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The Journal of Biological Chemistry Feb 2024The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of...
The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.
Topics: Humans; Biological Transport; Glycosylation; Mammals; Membrane Proteins; Nucleotide Transport Proteins; RNA
PubMed: 38237680
DOI: 10.1016/j.jbc.2024.105654 -
SLAS Discovery : Advancing Life... Jul 2021Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. oocytes provide a powerful heterologous...
Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities.Recently, Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in oocytes by acquiring confocal images of the quenching of injected fluorophore calcein.This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48-72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals.The method was tested with different metal transporters (SLC11), DMT1, Nramp1, and NrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.
Topics: Animals; Biological Transport; Cation Transport Proteins; Dictyostelium; Electrophysiological Phenomena; Female; Fluoresceins; Fluorescent Dyes; Membrane Potentials; Membrane Transport Proteins; Microscopy, Fluorescence; Oocytes; Patch-Clamp Techniques; Xenopus laevis
PubMed: 33825579
DOI: 10.1177/24725552211004123 -
Proceedings of the National Academy of... Jan 2023Secretory proteins are cotranslationally or posttranslationally translocated across lipid membranes via a protein-conducting channel named SecY in prokaryotes and Sec61...
Secretory proteins are cotranslationally or posttranslationally translocated across lipid membranes via a protein-conducting channel named SecY in prokaryotes and Sec61 in eukaryotes. The vast majority of secretory proteins in bacteria are driven through the channel posttranslationally by SecA, a highly conserved ATPase. How a polypeptide chain is moved by SecA through the SecY channel is poorly understood. Here, we report electron cryomicroscopy structures of the active SecA-SecY translocon with a polypeptide substrate. The substrate is captured in different translocation states when clamped by SecA with different nucleotides. Upon binding of an ATP analog, SecA undergoes global conformational changes to push the polypeptide substrate toward the channel in a way similar to how the RecA-like helicases translocate their nucleic acid substrates. The movements of the polypeptide substrates in the SecA-SecY translocon share a similar structural basis to those in the ribosome-SecY complex during cotranslational translocation.
Topics: SecA Proteins; Bacterial Proteins; SEC Translocation Channels; Models, Molecular; Protein Transport; Peptides; Escherichia coli Proteins
PubMed: 36598944
DOI: 10.1073/pnas.2208070120 -
Nutrients Mar 2021P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase...
P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.
Topics: Biological Transport; Caco-2 Cells; Hep G2 Cells; Humans; Hydroxymethylglutaryl CoA Reductases; Intestinal Mucosa; Lipid Metabolism; Lupinus; Peptides; Plant Proteins; Proprotein Convertase 9
PubMed: 33808034
DOI: 10.3390/nu13030863 -
Thyroid : Official Journal of the... Sep 2022Fetal brain development in the first half of pregnancy is dependent on maternal thyroid hormone (TH), highlighting the importance of trans-placental TH transport. It is...
Fetal brain development in the first half of pregnancy is dependent on maternal thyroid hormone (TH), highlighting the importance of trans-placental TH transport. It is yet unclear which transporters are involved in this process. We aimed to identify the major TH transporters in a human placental cell model (BeWo cells). Messenger RNA expression of the known TH transporters (the monocarboxylate transporter [MCT]8, MCT10, the L-type amino acid transporter [LAT]1, LAT2, the organic anion transporting peptide [OATP]1A2 and OATP4A1) in BeWo cells and human placenta were determined by quantitative PCR. To determine the specificity and efficacy of transporter inhibitors, we first determined TH uptake at different inhibitor concentrations in African green monkey kidney fibroblast-like cells (COS1 cells) overexpressing TH transporters. We then tested TH uptake in BeWo cells in the presence or absence of the optimal inhibitor concentrations. All tested TH transporters were expressed in human term placentas, whereas MCT8 was absent in BeWo cells. Both 2-amino-2-norbornanecarboxylic acid (BCH) and L-tryptophan at 1 mM inhibited LATs, whereas at the highest concentration (10 mM) L-tryptophan also inhibited MCT10. Verapamil inhibited OATP1A2 and less efficiently both MCTs, but not LATs. Both rifampicin and naringin reduced OATP1A2 activity. Finally, silychristin inhibited MCT8 at submicromolar concentrations and OATP1A2 partially only at the highest concentration tested (10 μM). In BeWo cells, verapamil reduced triiodothyronine (T3) uptake by 24%, BCH by 31%, and 1 mM L-tryptophan by 41%. The combination of BCH and verapamil additively decreased T3 uptake by 53% and the combination of BCH and 10 mM L-tryptophan by 60%, suggesting a major role for MCT10 and LATs in placental T3 uptake. Indeed, transfection of BeWo cells with MCT10-specific small interfering RNA significantly reduced T3 uptake. Only the combination of BCH and verapamil significantly reduced thyroxine (T4) uptake in BeWo cells, by 32%. Using pharmacological inhibitors, we show that MCT10 and LATs play a major role in T3 uptake in BeWo cells. T4 uptake appears independent of known TH transporters, suggesting the presence of, currently unknown, alternative transporter(s).
Topics: Amino Acid Transport Systems, Neutral; Animals; Chlorocebus aethiops; Female; Humans; Monocarboxylic Acid Transporters; Peptides; Placenta; Pregnancy; RNA, Messenger; RNA, Small Interfering; Rifampin; Symporters; Thyroid Hormones; Thyroxine; Triiodothyronine; Tryptophan; Verapamil
PubMed: 35699060
DOI: 10.1089/thy.2021.0503 -
Proceedings of the National Academy of... Mar 2024The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target...
The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.
Topics: Antigen Presentation; ATP-Binding Cassette Transporters; Cytomegalovirus; Degrons; Endoplasmic Reticulum-Associated Degradation; Membrane Transport Proteins; Peptides; Ubiquitin-Protein Ligases; Herpesviridae
PubMed: 38442151
DOI: 10.1073/pnas.2309841121 -
Pharmacological Research Oct 2023Solute carrier (SLC) transport proteins are fundamental for the translocation of endogenous compounds and drugs across membranes, thus playing a critical role in disease...
Solute carrier (SLC) transport proteins are fundamental for the translocation of endogenous compounds and drugs across membranes, thus playing a critical role in disease susceptibility and drug response. Because only a limited number of transporter substrates are currently known, the function of a large number of SLC transporters is elusive. Here, we describe the proof-of-concept of a novel strategy to identify SLC transporter substrates exemplarily for the proton-coupled peptide transporter (PEPT) 2 (SLC15A2) and multidrug and toxin extrusion (MATE) 1 transporter (SLC47A1), which are important renal transporters of drug reabsorption and excretion, respectively. By combining metabolomic profiling of mice with genetically-disrupted transporters, in silico ligand screening and in vitro transport studies for experimental validation, we identified nucleobases and nucleoside-derived anticancer and antiviral agents (flucytosine, cytarabine, gemcitabine, capecitabine) as novel drug substrates of the MATE1 transporter. Our data confirms the successful applicability of this new approach for the identification of transporter substrates in general, which may prove particularly relevant in drug research.
Topics: Animals; Mice; Ligands; Membrane Transport Proteins; Solute Carrier Proteins; Biological Transport
PubMed: 37775020
DOI: 10.1016/j.phrs.2023.106941 -
Nature Communications Sep 2022Adaptive immunity depends on cell surface presentation of antigenic peptides by major histocompatibility complex class I (MHC I) molecules and on stringent ER quality...
Adaptive immunity depends on cell surface presentation of antigenic peptides by major histocompatibility complex class I (MHC I) molecules and on stringent ER quality control in the secretory pathway. The chaperone tapasin in conjunction with the oxidoreductase ERp57 is crucial for MHC I assembly and for shaping the epitope repertoire for high immunogenicity. However, how the tapasin-ERp57 complex engages MHC I clients has not yet been determined at atomic detail. Here, we present the 2.7-Å crystal structure of a tapasin-ERp57 heterodimer in complex with peptide-receptive MHC I. Our study unveils molecular details of client recognition by the multichaperone complex and highlights elements indispensable for peptide proofreading. The structure of this transient ER quality control complex provides the mechanistic basis for the selector function of tapasin and showcases how the numerous MHC I allomorphs are chaperoned during peptide loading and editing.
Topics: HLA Antigens; Histocompatibility Antigens Class I; Humans; Membrane Transport Proteins; Molecular Chaperones; Peptides; Protein Disulfide-Isomerases
PubMed: 36104323
DOI: 10.1038/s41467-022-32841-9 -
ACS Chemical Biology Mar 2021PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)K, targeting essential bacterial genes, such as P, can inhibit bacterial growth at...
PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)K, targeting essential bacterial genes, such as P, can inhibit bacterial growth at one-digit micromolar concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)K-eg-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)K-eg-PNA by showing that the peptide part of (KFF)K-eg-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture ( < 5 min) and inside the bacteria. Analysis of peptide-PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)k-eg-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)K-eg-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).
Topics: Anti-Bacterial Agents; Cell Culture Techniques; Cell Membrane Permeability; Drug Discovery; Escherichia coli; Escherichia coli Proteins; Kinetics; Membrane Transport Proteins; Microbial Sensitivity Tests; Molecular Structure; Oligonucleotides, Antisense; Peptide Hydrolases; Peptide Nucleic Acids; Peptides; Protein Stability
PubMed: 33684286
DOI: 10.1021/acschembio.0c00822 -
Current Opinion in Immunology Jun 2020Major histocompatibility complex encoded class I (MHC-I) molecules bind a broad spectrum of peptides generated in the cytoplasm and encountered during protein folding... (Review)
Review
Major histocompatibility complex encoded class I (MHC-I) molecules bind a broad spectrum of peptides generated in the cytoplasm and encountered during protein folding and maturation in the endoplasmic reticulum (ER). For cell surface expression and recognition by T cell receptors (TCR) and natural killer (NK) receptors, MHC-I require loading with high affinity peptides. Peptide optimization is catalyzed by either of two pathways. The first is via the peptide-loading complex (PLC) which consists of the transporter associated with antigen processing (TAP)1/TAP2 heterodimer, tapasin (an ER resident chaperone, also known as TAP-binding protein (TAPBP)), ERp57 (an oxidoreductase), and calreticulin (a sugar-binding chaperone) [1]. The second pathway depends on TAP-binding protein, related (TAPBPR), a PLC-independent chaperone, that is similar in amino acid sequence and structure to tapasin [2]. Until recently, mechanistic understanding of how the PLC or TAPBPR influences MHC-I peptide loading has been hampered by a lack of detailed structural information on the modification of the MHC-I peptide-binding site by chaperone interactions. Here we review recent functional, structural, and computational dynamic studies of tapasin and TAPBPR that contribute to a vivid description of the molecular changes in MHC-I molecules that accompany tapasin or TAPBPR interaction.
Topics: Antigen Presentation; Histocompatibility Antigens Class I; Humans; Immunoglobulins; Membrane Transport Proteins; Peptides
PubMed: 32402827
DOI: 10.1016/j.coi.2020.04.004