A practical approach to crosslinking

Mattson, Gail K.; Conklin, Edwin G.; Desai, Surbhi; Nielander, G; Savage, M D; Morgensen, S

doi:10.1007/bf00986726

Cited by 269 publications

(287 citation statements)

References 32 publications

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“…One method to determine the functional role of individual amino acids of a transmembrane segment of a membrane protein is cysteine-scanning mutagenesis in combination with reaction with sulfhydryl reagents. Cysteine-scanning mutagenesis takes advantage of the fact that the sulfhydryl moiety is the most reactive functional group in a protein (29). It has been used to determine pore-lining residues in numerous membrane proteins (30 -32), including the lactose permease of E. coli (33), the mouse acetylcholine receptor (34), the human glucose transporter Glut1, and the human anion exchanger isoform 1 (AE1) (35).…”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

Slepkov¹,

Rainey

Li³

et al. 2005

Journal of Biological Chemistry

150

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The Na ؉ /H ؉ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteineless NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe 161 3 Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD 3 OH:CDCl 3 :H 2 O mixture and in Me 2 SO. In CD 3 OH: CDCl 3 :H 2 O, TM IV was structured but not as a canonical ␣-helix. Residues Asp 159 -Leu 162 were a series of ␤-turns; residues Leu 165 -Pro 168 showed an extended structure, and residues Ile 169 -Phe 176 were helical in character. These three structured regions rotated quite freely with respect to the others. In Me 2 SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe 161 is a pore-lining residue.

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Section: Discussionmentioning

confidence: 99%

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

Slepkov¹,

Rainey

Li³

et al. 2005

Journal of Biological Chemistry

150

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“…Lipofectamine 2000 (Invitrogen) was used for transfection. For immunoprecipitation experiments, PDX-1 antibody (generated in the Montminy's laboratory) was covalently cross-linked to protein A beads by using a standard protocol (25). Immunoprecipitation of FLAG-tagged proteins was performed with the FLAG-agarose beads from Promega (Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Pancreatic Duodenal Homeobox-1 Protein by DNA-dependent Protein Kinase

Lebrun

Montminy

Obberghen

2005

Journal of Biological Chemistry

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The transcription factor PDX-1 plays a crucial role during pancreatic development and in the function of insulin-producing beta cells. Disruption of the pdx-1 gene in these cells induces overt diabetes in mice, and this gene is modified in several type 2 diabetic families. It is thus crucial to determine the molecular mechanisms involved in the regulation of PDX-1 expression and/or activation. We identified new proteins associated with PDX-1 by mass spectrometry. These proteins, Ku70 and Ku80, are regulatory subunits of DNA-dependent protein kinase (DNA-PK). We determined that the interaction between PDX-1 and Ku70 or Ku80 is dependent on the homeodomain of PDX-1. Most interestingly, we demonstrated in vitro that the DNA-PK phosphorylates PDX-1 on threonine 11. Although this residue is located in the transactivation domain, this phosphorylation does not seem to be implicated in the transcriptional activation of PDX-1. However, in response to radiation, which activates DNA-PK, a second form of the PDX-1 protein appears rapidly. This form is phosphorylated on threonine and seems to drive PDX-1 degradation by the proteosome. In correlation with this degradation, we observed a subsequent reduction in the activation of the insulin promoter and a decrease in PDX-1-mediated gene expression, i.e. glut2 and glucokinase. Our study demonstrates that radiation, through the activation of DNA-PK, may regulate PDX-1 protein expression.First characterized as an endoderm-specific homeobox protein (1) and later as a transcription factor for somatostatin (2, 3) and insulin (4) genes, PDX-1 (pancreatic duodenal homeobox-1 protein) (also termed IPF-1, STF-1, and IDX-1) plays a critical role in pancreatic development. Although initially produced in both exocrine and endocrine compartments of the developing pancreas, PDX-1 expression shifts to beta cells in the fully formed pancreas (5), where it functions in glucose homeostasis. Targeted disruption of the pdx-1 gene leads to pancreatic agenesis in Pdx-1 Ϫ/Ϫ homozygotes (6). Pdx-1 ϩ/Ϫ mice develop normally and have wild type islet cell mass by morphometric analysis, but they display abnormal serum glucose levels after intraperitoneal glucose injection (7). Indeed, disruption of the pdx-1 gene in insulin-producing beta cells induces overt diabetes in these animals, with reduced expression of insulin and glucose transporter genes (8). Previous studies have demonstrated that the pdx-1 gene is modified in several type 2 diabetic families, leading to the establishment of MODY 4 (maturity onset diabetes of the young 4) (9, 10). Thus, PDX-1 is a key player in the development of the pancreas and in the maintenance of the function of pancreatic beta cells. Although the role of PDX-1 in beta cells is now well established, the mechanisms triggering its activation remain to be clarified. Several studies have suggested that its activation involves post-translational modifications of PDX-1. Taken together, the reported data suggest that serine/threonine phosphorylation could play a major role in th...

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“…In essence EDC is a heterobifunctional cross-linking reagent that reacts with available ¹COOH groups (found on the terminal residues and aspartic acid and glutamic acid) to form an unstable active O-acylisourea intermediate; this intermediate subsequently reacts with free ¹NH 2 groups on amino acids (e.g. lysine) to form an amide bond with the release of a soluble urea derivative as a by-product (32)(33)(34). These by-products are unlikely to have a role in the enhancement as the reaction mixtures were thoroughly dialysed and thus removed prior to administration into dwarf mice.…”

Section: Discussionmentioning

confidence: 99%

Cross-linked growth hormone dimers have enhanced biological activity

Mockridge

Aston²,

Morrell³

et al. 1998

European Journal of Endocrinology

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In this study we have investigated the effect on the bioactivity of pituitary-derived human growth hormone (hGH) and recombinant bovine (b) GH after the addition of various concentrations of the water soluble cross-linking agent 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC; 6.25-100 mg/ml). The biological activity of resulting cross-linked reactions were determined by its ability to promote incorporation of 35 2¹ 4 into costal cartilage of hypopituitary Snell dwarf mice in vivo. Administration of EDC-treated hGH solutions resulted in a significant enhancement of hormone activity in vivo compared with non-cross-linked samples. A similar significant enhancement of bGH activity in vivo was also observed when solutions containing recombinant bGH were cross-linked using EDC. For both hGH and bGH the degree of enhancement appears to be dose-dependent for the concentration of EDC (6.25-100 mg/ml for hGH; 6.25-50 mg/ml for bGH) present in the crosslinking reactions. SDS-PAGE analysis of EDC cross-linked solutions containing hGH and bGH spiked with 125 I-hGH and 125 I-bGH respectively revealed that dimeric GH was the primary cross-linked component. Increasing the concentration of EDC in cross-linking reactions resulted in increased formation of dimeric hGH and bGH. There was a significant correlation between the amount of GH dimer present and the increase in biological activity, suggesting that GH dimers were responsible for the enhanced biological activity. This was confirmed by the enhanced biological activity of a purified preparation of EDC cross-linked dimeric hGH. SOIn conclusion, covalently cross-linked GH dimers reported here have enhanced bioactivity in vivo. However, since naturally occurring GH dimers are known to have reduced biological activity, this work suggests that the structure of EDC cross-linked GH dimers differs fundamentally from that of native dimeric hGH.

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A practical approach to crosslinking

Cited by 269 publications

References 32 publications

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

Regulation of the Pancreatic Duodenal Homeobox-1 Protein by DNA-dependent Protein Kinase

Cross-linked growth hormone dimers have enhanced biological activity

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