl-Hsp90 occurred in the heart during I/R and in primary cardiomyocytes during oxygen-glucose deprivation/reoxygenation (OGD/R). Moreover, the nonacetylated mutant of Hsp90 (Hsp90-K284R), through the regulation of ATPase activities within its N-terminal domain (NTD), indirectly or directly increases its interaction with NF-κB p65. This led to a reduction in the activation of the NF-κB p65 pathway, thereby attenuating inflammation, apoptosis, and fibrosis, ultimately leading to an improvement in cardiac function. Furthermore, we demonstrated that recombinant human interleukin-37 (rIL-37) exerts a similar cardioprotective effect by reducing acetylation at K284 of Hsp90 after inhibiting the expression of KAT2A.

A SAM-key domain required for enzymatic activity of the Fun30 nucleosome remodeler.

Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30Δ mutant. In vitro, Fun30 protein lacking the SAM-key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity and nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome-remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes.

Differential regulation of Shigella Spa47 ATPase activity by a native C-terminal product of Spa33.

Shigella is a Gram-negative bacterial pathogen that relies on a single type three secretion system (T3SS) as its primary virulence factor. The T3SS includes a highly conserved needle-like apparatus that directly injects bacterial effector proteins into host cells, subverting host cell function, initiating infection, and circumventing resulting host immune responses. Recent findings have located the T3SS ATPase Spa47 to the base of the Shigella T3SS apparatus and have correlated its catalytic function to apparatus formation, protein effector secretion, and overall pathogen virulence. This critical correlation makes Spa47 ATPase activity regulation a likely point of native control over Shigella virulence and a high interest target for non-antibiotic- based therapeutics. Here, we provide a detailed characterization of the natural 11.6 kDa C-terminal translation product of the Shigella T3SS protein Spa33 (Spa33C), showing that it is required for proper virulence and that it pulls down with several known T3SS proteins, consistent with a structural role within the sorting platform of the T3SS apparatus. In vitro binding assays and detailed kinetic analyses suggest an additional role, however, as Spa33C differentially regulates Spa47 ATPase activity based on Spa47s oligomeric state, downregulating Spa47 monomer activity and upregulating activity of both homo-oligomeric Spa47 and the hetero-oligomeric MxiN2Spa47 complex. These findings identify Spa33C as only the second known differential T3SS ATPase regulator to date, with the Shigella protein MxiN representing the other. Describing this differential regulatory protein pair begins to close an important gap in understanding of how Shigella may modulate virulence through Spa47 activity and T3SS function.

N-glycosylation modification of heat shock protein gp96 affects its immunological function

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

Phosphoproteomic Comparison of Four Eimeria tenella Life Cycle Stages.

Protein phosphorylation is an important post-translational modification (PTM) involved in diverse cellular functions. It is the most prevalent PTM in both Toxoplasma gondii and Plasmodium falciparum, but its status in Eimeria tenella has not been reported. Herein, we performed a comprehensive, quantitative phosphoproteomic profile analysis of four stages of the E. tenella life cycle: unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), and sporozoites (S). A total of 15,247 phosphorylation sites on 9514 phosphopeptides corresponding to 2897 phosphoproteins were identified across the four stages. In addition, 456, 479, and 198 differentially expressed phosphoproteins (DEPPs) were identified in the comparisons SO7h vs. USO, SO vs. SO7h, and S vs. SO, respectively. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEPPs suggested that they were involved in diverse functions. For SO7h vs. USO, DEPPs were mainly involved in cell division, actin cytoskeleton organization, positive regulation of transport, and pyruvate metabolism. For SO vs. SO7h, they were related to the peptide metabolic process, translation, and RNA transport. DEPPs in the S vs. SO comparison were associated with the tricarboxylic acid metabolic process, positive regulation of ATPase activity, and calcium ion binding. Time course sequencing data analysis (TCseq) identified six clusters with similar expression change characteristics related to carbohydrate metabolism, cytoskeleton organization, and calcium ion transport, demonstrating different regulatory profiles across the life cycle of E. tenella. The results revealed significant changes in the abundance of phosphoproteins during E. tenella development. The findings shed light on the key roles of protein phosphorylation and dephosphorylation in the E. tenella life cycle.

Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity.

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Å away.

Functions of RPM1-interacting protein 4 in plant immunity.

We reviewed recent advances related to RIN4, including its involvement in the immune process through posttranslational modifications, PM H+-ATPase activity regulation, interaction with EXO70 and identification of RIN4-associated NLR proteins. RPM1-interacting protein 4 (RIN4) is a conserved plant immunity regulator that has been extensively studied and can be modified by pathogenic effector proteins. RIN4 plays an important role in both PTI and ETI. In this article, we review the functions of the two conserved NOI domains of RIN4, the C-terminal cysteine residues required for membrane localization and the sites targeted and modified by effector proteins during plant immunity. In addition, we discuss the effect of RIN4 on the stomatal virulence of pathogens via the regulation of PM H+-ATPase activity, which is involved in the immune process through interactions with the exocyst subunit EXO70, and progress in the identification of RIN4-related R proteins in multiple species. This review provides new insights enhancing the current understanding of the immune function of RIN4.

Monomeric bile acids modulate the ATPase activity of detergent-solubilized ABCB4/MDR3

ABCB4, also called multidrug-resistant protein 3 (MDR3), is an ATP binding cassette transporter located in the canalicular membrane of hepatocytes that specifically translocates phosphatidylcholine (PC) lipids from the cytoplasmic to the extracellular leaflet. Due to the harsh detergent effect of bile acids, PC lipids provided by ABCB4 are extracted into the bile. While it is well known that bile acids are the major extractor of PC lipids from the membrane into bile, it is unknown whether only PC lipid extraction is improved or whether bile acids also have a direct effect on ABCB4. Using in vitro experiments, we investigated the modulation of ATP hydrolysis of ABC by different bile acids commonly present in humans. We demonstrated that all tested bile acids stimulated ATPase activity except for taurolithocholic acid, which inhibited ATPase activity due to its hydrophobic nature. Additionally, we observed a nearly linear correlation between the critical micelle concentration and maximal stimulation by each bile acid, and that this modulation was maintained in the presence of PC lipids. This study revealed a large effect of 24-nor-ursodeoxycholic acid, suggesting a distinct mode of regulation of ATPase activity compared with other bile acids. In addition, it sheds light on the molecular cross talk of canalicular ABC transporters of the human liver.

Cancer-Associated Gain-of-Function Mutations Activate a SWI/SNF-Family Regulatory Hub

SummarySWI/SNF-family remodelers (BAF/PBAF in mammals) are essential chromatin regulators, and mutations in human BAF/PBAF components are associated with ∼20% of cancers. Cancer-associated missense mutations in human BRG1 (encoding the catalytic ATPase) have been characterized previously as conferring loss-of-function. Here, we show that cancer-associated missense mutations in BRG1, when placed into the orthologous Sth1 ATPase of the yeast RSC remodeler, separate into two categories: loss-of-function enzymes, or instead, gain-of-function enzymes that greatly improve DNA translocation efficiency and nucleosome remodeling in vitro. Our work identifies a structural “hub,” formed by the association of several Sth1 domains, that regulates ATPase activity and DNA translocation efficiency. Remarkably, all gain-of-function cancer-associated mutations and all loss-of-function mutations physically localize to distinct adjacent regions in the hub, which specifically regulate and implement DNA translocation, respectively. In vivo, only gain-of-function cancer-associated mutations conferred precocious chromatin accessibility. Taken together, we provide a structure-function mechanistic basis for cancer-associated hyperactivity.

Integrated analysis of quantitative proteome and transcriptional profiles reveals abnormal gene expression and signal pathway in bladder cancer.

Bladder cancer (BCa) is a tumor associated with high morbidity and mortality and its incidence is increasing worldwide. However, the pathogenesis of bladder cancer is not well understood. To further illustrate the molecular mechanisms involved in the pathogenesis of BCa and identify potential therapeutic targets, we combined the transcriptomic analysis with RNA sequencing and tandem mass tags (TMT)-based proteomic methods to quantitatively screen the differentially expressed genes and proteins between bladder cancer tissues (BC) and adjacent normal tissues (AN). Transcriptome and proteome studies indicated 7094 differentially expressed genes (DEGs) and 596 differentially expressed proteins (DEPs) between BC and AN, respectively. GO enrichment analyses revealed that cell adhesion, calcium ion transport, and regulation of ATPase activity were highly enriched in BCa. Moreover, several key signaling pathway were identified as of relevance to BCa, in particular the ECM-receptor interaction, cell adhesion molecules (CAMs), and PPAR signaling pathway. Interestingly, 367 genes were shared by DEGs and DEPs, and a significant positive correlation between mRNA and translation profiles was found. In summary, this joint analysis of transcript and protein profiles provides a comprehensive reference map of gene activity regarding the disease status of BCa.

Integrative analysis of transcriptome-wide association study and gene expression profiling identifies candidate genes associated with stroke.

BackgroundStroke is a major public health burden worldwide. Although genetic variation is known to play a role in the pathogenesis of stroke, the specific pathogenic mechanisms are still unclear. Transcriptome-wide association studies (TWAS) is a powerful approach to prioritize candidate risk genes underlying complex traits. However, this approach has not been applied in stroke.MethodsWe conducted an integrative analysis of TWAS using data from the MEGASTROKE Consortium and gene expression profiling to identify candidate genes for the pathogenesis of stroke. Gene ontology (GO) enrichment analysis was also conducted to detect functional gene sets.ResultsThe TWAS identified 515 transcriptome-wide significant tissue-specific genes, among which SLC25A44 (P = 5.46E−10) and LRCH1 (P = 1.54E−6) were significant by Bonferroni test for stroke. After validation with gene expression profiling, 19 unique genes were recognized. GO enrichment analysis identified eight significant GO functional gene sets, including regulation of cell shape (P = 0.0059), face morphogenesis (P = 0.0247), and positive regulation of ATPase activity (P = 0.0256).ConclusionsOur study identified multiple stroke-associated genes and gene sets, and this analysis provided novel insights into the genetic mechanisms underlying stroke.

Genetic analysis and identification of a candidate gene associated with in vitro regeneration ability of cucumber.

Candidate genes associated with in vitro regeneration were identified in cucumber. The ability to regenerate shoots or whole plants from differentiated plant tissues is essential for plant transformation. In cucumber (Cucumis sativus L.), regeneration ability varies considerably across accessions, but the genetic mechanism has not yet been demonstrated. In the present study, 148 recombinant inbred lines and a core collection were examined to identify candidate genes involved in cucumber regeneration. Four QTL for cotyledon regeneration that explained 9.7-16.6% of the phenotypic variation in regeneration were identified on cucumber chromosomes 1, 3, and 6. The loci Fcrms1.1 and Fcrms+1.1 were consistently detected in the same genetic interval on two regeneration media. A genome-wide association study revealed 18 SNPs (- log(p) > 5) significantly associated with cotyledon regeneration. Three candidate genes in this region were identified. RT-PCR analyses revealed that Csa1G642540 was significantly more highly expressed in genotypes with high cotyledon regeneration rates than in those with low regeneration. The Csa1G642540 CDS driven by its native promoter was transformed into cucumber line 9110Gt; molecular analyses showed that the T-DNA had integrated into the genomes of 8.6% of regenerated plantlets. The seeds from T0 plants expressing Csa1G642540 were tested for regeneration from cotyledon explants, and the segregate ratio in regeneration frequency is 3:1. The AT3G44110.1, the homologue gene of Csa1G642540 in Arabidopsis, has been reported as PM H+-ATPase activity regulation, integrating flowering signals and enlarging meristem function. These results demonstrate that Csa1G642540 might play an important role in regeneration in cucumber and could serve as a selectable marker for regeneration from cotyledons.

X-ray structure of full-length human RuvB-Like 2 \u2013 mechanistic insights into coupling between ATP binding and mechanical action

RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia. The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown. We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology. The ring-shaped hexameric RuvBL2 structure presented here resolves for the first time the mobile domain II of the human protein, which is responsible for protein-protein interactions and ATPase activity regulation. Structural analysis suggests how ATP binding may lead to domain II motion through interactions with conserved N-terminal loop histidine residues. Furthermore, a comparison between hsRuvBL1 and 2 shows differences in surface charge distribution that may account for previously described differences in regulation. Analytical ultracentrifugation and cryo electron microscopy analyses performed on hsRuvBL2 highlight an oligomer plasticity that possibly reflects different physiological conformations of the protein in the cell, as well as that single-stranded DNA (ssDNA) can promote the oligomerization of monomeric hsRuvBL2. Based on these findings, we propose a mechanism for ATP binding and domain II conformational change coupling.

The AAA protein spastin possesses two levels of basal ATPase activity.

The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrate the necessity of intersubunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreases the ATPase activity at low concentration, although the ATP-hydrolyzing potential is not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected.

Limited Proteolysis Analysis of a DEAD‐box Protein and its Domain Truncated Variants

RNA helicases play a crucial role in virtually all aspects of RNA metabolism, and although they share a highly conserved structure, the enzymes exhibit a wide variety of biochemical activity. One family of RNA helicases is DEAD‐box proteins, and while their functions are diverse, the one defining activity of these proteins is their ability to hydrolyze ATP in the presence of single‐stranded or double‐stranded RNA.1 ATPase activity leads to changes in RNA affinity, enabling ligand dissociation and cyclic rebinding to promote correct RNA folding in the cell. For example, Rok1p is a yeast DEAD‐box protein essential in ribosomal RNA folding, and is characterized by a catalytic core where ATP and RNA bind, as well as two peripheral domains.2 The peripheral domains (N‐terminal domain or NTD, and the C‐terminal domain, or CTD), are proposed to regulate ATPase activity as well as provide a region for RNA specificity. Thus, peripheral domains could play a role in regulating the overall structure of the protein and its mechanism of RNA refolding. To monitor peripheral effects, the structural changes of Rok1p in the presence and absence of a ligand were analyzed using limited proteolysis under various conditions. These results were subsequently compared to the truncated variant, Rok1p‐ΔCTD. This comparative analysis determined the structural role of the NTD, and to some extent, the CTD. Time dependent proteolysis suggests that the protein is more dynamic at higher temperatures. In the presence of RNA or a nucleotide, the level of structure fluctuation increases. The observed level of conformational changes is minimal yet has dramatic effects on the ATPase activity of the protein. Specifically, the results show that the protein does not occupy a fully extended core state, which accounts for the unique catalytic activity of Rok1p.Support or Funding InformationThe Thomas Lord Charitable Trust FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Calcium and ATPase Activity in Slow Adaptation and Set Point Regulation in Cochlear Mechanotransduction

Role of Calcium and ATPase Activity in Slow Adaptation and Set Point Regulation in Cochlear Mechanotransduction

Structure-activity relationships of cryptopleurine analogs with E-ring modifications as anti-hepatitis C virus agents

The tylophorine analog rac-cryptopleurine exhibited potent anti-hepatitis C virus (HCV) activity through allosteric regulation of ATPase activity of heat shock cognate protein 70 (Hsc70). We evaluated the impact of modifications on the E-ring of rac-cryptopleurine to the inhibitory activity against HCV replication and regulation of ATPase activity of Hsc70. Cryptopleurine analog YXM-110 with a 13α-hydroxyl group maintained activity against HCV and promoted ATP/ADP turnover of Hsc70; however, compounds with hydroxyl groups at other positions or with other orientations (YXM-109, YXM-139, and YXM-140) did not exhibit similar activities. Size modification or heteroatom incorporation of the E-ring led to loss of anti-HCV activity. Promotion of the chaperone activity of Hsc70 with carboxyl terminus Hsc70 interacting protein (CHIP) further enhanced the anti-HCV activity of rac-cryptopleurine and XYM-110. This structure-activity relationship (SAR) study refined structural design and optimization for developing rac-crytopleurine analogs as potent anti-HCV agents targeted against the host factor involved in HCV replication.