Mutagenesis and Cysteine Scanning of Transmembrane Domain 10 of the Human Dipeptide Transporter

Xu, Liting; Haworth, Ian S.; Kulkarni, Ashutosh A.; Bolger, Michael B.; Davies, Daryl L.

doi:10.1007/s11095-009-9952-9

Cited by 23 publications

(24 citation statements)

References 35 publications

(53 reference statements)

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“…In contrast, several studies have emphasized the importance of a negative charge in position 419 [11], [16], [17]. Lys117 (Lys127 in PepT So ) is flanked by the highly conserved Glu20 and two semi conserved residues Tyr25 and 58 (29 and 68 in PepT So , respectively).…”

Section: Resultsmentioning

confidence: 99%

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

et al. 2012

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YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.

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Section: Resultsmentioning

confidence: 99%

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

et al. 2012

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“…In fact, the rapid modernization and the consequent decline in routine load of physical activity induced an increased fracture incidence of the hip in Hong Kong [69] and Beijing [70] . Moreover urban settings have higher incidence rates than rural ones (i.e., Oslo, Norway ) [56,71,72] .…”

Section: Geographical Factorsmentioning

confidence: 99%

Major osteoporotic fragility fractures: Risk factor updates and societal impact

Pisani¹,

Renna²,

Conversano³

et al. 2016

WJO

390

243

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Osteoporosis is a silent disease without any evidence of disease until a fracture occurs. Approximately 200 million people in the world are affected by osteoporosis and 8.9 million fractures occur each year worldwide. Fractures of the hip are a major public health burden, by means of both social cost and health condition of the elderly because these fractures are one of the main causes of morbidity, impairment, decreased quality of life and mortality in women and men. The aim of this review is to analyze the most important factors related to the enormous impact of osteoporotic fractures on population. Among the most common risk factors, low body mass index; history of fragility fracture, environmental risk, early menopause, smoking, lack of vitamin D, endocrine disorders (for example insulin-dependent diabetes mellitus), use of glucocorticoids, excessive alcohol intake, immobility and others represented the main clinical risk factors associated with augmented risk of fragility fracture. The increasing trend of osteoporosis is accompanied by an underutilization of the available preventive strategies and only a small number of patients at high fracture risk are recognized and successively referred for therapy. This report provides analytic evidences to assess the best practices in osteoporosis management and indications for the adoption of a correct healthcare strategy to significantly reduce the osteoporosis burden. Early diagnosis is the key to resize the impact of osteoporosis on healthcare system. In this context, attention must be focused on the identification of high fracture risk among osteoporotic patients. It is necessary to increase national awareness campaigns across countries in order to reduce the osteoporotic fractures incidence.

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“…The site-directed mutagenesis protocol and transient transfection of cDNAs into HEK293 cells were performed as previously described (Xu et al 2009). The pcDNA3-hPEPT1 plasmid was used as a template for all mutagenesis reactions.…”

Section: Methodsmentioning

confidence: 99%

“…hPEPT1 is a proton-coupled symporter with 12 α -helical transmembrane domains (TMDs) (Covitz et al 1998), of which TMDs 3, 5, 7 and 10 have been proposed to form part of the substrate translocation pathway (Links et al 2007; Kulkarni et al 2003a, b; Xu et al 2009). As might be expected, charged residues in the TMDs play crucial roles in substrate transport.…”

Section: Introductionmentioning

confidence: 99%

“…As might be expected, charged residues in the TMDs play crucial roles in substrate transport. E595 in TMD 10 is essential for function and R282 in TMD 7 also has a key role (Xu et al 2009). In rabbit PEPT1, R282 links transport of the substrate and proton (Meredith 2004), and findings in the human and rabbit proteins suggest that a salt bridge forms between R282 and D341 in TMD 8 (Kulkarni et al 2007; Meredith 2009).…”

Section: Introductionmentioning

confidence: 99%

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Functional Role of the Intracellular Loop Linking Transmembrane Domains 6 and 7 of the Human Dipeptide Transporter hPEPT1

Haworth

et al. 2010

J Membrane Biol

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The human intestinal di-/tripeptide transporter (hPEPT1) is a 12-transmembrane protein that facilitates transport of peptides from the intestine into the circulation. hPEPT1 is also an important target in oral delivery of drugs, but mechanistic and structural data for the protein are limited. In particular, there is little information on the function of the loops of the transporter. In this study, we show that mutation of several charged residues in the largest intracellular loop of hPEPT1 (loop 6-7, amino acids 224-278) significantly reduces hPEPT1 function. This loop has an asymmetric distribution of charged residues, with only positive charges in the N-terminal half and all five negative charges in the loop located in a small part of the C-terminal half. Point mutagenesis to alanine of three positive residues in the N-terminal half of loop 6-7 and four negative residues in the C-terminal half of the loop significantly reduced glycylsarcosine uptake. E267 was particularly sensitive to mutation, and kinetic analyses of E267A-and E267K-hPEPT1 gave V max values 10-fold lower than that for the wild-type protein. Secondary structure prediction suggested that loop 6-7 includes two amphipathic α-helices, with net positive and negative charges, respectively. We interpret the mutagenesis data in terms of interactions of the charged residues in loop 6-7 that may influence conformational changes of hPEPT1 during and after substrate transport.

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Mutagenesis and Cysteine Scanning of Transmembrane Domain 10 of the Human Dipeptide Transporter

Cited by 23 publications

References 35 publications

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

Major osteoporotic fragility fractures: Risk factor updates and societal impact

Functional Role of the Intracellular Loop Linking Transmembrane Domains 6 and 7 of the Human Dipeptide Transporter hPEPT1

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