scholarly journals Structural basis of the P4B ATPase lipid flippase activity

2021 ◽  
Author(s):  
Lin Bai ◽  
Bhawik K. Jain ◽  
Qinglong You ◽  
H. Diessel Duan ◽  
Todd R. Graham ◽  
...  
Keyword(s):  
Transport Mechanism ◽  
Structural Basis ◽  
Β Subunit ◽  
Α Subunit ◽  
Substrate Transport ◽  
Membrane Asymmetry ◽  
Intermediate States ◽  
Lipid Flippase

ABSTRACTP4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory β-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a β-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the first structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1’s ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

scholarly journals H,K-ATPase α subunit C-terminal membrane topology: epitope tags in the insect cell expression system

1999 ◽  
Vol 340 (3) ◽  
pp. 601-611
Author(s):  
Adam J. SMOLKA ◽  
Kellie A. LARSEN ◽  
Clifford W. SCHWEINFEST ◽  
Charles E. HAMMOND
Keyword(s):  
Expression System ◽  
Recombinant Baculovirus ◽  
Transmembrane Domains ◽  
Terminal Region ◽  
Structural Basis ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Α Subunit ◽  
Baculovirus Infection ◽  
P Type

The H,K-ATPase responsible for gastric acidification is a heterodimeric (α and β subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-α]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic α subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the β subunit (HKβ), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the α subunit (HKα) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKα and HKβ cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKα- and HKβ-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKα and HKβ in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKα residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKα.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Thyroid hormone regulation of mRNAs encoding thyrotropin β-subunit, glycoprotein α-subunit, and thyroid hormone receptors α and β in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas

2009 ◽  
Vol 202 (1) ◽  
pp. 43-54 ◽  
Author(s):  
Sean C Lema ◽  
Jon T Dickey ◽  
Irvin R Schultz ◽  
Penny Swanson
Keyword(s):  
Thyroid Hormone ◽  
Pituitary Gland ◽  
Teleost Fish ◽  
Hormone Receptors ◽  
Pimephales Promelas ◽  
Early Gene ◽  
Thyroid Hormone Receptors ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3′-triiodo-l-thyronine (T3) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T3 elevated circulating total T3, while methimazole depressed plasma levels of total thyroxine (T4). In both sexes, this methimazole-induced reduction in T4 led to elevated mRNA abundance for thyrotropin β-subunit (tshβ) in the pituitary gland. Fish treated with T3 had elevated transcript levels for TR isoforms α and β (trα and trβ) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T3 elevated gene transcripts for tshβ, glycoprotein hormone α-subunit (gphα), and trβ, while not affecting trα levels. Taken together, these results demonstrate negative feedback of T4 on pituitary tshβ, identify trα and trβ as T3-autoinduced genes in the brain and liver, and provide new evidence that tshβ, gphα, and trβ are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

scholarly journals Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

2017 ◽  
Vol 474 (5) ◽  
pp. 751-769 ◽  
Author(s):  
M. Kristian Koski ◽  
Jothi Anantharajan ◽  
Petri Kursula ◽  
Prathusha Dhavala ◽  
Abhinandan V. Murthy ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Coiled Coil ◽  
Β Subunit ◽  
Dimerization Domain ◽  
Α Subunit ◽  
Protein Dimer ◽  
Symmetric Molecule ◽  
Asymmetric Shape ◽  
Proline Rich Peptide ◽  
Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

Differential Regulation of the Epithelial Sodium Channel (ENaC) in Rat Kidney, Lung and Distal Colon in Response to Aldosterone.

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 724-724
Author(s):  
Shyama M E Masilamani ◽  
Gheun-Ho Kim ◽  
Mark A Knepper
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Distal Colon ◽  
Β Subunit ◽  
Dietary Salt ◽  
Α Subunit ◽  
Salt Restriction ◽  
Γ Subunit ◽  
Dietary Salt Restriction ◽  
Epithelial Sodium Channel Enac

P170 The mineralocorticoid hormone, aldosterone increases renal tubule Na absorption via increases in the protein abundances of the α-subunit of the epithelial sodium channel (ENaC) and the 70 kDa form of the γ- subunit of ENaC (JCI 104:R19-R23). This study assesses the affect of dietary salt restriction on the regulation of the epithelial sodium channel (ENaC) in the lung and distal colon, in addition to kidney, using semiquantitative immunoblotting. Rats were placed initially on either a control Na intake (0.02 meq/day), or a low Na intake (0.2 meq/day) for 10 days. The low salt treated rats demonstrated an increase in plasma aldosterone levels at day 10 (control = 0.78 + 0.32 nM; Na restricted = 3.50 + 1.30 nM). In kidney homogenates, there were marked increases in the band density of the α-subunit of ENaC (286 % of control) and the 70 kDa form of γ-subunit of ENaC (262 % of control), but no increase in the abundance of the β-subunit of ENaC. In lung homogenates, there was no significant change in the band densities of the α, β, or γ subunits of ENaC. In distal colon, there was an increase in the band density of the β-subunit of ENaC (311 % of control) and an increase in both the 85 kDa (2355% of control) and 70 kDa (843 % of control) form of the γ subunit of ENaC in response to dietary Na restriction. However, there was no significant difference in the band density of the α-subunit of ENaC. These findings demonstrate tissue specific regulation of the three subunits of ENaC in response to dietary salt restriction.

scholarly journals The Extracellular Domain of the β2Integrin β Subunit (CD18) Is Sufficient forEscherichia coliHemolysin andAggregatibacter actinomycetemcomitansLeukotoxin Cytotoxic Activity

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Laura C. Ristow ◽  
Vy Tran ◽  
Kevin J. Schwartz ◽  
Lillie Pankratz ◽  
Andrew Mehle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Integrin Signaling ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Content Type ◽  
Animal Pathogens ◽  
Tract Infections

ABSTRACTTheEscherichia colihemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA fromAggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the β2integrin β subunit. Δβ2cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δβ2cells are more resistant than wild-type U-937 cells to LtxA, as Δβ2cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single β2integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the β2subunit, but not to αL, αM, or αXin far-Western blots. Genetic complementation of Δβ2cells with either β2or β2with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that β2integrin signaling is not required for cytotoxicity. Finally, β2mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δβ2cells are equally sensitive toStaphylococcus aureusα-toxin andProteus mirabilisHpmA. Our studies show two RTX toxins use the β2integrin β subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.IMPORTANCEUrinary tract infections are one of the most common bacterial infections worldwide. UropathogenicEscherichia colistrains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenicEscherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the β2integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. TheE. colihemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.

scholarly journals Asssembly of the chimeric Na+K+-ATPase and H+K+-ATPase β-subunit with the Na+K+-ATPase α-subunit

1997 ◽  
Vol 1330 (2) ◽  
pp. 217-224 ◽  
Author(s):  
Susumu Ueno ◽  
Kazuo Takeda ◽  
Izumi Futoshi ◽  
Futai Masamitsu ◽  
Wolfgang Schwarz ◽  
...  
Keyword(s):  
Β Subunit ◽  
Α Subunit

scholarly journals Cytoplasmic Sequestration of the Polyomavirus Enhancer Binding Protein 2 (PEBP2)/Core Binding Factor α (CBFα) Subunit by the Leukemia-Related PEBP2/CBFβ-SMMHC Fusion Protein Inhibits PEBP2/CBF-Mediated Transactivation

1998 ◽  
Vol 18 (7) ◽  
pp. 4252-4261 ◽  
Author(s):  
Yuka Kanno ◽  
Tomohiko Kanno ◽  
Chohei Sakakura ◽  
Suk-Chul Bae ◽  
Yoshiaki Ito
Keyword(s):  
Nuclear Translocation ◽  
Binding Protein ◽  
Myelogenous Leukemia ◽  
Luciferase Assay ◽  
Β Subunit ◽  
Core Binding Factor ◽  
Α Subunit ◽  
Enhancer Binding Protein ◽  
Binding Factor ◽  
Minimal Region

ABSTRACT The polyomavirus enhancer binding protein 2 (PEBP2)/core binding factor (CBF) is a transcription factor composed of two subunits, α and β. The gene encoding the β subunit is disrupted by inv(16), resulting in the formation of a chimeric protein, β-SMMHC, which is associated with acute myelogenous leukemia. To understand the effect of β-SMMHC on PEBP2-mediated transactivation, we used a luciferase assay system in which contribution of both the α and β subunits was absolutely required to activate transcription. Using this system, we found that the minimal region of the β subunit required for transactivation resides between amino acid 1 and 135, which is known to dimerize with the α subunit. In contrast, β-SMMHC, despite having this minimal region for dimerization and transactivation, failed to support transcription with the α subunit. Furthermore β-SMMHC blocked the synergistic transcription achieved by PEBP2 and CCAAT/enhancer binding protein α. By using a construct in which the PEBP2 α subunit was fused to the glucocorticoid receptor ligand binding domain, we demonstrated that coexpressed β-SMMHC tightly sequestered the α subunit in the cytoplasm and blocked dexamethasone-dependent nuclear translocation of the α subunit. Thus, the result suggess that β-SMMHC inhibits PEBP2-mediated transcription via cytoplasmic sequestration of the α subunit. Lastly proliferation of ME-1 cells that harbor inv(16) was blocked by an antisense oligonucleotide complementary to the junction of the chimeric mRNA, suggesting that β-SMMHC contributes to leukemogenesis by blocking the differentiation of myeloid cells.

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