Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers

Chinery, Rebecca; Bates, Paul A.; De, Amitabha; Freemont, Paul S.

doi:10.1016/0014-5793(94)01297-e

Cited by 57 publications

(39 citation statements)

References 20 publications

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“…is in agreement with typical values for protein solutions [45]. It has been reported by others [46] and observed by ourselves (Chadwick, M., May, F. and Westley, B., unpublished results) that native pNR-2/pS2 forms disulphide-linked dimers through Cys58. This mechanism for dimerisation is not available in the Ser58 variant examined here.…”

Section: Resultssupporting

confidence: 77%

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NMR‐Based Structural Studies of the pNR‐2/pS2 Single Domain Trefoil Peptide

Polshakov

Frenkiel

Westley

et al. 1995

European Journal of Biochemistry

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NMR spectroscopy measurements have been used to obtain structural information about the pNR-2/ pS2 single-domain trefoil peptide. NMR data from 2D (two dimensional) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), NOE spectroscopy (NOESY), rotating frame NOE spectroscopy (ROESY) and 2D "C-'H heteronuclear single-quantum coherence (HSQC) and ' C ' H HSQC-TOCSY spectra have been analysed to provide essentially complete 'H and "C sequence-specific assignments for the pNR-2/pS2 protein. From a consideration of the NOE intensities, 3J(NH-aCH) coupling constants, 'H and 13C chemical shifts of backbone atoms and amide-proton exchange rates, the pNR-2/pS2 was found to contain two short antiparallel P-strands (32-35 and 43-46), a short helix (2.5-30) and a type I p-turn (11 -1.5). These elements of secondary structure are very similar to those found in the two trefoil domains of pSP for which detailed structural information is already available. Similar 'H chemical shifts were noted for several conserved residues in pNR-2/pS2 and pSP and a characteristic Phe residue with a slowly flipping ring was found in the pNR-2/pS2 variant and in both domains of pSP. The tertiary structures of the domains therefore appear to be very similar in the two proteins and it is likely that the pNR-2/pS2 has the same pattern of disulphide bonds (1-5, 2-4, 3-6) as pSP. Correlation time measurements derived from 'H-'H NOE measurements indicate that the Cys58-+Ser form of the pNR-2/pS2 protein used in this study is monomeric in solution at approximately 2 mM.Keywords; trefoil peptide ; pNR-2/pS2 ; secondary structure ; NMR ; monomeric structure. pNR-2/pS2 (plasmid Newcastle regulated-2/plasmid stimulated-2) is a small secreted protein (6.5 kDa) which was originally discovered by virtue of its oestrogen regulation in cultured breast cancer cells [l-41. It was subsequently detected in some breast tumours, where it has been shown to be a marker of oestrogen responsiveness [5 -71, and in other tumours including carcinoma of the pancreas, large intestine, stomach, endometrium and ovary [8]. pNR-2/pS2 is expressed at high levels in normal foveolar epithelium of the gastric antrum and fundus [9-111 and at low levels in normal breast epithelial cells 19, 121. It is also expressed within the upper duct and surface cells of the ulcer-associated cell lineage which is involved in the repair of damaged endodermal tissues [13, 141. pNR-2/pS2 is very similar to a number of other polypeptides referred to as trefoil proteins, a name derived from the pattern of Abbreviations. 2D, two-dimensional ; COSY, two-dimensional correlation spectroscopy; DQF-COSY, double-quantum-filtered correlation spectroscopy ; hITF, human intestinal trefoil factor; hSP, human spasmolytic polypeptide; HSQC, heteronuclear single quantum coherence : MLEV17, M. Levitt-I 7 pulse sequence; NOESY, two-dimensional nuclear Overhauser effect spectroscopy ; pNR-2/pS2, estrogen regulated peptide isolated from breast cancer cell line (plasmid N...

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Section: Resultssupporting

confidence: 77%

“…It is also clear that the location of the two-stranded antiparallel jj-sheet in pNR-2/ pS2 [ (32)(33)(34)(35) and (43)(44)(45)(46)] is identical to similar sheets seen in the domains of pSP. Similarly, the type I P-turn for residues (11 -14) has direct counterparts in pSP (being described as a 3,,,-helix in the crystal structure of pSP [21]).…”

Section: Resultsmentioning

confidence: 79%

NMR‐Based Structural Studies of the pNR‐2/pS2 Single Domain Trefoil Peptide

Polshakov

Frenkiel

Westley

et al. 1995

European Journal of Biochemistry

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“…Another example is intestinal trefoil factor, which contains seven cysteines, one of which is involved in dimer formation (13,14). Dimerization is required for some but not all functions of the trefoil peptides.…”

Section: Discussionmentioning

confidence: 99%

Dimerization of Resistin and Resistin-like Molecules Is Determined by a Single Cysteine

Banerjee¹,

Lazar²

2001

Journal of Biological Chemistry

108

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Resistin is a peptide hormone secreted by adipocytes. Cysteine residues comprise 11 of 94 (12%) amino acids in resistin. The arrangement of these cysteines is unique to resistin and its recently discovered family of tissue-specific secreted proteins, which have been independently termed resistin-like molecules (RELMs) and the FIZZ (found in inflammatory zone) family. Here we show that resistin is a disulfide-linked homodimer that can be converted to a monomer by reducing conditions. The intestine-specific RELM␤ has similar characteristics. Remarkably, however, the adipose-enriched RELM␣ is a monomer under non-reducing conditions. We note that RELM␣ lacks a cysteine residue, closest to the cleaved N terminus, that is present in resistin and RELM␤ in multiple species. Conversion of this cysteine to alanine abolishes dimerization of resistin. Thus, a single disulfide bond is necessary to connect two resistin subunits in a homodimer. The additional 10 cysteines most likely participate in intramolecular disulfide bonds that define the conserved structure of the family members. The monomeric nature of RELM␣ suggests structural and potentially functional divergence between resistin and this close family member.Resistin is a recently described peptide hormone belonging to a novel class of cysteine-rich secreted proteins termed the RELM 1 /FIZZ (found in inflammatory zone) family (1-3). Resistin (also known as FIZZ3 and adipose tissue-specific secretory factor (1, 4)) is adipocyte-specific, down-regulated by antidiabetic drugs, and functionally antagonistic to insulin (2). RELM␣ (also known as FIZZ1) is expressed in white adipose tissue, tongue, and lung, where it appears to be involved in inflammatory processes (1, 3). RELM␤ (also known as FIZZ2) is intestine-specific and of unknown function (1, 3). The family is characterized by a highly conserved, cysteine-rich C terminus in which the spacing of the cysteines is invariant. Although resistin and the RELMs have interesting biological properties, little is known about their structure and signaling mechanisms.Formation of disulfide bonds, both intramolecular and intermolecular, is an important process in protein folding (5). Disulfide bonding is required for proper folding and secretion of proteins (6), and it is important for creation and maintenance of tertiary structure of polypeptide growth factors and hormones (7). Given the high percentage of cysteine residues in the amino acid sequences of resistin and the RELM family, we hypothesized that disulfide bonding might have important structural consequences in these molecules. Here we show that resistin is a disulfide-linked dimer whether expressed recombinantly or endogenously secreted by 3T3-L1 adipocytes. RELM␤ is also a homodimer. By contrast, RELM␣ is secreted as a monomer. This fundamental difference is because of a single cysteine residue that is present in resistin and RELM␤ in multiple species but is not conserved in RELM␣. The biological functions of resistin and the RELMs are likely to be influenced by this st...

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“…Human intestinal trefoil factor (TFF3, gene symbol hITF) is a member of a family of polypeptides characterized by containing at least one copy of the trefoil motif, a 3-leaved structure formed by three conserved disulfides within a 40-amino acid segment [1,2]. Trefoil peptides are expressed mainly in gastrointestinal and other selected epithelial tissues and are packaged along with mucins and secreted [3][4][5].…”

Section: Introductionmentioning

confidence: 99%

“…Trefoil peptides are expressed mainly in gastrointestinal and other selected epithelial tissues and are packaged along with mucins and secreted [3][4][5]. TFF3 was first identified in the rat intestine [6] and is constitutively expressed in the goblet cells of the small and large intestine [1,3,7]. TFF3 was later characterized for its role in reconstitution of an epithelial barrier after mucosal injury in the jejunum [8].…”

Section: Introductionmentioning

confidence: 99%