Structural insights into sodium transport by the oxaloacetate decarboxylase sodium pump

The oxaloacetate decarboxylase sodium pump (OAD) is a unique primary-active transporter that utilizes the free energy derived from oxaloacetate decarboxylation for sodium transport across the cell membrane. It is composed of 3 subunits: the α subunit catalyzes carboxyl-transfer from oxaloacetate to biotin, the membrane integrated β subunit catalyzes the subsequent carboxyl-biotin decarboxylation and the coupled sodium transport, the γ subunit interacts with the α and β subunits and stabilizes the OAD complex. We present here structure of the Salmonella typhimurium OAD βγ sub-complex. The structure revealed that the β and γ subunits form a β3γ3 hetero-hexamer with extensive interactions between the subunits and shed light on the OAD holo-enzyme assembly. Structure-guided functional studies provided insights into the sodium binding sites in the β subunit and the coupling between carboxyl-biotin decarboxylation and sodium transport by the OAD β subunit.

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Author response: Structural insights into sodium transport by the oxaloacetate decarboxylase sodium pump

10.7554/elife.53853.sa2 ◽

2020 ◽

Author(s):

Xin Xu ◽

Huigang Shi ◽

Xiaowen Gong ◽

Pu Chen ◽

Ying Gao ◽

...

Keyword(s):

Sodium Transport ◽

Sodium Pump ◽

Author Response ◽

Oxaloacetate Decarboxylase ◽

Structural Insights

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Decision letter: Structural insights into sodium transport by the oxaloacetate decarboxylase sodium pump

10.7554/elife.53853.sa1 ◽

2020 ◽

Author(s):

Lan Guan

Keyword(s):

Sodium Transport ◽

Sodium Pump ◽

Oxaloacetate Decarboxylase ◽

Structural Insights

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Evolution of the epithelial sodium channel and the sodium pump as limiting factors of aldosterone action on sodium transport

Physiological Genomics ◽

10.1152/physiolgenomics.00002.2011 ◽

2011 ◽

Vol 43 (13) ◽

pp. 844-854 ◽

Cited By ~ 29

Author(s):

Romain A. Studer ◽

Emilie Person ◽

Marc Robinson-Rechavi ◽

Bernard C. Rossier

Keyword(s):

Sodium Channel ◽

Sodium Transport ◽

Salt Intake ◽

Epithelial Sodium Channel ◽

Cell Junctions ◽

Limiting Factors ◽

Last Common Ancestor ◽

Β Subunit ◽

Α Subunit ◽

Additional Function

Despite large changes in salt intake, the mammalian kidney is able to maintain the extracellular sodium concentration and osmolarity within very narrow margins, thereby controlling blood volume and blood pressure. In the aldosterone-sensitive distal nephron (ASDN), aldosterone tightly controls the activities of epithelial sodium channel (ENaC) and Na,K-ATPase, the two limiting factors in establishing transepithelial sodium transport. It has been proposed that the ENaC/degenerin gene family is restricted to Metazoans, whereas the α- and β-subunits of Na,K-ATPase have homologous genes in prokaryotes. This raises the question of the emergence of osmolarity control. By exploring recent genomic data of diverse organisms, we found that: 1) ENaC/degenerin exists in all of the Metazoans screened, including nonbilaterians and, by extension, was already present in ancestors of Metazoa; 2) ENaC/degenerin is also present in Naegleria gruberi , an eukaryotic microbe, consistent with either a vertical inheritance from the last common ancestor of Eukaryotes or a lateral transfer between Naegleria and Metazoan ancestors; and 3) The Na,K-ATPase β-subunit is restricted to Holozoa, the taxon that includes animals and their closest single-cell relatives. Since the β-subunit of Na,K-ATPase plays a key role in targeting the α-subunit to the plasma membrane and has an additional function in the formation of cell junctions, we propose that the emergence of Na,K-ATPase, together with ENaC/degenerin, is linked to the development of multicellularity in the Metazoan kingdom. The establishment of multicellularity and the associated extracellular compartment (“internal milieu”) precedes the emergence of other key elements of the aldosterone signaling pathway.

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Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)

Scientific Reports ◽

10.1038/s41598-021-02860-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Prakash G. Kshirsagar ◽

Mansi Gulati ◽

Wade M. Junker ◽

Abhijit Aithal ◽

Gaelle Spagnol ◽

...

Keyword(s):

Structure Prediction ◽

Biologically Active ◽

Β Subunit ◽

Α Subunit ◽

Functional Studies ◽

Domain Specific ◽

Random Coils ◽

Simple Strategy ◽

Egfr Family

AbstractMUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

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Characterization of Recombinant β Subunit of Human MUC4 Mucin (rMUC4β)

10.21203/rs.3.rs-812058/v1 ◽

2021 ◽

Author(s):

Prakash G. Kshirsagar ◽

Mansi Gulati ◽

Wade M. Junker ◽

Abhijit Aithal ◽

Gaelle Spagnol ◽

...

Keyword(s):

Structure Prediction ◽

Biologically Active ◽

Β Subunit ◽

Α Subunit ◽

Functional Studies ◽

Domain Specific ◽

Random Coils ◽

Simple Strategy ◽

Egfr Family

Abstract MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant rMUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

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Binary architecture of the Nav1.2-β2 signaling complex

eLife ◽

10.7554/elife.10960 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 28

Author(s):

Samir Das ◽

John Gilchrist ◽

Frank Bosmans ◽

Filip Van Petegem

Keyword(s):

Crystal Structure ◽

Disulfide Bonds ◽

Unique Feature ◽

Β Subunit ◽

Functional Studies ◽

Signaling Complex ◽

Flexible Loop ◽

Spider Toxins ◽

Subunit Isoforms ◽

Structural Insights

To investigate the mechanisms by which β-subunits influence Nav channel function, we solved the crystal structure of the β2 extracellular domain at 1.35Å. We combined these data with known bacterial Nav channel structural insights and novel functional studies to determine the interactions of specific residues in β2 with Nav1.2. We identified a flexible loop formed by 72Cys and 75Cys, a unique feature among the four β-subunit isoforms. Moreover, we found that 55Cys helps to determine the influence of β2 on Nav1.2 toxin susceptibility. Further mutagenesis combined with the use of spider toxins reveals that 55Cys forms a disulfide bond with 910Cys in the Nav1.2 domain II pore loop, thereby suggesting a 1:1 stoichiometry. Our results also provide clues as to which disulfide bonds are formed between adjacent Nav1.2 912/918Cys residues. The concepts emerging from this work will help to form a model reflecting the β-subunit location in a Nav channel complex.

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

Endocrinology ◽

10.1210/en.2006-1499 ◽

2007 ◽

Vol 148 (8) ◽

pp. 3977-3986 ◽

Cited By ~ 12

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.

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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

Journal of Endocrinology ◽

10.1677/joe-08-0472 ◽

2009 ◽

Vol 202 (1) ◽

pp. 43-54 ◽

Cited By ~ 56

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.

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

Biochemical Journal ◽

10.1042/bcj20161000 ◽

2017 ◽

Vol 474 (5) ◽

pp. 751-769 ◽

Cited By ~ 7

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.

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