scholarly journals Glycans in the intestinal peptide transporter PEPT1 contribute to function and protect from proteolysis

2017 ◽  
Vol 312 (6) ◽  
pp. G580-G591 ◽  
Author(s):  
Tamara Stelzl ◽  
Kerstin E. Geillinger-Kästle ◽  
Jürgen Stolz ◽  
Hannelore Daniel
Keyword(s):  
Protein Function ◽  
Proteolytic Cleavage ◽  
Glycosylation Site ◽  
Proteinase K ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Electrophysiological Recordings ◽  
Transport Velocity ◽  
Flux Measurements ◽  
The Individual

Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, and N532). Mutagenesis-induced disruption of the individual N-glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1-mediated inward transport of peptides. Here, we show that for the murine protein the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine, or cysteine or by replacing S52 with alanine equally altered PEPT1 transport kinetics in oocytes. Furthermore, we provide evidence that the uptake of [14C]glycyl-sarcosine in immortalized murine small intestinal (MODE-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild-type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight with two to four monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, whereas a remarkable intrinsic stability against trypsin, even in the absence of N-linked glycans, was detected. NEW & NOTEWORTHY This study highlights the role of N50-linked glycans in modulating the bidirectional transport activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux measurements in Xenopus oocytes have shown that removal of the N50 glycans increases the maximal peptide transport rate in the inward and outward directions. This effect could be largely reversed by replacement of N50 glycans with structurally dissimilar biotin derivatives. In addition, N-glycans were detected to stabilize PEPT1 against proteolytic cleavage.

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family

2011 ◽  
Vol 39 (5) ◽  
pp. 1353-1358 ◽  
Author(s):  
Simon Newstead
Keyword(s):  
High Resolution ◽  
Anticancer Agents ◽  
Targeted Delivery ◽  
Drug Transport ◽  
Structural Information ◽  
Sequence Similarity ◽  
The Body ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Starting Point

One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as ‘natural’ compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug–transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.

Differential Roles of NMDA Receptor Subtypes NR2A and NR2B in Dendritic Branch Development and Requirement of RasGRF1

2010 ◽  
Vol 103 (4) ◽  
pp. 1758-1770 ◽  
Author(s):  
Fernando J. Sepulveda ◽  
Fernando J. Bustos ◽  
Eveling Inostroza ◽  
Felipe A. Zúñiga ◽  
Rachael L. Neve ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Receptor Subtypes ◽  
Dendritic Branch ◽  
Spinal Cord Neurons ◽  
Whole Cell Patch Clamp ◽  
Electrophysiological Recordings ◽  
Branch Formation ◽  
The Individual

N-methyl-d-aspartate receptors (NMDARs) are known to regulate axonal refinement and dendritic branching. However, because NMDARs are abundantly present as tri-heteromers (e.g., NR1/NR2A/NR2B) during development, the precise role of the individual subunits NR2A and NR2B in these processes has not been elucidated. Ventral spinal cord neurons (VSCNs) provide a unique opportunity to address this problem, because the expression of both NR2A and NR2B (but not NR1) is downregulated in culture. Exogenous NR2A or NR2B were introduced into these naturally NR2-null neurons at 4 DIV, and electrophysiological recordings at 11 DIV confirmed that synaptic NR1NR2A receptors and NR1NR2B receptors were formed, respectively. Analysis of the dendritic architecture showed that introduction of NR2B, but not NR2A, dramatically increased the number of secondary and tertiary dendritic branches of VSCNs. Whole cell patch-clamp recordings further indicated that the newly formed branches in NR2B-expressing neurons were able to establish functional synapses because the frequency of miniature AMPA-receptor synaptic currents was increased. Using previously described mutants, we also found that disruption of the interaction between NR2B and RasGRF1 dramatically impaired dendritic branch formation in VSCNs. The differential role of the NR2A and NR2B subunits and the requirement for RasGRF1 in regulating branch formation was corroborated in hippocampal cultures. We conclude that the association between NR1NR2B-receptors and RasGRF1 is needed for dendritic branch formation in VSCNs and hippocampal neurons in vitro. The dominated NR2A expression and the limited interactions of this subunit with the signaling protein RasGRF1 may contribute to the restricted dendritic arbor development in the adult CNS.

H+-peptide cotransport in the human bile duct epithelium cell line SK-ChA-1

2002 ◽  
Vol 283 (1) ◽  
pp. G222-G229 ◽  
Author(s):  
Ilka Knütter ◽  
Isabel Rubio-Aliaga ◽  
Michael Boll ◽  
Gerd Hause ◽  
Hannelore Daniel ◽  
...  
Keyword(s):  
Bile Duct ◽  
Apical Membrane ◽  
Aminolevulinic Acid ◽  
Intestinal Type ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Clinical Aspects ◽  
Biliary Duct ◽  
Human Bile ◽  
In Cells

This study describes for the first time the presence of H+-peptide cotransport in cells of the bile duct. Uptake of [glycine-1-14C]glycylsarcosine ([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma SK-ChA-1 cells was stimulated sevenfold by an inwardly directed H+ gradient. Transport was mediated by a low-affinity system with a transport constant ( K t) value of 1.1 mM. Several dipeptides, cefadroxil, and δ-aminolevulinic acid, but not glycine and glutathione, were strong inhibitors of Gly-Sar uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable membranes. The transepithelial electrical resistance was 856 ± 29 Ω × cm2. The transepithelial flux of [14C]Gly-Sar in apical-to-basolateral direction exceeded the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from the apical side. RT-PCR analysis using primer pairs specific for the intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2) revealed that the transport system expressed in SK-ChA-1 and also in cells of the native rabbit bile duct is PEPT1. Immunohistochemistry localized PEPT1 to the apical membrane of cholangiocytes of mouse extrahepatic biliary duct. We conclude that the cells of the mammalian extrahepatic biliary tract epithelium express the intestinal-type H+-peptide cotransporter in their apical membrane. SK-ChA-1 cells represent a convenient model to study the physiological and clinical aspects of peptide transport in cholangiocytes.

scholarly journals Bioinformatic characterization of the Transmembrane protein95 gene (TMEM95) in Murrah buffalo (Bubalus bubalis)

2021 ◽  
Vol 91 (1) ◽  
pp. 11-18
Author(s):  
Sampathirao Shireesha ◽  
◽  
Krovvidi Sudhakar ◽  
Regula Vinoo ◽  
Chappidi V. Seshaiah ◽  
...  
Keyword(s):  
Protein Function ◽  
Male Fertility ◽  
Leucine Zipper ◽  
Phosphorylation Site ◽  
Glycosylation Site ◽  
Sequence Information ◽  
Missense Mutations ◽  
Murrah Buffalo ◽  
Synonymous Mutations ◽  
Male Subfertility

Idiopathic male subfertility is often a neglected phenotype with respect to male fertility in bovines. The gene TMEM95 plays a crucial role in idiopathic male subfertility in cattle. Using the DNA sequence information from cattle TMEM95 gene, we characterized the gene in Murrah buffalo. A total of 2.6 kb of a fragment orthologous to cattle was sequenced from Murrah buffalo and Gir cattle. A 2 bp deletion is present in Murrah buffalo, causing missense mutations in three isoforms that are present in cattle. The functional effects of various non-synonymous mutations were predicted using the SNAP2 program, and showed that the non-synonymous SNPs could affect the protein function. Functional motif annotation revealed the presence of a Casein kinase II phosphorylation site that plays an important role in sperm morphology, Leucine zipper pattern, N-myristoylation site, protein kinase C phosphorylation site, CHRD domain profile, N-glycosylation site and HIT zinc finger motifs in cattle. The HIT ZF motif is absent in all of the functional isoforms in buffalo. The results together suggest that the subfertility gene TMEM95 in cattle and buffalo must have evolved with different functions but plays a role in male fertility as in other mammals.

scholarly journals Genomic Profiles of Bone Marrow (BM) Clonal Plasma Cells (PCs) Vs Circulating Tumor Cells (CTCs) and Extramedullary (EM) Plasmacytomas in Multiple Myeloma (MM)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4442-4442
Author(s):  
Gabriel Bretones ◽  
Bruno Paiva ◽  
Rafael Valdes-Mas ◽  
Diego Alignani ◽  
Miguel Garcia ◽  
...  
Keyword(s):  
Protein Function ◽  
Research Funding ◽  
Somatic Mutations ◽  
Stop Codon ◽  
Clonal Evolution ◽  
Dna Amplification ◽  
Individual Patient Level ◽  
Patient Level ◽  
Recurrent Mutations ◽  
The Individual

Abstract EM disease in MM increases from newly-diagnosed into relapsed patients, and typically predicts for inferior survival. In fact, no treatment seems to be effective in cases with plasma cell (PC) leukemia, which represents the most aggressive form of EM MM. Unfortunately, the mechanisms of extramedullary spread in MM are not well understood, and there is almost no data about the genetic landscape of both forms of EMD (plasmacytomas and peripheral blood CTCs). Here, we performed whole exome sequencing (WES) to analyze the genomic profiles of highly purified FACS sorted BM and EM clonal PCs from 6 patients with relapsed MM. In 5/6 cases we had all three tissue specific clones, whereas in the remaining case no CTCs were detectable. Depending on the amount of genomic DNA from each clone, whole-genome amplification was performed and in such cases, triplicates of 10 ng of DNA were amplified up to 10 µg using the Illustra GenomiPhi HY DNA amplification kit (GE healthcare). Enrichment of exonic sequences was performed for each library using the SureSelectXT Human All Exon V5+UTRs capture kit (Agilent). To identify somatic mutations we used the mutation caller named Varscan. Variants potentially affecting protein function, including non-synonymous variants, frameshifts in the coding sequence, stop codon-introducing (nonsense) or variants potentially affecting splicing, were analyzed. Only those mutations present in 2/3 libraries analyzed per sample were considered positive. Overall, a median of 89 (67 - 474) somatic mutations were detected. EM plasmacytomas showed the highest mutation load, followed by CTCs and BM clonal PCs (85 vs 77 vs 75, respectively; P=.07), supporting a higher genomic instability/evolution of EM clones. Strikingly, all 6 cases showed lack of concordance in the mutation profiles of the three tissue related clones; even 2x2 comparisons between BM clonal PCs vs CTCs or plasmacytomas, or between the two forms of EM MM (ie. plasmacytomas vs CTCs) showed lack of 100% concordance in every single patient. Despite high inter-tissue heterogeneity, it should be noted that whenever present, recurrent and potentially actionable mutations in genes such as KRAS (n=3), KDM4A (n=2), KMT2A (n=2), ARID5B (n=2), TRIO (n=2), BRAF (n=1) or CCND1 (n=1) were detected in all three clones, with the exception of one patient in which CTCs lacked a KRAS mutation. Only one and less frequent actionable mutation in the EPHB2 gene (eg. Herceptin) was exclusively noted in the EM plasmacytoma from one patient. In order to understand the cellular origin of the three tissue related subclones, we then investigated the degree of similarity between each pair of clones at the individual patient level. The pair of clones showing the highest similarity in their mutation profiles were EM plasmacytomas with CTCs (60%, 30% - 94%), followed by BM clonal PCs with CTCs (58%, 35% - 92%), and by BM clonal PCs with EM plasmacytomas (57%, 37% - 91%). Thus, these data suggests that while CTC clones may represent a cellular bridge in between BM and EM plasmacytomas, there is continuous genomic evolution once the different clones have seeded in their respective tissue niches. Accordingly, EM plasmacytomas showed the highest number of specific mutations, followed by BM clonal PCs and CTCs (medians of 9, 4 and 3, respective). We then looked for recurrent mutations specifically present in EM clones, and found that CTCs showed exclusive and recurrent mutations in the CEP152, FSIP2, SYNE1 and TENM1 and ZNF585A genes, whereas EM plasmacytomas showed exclusive and recurrent mutations in the PITRM1 (Metalloprotease 1) gene. Furthermore, ATP7B, MTOR, TBC1D21 and ZNF717 mutations were present in both CTCs and plasmacytomas. In summary, we compared for the first time the genomic profiles of BM vs EM CTCs and plasmacytoma clones, and showed at the individual patient level, the existence of high mutation heterogeneity in between all three tissue related clones. However, actionable mutations in genes such as KRAS or BRAF were typically shared by BM and EM clones. According to the level of similarity between each clone, CTCs would be the most plausible precursor of EM plasmacytomas. Nevertheless, based on the number of specific mutations present in EM clones, it is likely that EM spreading followed by continuous clonal evolution starts much earlier prior to its clinical detection, which emphasizes the need for sensitive techniques to capture the early stages of EM spreading. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

The nature and development of cancer: Cancer mutations and their implications

2020 ◽  
pp. 445-455
Author(s):  
James D. Brenton ◽  
Tim Eisen
Keyword(s):  
Protein Function ◽  
Chromosomal Rearrangements ◽  
Regulation Of Gene Expression ◽  
Tumour Suppressor Genes ◽  
Cancer Type ◽  
Loss Of Function ◽  
Physiological Control ◽  
Cellular Processes ◽  
Cancer Mutations ◽  
The Individual

Cancer is a genetic disease in which progressive accumulation of mutations in the genome of somatic cells induces abnormal biological capabilities. Cancer-inducing mutations may originate from single base substitutions or large chromosomal rearrangements; but ultimately they disrupt normal cellular processes by altering protein function or disturbing the regulation of gene expression. Loss-of-function mutations in tumour suppressor genes inactivate physiological control of cell processes, whereas gain-of-function mutations directly affect physiological networks and, for example, induce pathological activation of signalling pathways. For many common cancers, we are now close to defining unique sets of somatic alterations which confer a specific signature of the cancer type and are also highly specific to the individual patient.

scholarly journals The Protein Interactome of Streptococcus pneumoniae and Bacterial Meta-interactomes Improve Function Predictions

mSystems ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
S. Wuchty ◽  
S. V. Rajagopala ◽  
S. M. Blazie ◽  
J. R. Parrish ◽  
S. Khuri ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Experimental Approach ◽  
Bacterial Species ◽  
Human Pathogen ◽  
Content Type ◽  
Predict Protein Function ◽  
Cellular Pathways ◽  
The Individual ◽  
New Protein

ABSTRACT Identification of protein interactions in bacterial species can help define the individual roles that proteins play in cellular pathways and pathogenesis. Very few protein interactions have been identified for the important human pathogen S. pneumoniae. We used an experimental approach to identify over 2,000 new protein interactions for S. pneumoniae, the most extensive interactome data for this bacterium to date. To predict protein function, we used our interactome data augmented with interactions from other closely related bacteria. The combination of the experimental data and meta-interactome data significantly improved the prediction results, allowing us to assign possible functions to a large number of poorly characterized proteins. The functions of roughly a third of all proteins in Streptococcus pneumoniae, a significant human-pathogenic bacterium, are unknown. Using a yeast two-hybrid approach, we have determined more than 2,000 novel protein interactions in this organism. We augmented this network with meta-interactome data that we defined as the pool of all interactions between evolutionarily conserved proteins in other bacteria. We found that such interactions significantly improved our ability to predict a protein’s function, allowing us to provide functional predictions for 299 S. pneumoniae proteins with previously unknown functions. IMPORTANCE Identification of protein interactions in bacterial species can help define the individual roles that proteins play in cellular pathways and pathogenesis. Very few protein interactions have been identified for the important human pathogen S. pneumoniae. We used an experimental approach to identify over 2,000 new protein interactions for S. pneumoniae, the most extensive interactome data for this bacterium to date. To predict protein function, we used our interactome data augmented with interactions from other closely related bacteria. The combination of the experimental data and meta-interactome data significantly improved the prediction results, allowing us to assign possible functions to a large number of poorly characterized proteins.

scholarly journals A novel homozygous missense mutation p.P388S in TULP1 causes protein instability and retinitis pigmentosa

2020 ◽  
Author(s):  
DaNae R. Woodard ◽  
Chao Xing ◽  
Pratyusha Ganne ◽  
Hanquan Liang ◽  
Avinash Mahindrakar ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Er Stress ◽  
Subcellular Distribution ◽  
Protein Function ◽  
Cultured Cells ◽  
Retinal Pigment ◽  
Photoreceptor Cells ◽  
Causative Mutation ◽  
The Individual ◽  
Systemic Examination

ABSTRACTPurposeRetinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein an individual in his 20’s presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function.MethodsWe performed a complete ophthalmologic examination of the proband followed by exome sequencing. The identified mutation was then modeled in cultured cells, evaluating its expression, solubility (both by western blot), subcellular distribution (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (qPCR and western blotting).ResultsThe proband presented with generalized and parafoveal retinal pigment epithelial atrophy with bone spicule pigmentation in the mid periphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of outer retinal layers with loss of the ellipsoid zone, whereas systemic examination of this individual was normal. The proband’s parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated by steady-state and cycloheximide-chase experiments.ConclusionsThese results add to the list of known TULP1 mutations associated with RP and suggest a unique pathogenic mechanism in TULP1-induced RP, which may be shared amongst select mutations in TULP1.

scholarly journals Benzotropolone moiety in theaflavins is responsible for inhibiting peptide-transport and activating AMP-activated protein kinase in Caco-2 cells

2013 ◽  
Vol 3 (5) ◽  
pp. 111 ◽  
Author(s):  
Ha-Young Park ◽  
Yuri Kunitake ◽  
Toshiro Matsui
Keyword(s):  
Protein Kinase ◽  
Ussing Chamber ◽  
Fixed Time ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Apparent Permeability ◽  
Structural Requirement ◽  
Compound C ◽  
Apparent Permeability Coefficient ◽  
Amp Activated Protein Kinase

Objective: In the small intestine, peptide transporter 1 (PEPT1) plays a role in the transport of di- and tri-peptides. Recently, we found that theaflavins (TFs), dimeric catechins, inhibited the transport of di-peptides across Caco-2 monolayers by suppressing the expression of PEPT1 through AMP-activated protein kinase (AMPK) activation. In this study, we investigated the structural requirement of theaflavins for the effect, and the mechanism(s) underling theaflavin-induced AMPK activation.Methods: Theaflavin-3’-O-gallate (TF3’G) was used for this study, since it possessed the most potent inhibition power for peptide-transport among theaflavins. Absorption ability was measured with Caco-2 cell monolayers treated with or without 20 μM sample (TF3’G or its related compounds) in an Ussing Chamber. The amount of Gly-Sar (a model of PEPT1-transporing peptide) transport at fixed time-points to 60 min was determined by fluorescent naphthalene-2,3-dicarboxaldehyde-derivatized assay (Ex/Em: 405 nm/460 nm). The apparent permeability coefficient (Papp) was used to evaluate the permeability. Expression of PEPT1 protein in Caco-2 cells treated with or without 20 μM TF3’G in the presence or absence of inhibitor (10 μM compound C as AMPK inhibitor or 25 μM STO-609 as CaMKK inhibitor) was evaluated by Western blot.Results: The Papp value of Gly-Sar significantly (P < 0.05) decreased in 20 μM purprogallin-treated Caco-2 cells as well as in TF3’G-treated cells, together with the reduction of PEPT1 expression, while their monomeric catechins did not show any Papp reduction. In TF3'G-treated Caco-2 cells, the recovery of the reduced PEPT1 expression was found by 10 μM compound C, but not STO-609.Conclusion: The study demonstrated that the benzotropolone moiety in theaflavins was a crucial structural requirement for exerting the inhibition of intestinal peptide-transport, and the suppression of PEPT1 expression by theaflavins would be caused by activating LKB1/AMPK pathway, but not CaMKK/AMPK pathway.Keywords: Theaflavin-3’-Ο-gallate, Peptide transport, PEPT1, Benzotropolone, AMP-activated protein kinase, Calmodulin-dependent protein kinase kinase  

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