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Protein and Peptide Letters 2020Currently, chemotherapy is one of the mainstays of oncologic therapies. But the efficacy of chemotherapy is often limited by drug resistance and severe side effects.... (Review)
Review
Currently, chemotherapy is one of the mainstays of oncologic therapies. But the efficacy of chemotherapy is often limited by drug resistance and severe side effects. Consequently, it is becoming increasingly important to investigate the underlying mechanism and overcome the problem of anticancer chemotherapy resistance. The solute carrier organic anion transporter family member 1B3 (SLCO1B3), a functional transporter normally expressed in the liver, transports a variety of endogenous and exogenous compounds, including hormones and their conjugates as well as some anticancer drugs. The extrahepatic expression of SLCO1B3 has been detected in different cancer cell lines and cancer tissues. Recently, accumulating data indicates that the abnormal expression and function of SLCO1B3 are involved in resistance to anticancer drugs, such as taxanes, camptothecin and its analogs, SN-38, and Androgen Deprivation Therapy (ADT) in breast, prostate, lung, hepatic, and colorectal cancer, respectively. Thus, more investigations have been implemented to identify the potential SLCO1B3-related mechanisms of cancer drug resistance. In this review, we focus on the emerging roles of SLCO1B3 protein in the development of cancer chemotherapy resistance and briefly discuss the mechanisms of resistance. Elucidating the function of SLCO1B3 in chemoresistance may bring out novel therapeutic strategies for cancer treatment.
Topics: Androgen Antagonists; Antineoplastic Agents; Biological Transport; Camptothecin; Drug Resistance, Neoplasm; Humans; Irinotecan; Protein Conformation; Signal Transduction; Solute Carrier Organic Anion Transporter Family Member 1B3; Structure-Activity Relationship; Taxoids
PubMed: 31556849
DOI: 10.2174/0929866526666190926154248 -
Microbiology (Reading, England) Nov 2023The controlled entry and expulsion of small molecules across the bacterial cytoplasmic membrane is essential for efficient cell growth and cellular homeostasis. While... (Review)
Review
The controlled entry and expulsion of small molecules across the bacterial cytoplasmic membrane is essential for efficient cell growth and cellular homeostasis. While much is known about the transcriptional regulation of genes encoding transporters, less is understood about how transporter activity is modulated once the protein is functional in the membrane, a potentially more rapid and dynamic level of control. In this review, we bring together literature from the bacterial transport community exemplifying the extensive and diverse mechanisms that have evolved to rapidly modulate transporter function, predominantly by switching activity off. This includes small molecule feedback, inhibition by interaction with small peptides, regulation through binding larger signal transduction proteins and, finally, the emerging area of controlled proteolysis. Many of these examples have been discovered in the context of metal transport, which has to finely balance active accumulation of elements that are essential for growth but can also quickly become toxic if intracellular homeostasis is not tightly controlled. Consistent with this, these transporters appear to be regulated at multiple levels. Finally, we find common regulatory themes, most often through the fusion of additional regulatory domains to transporters, which suggest the potential for even more widespread regulation of transporter activity in biology.
Topics: Membrane Transport Proteins; Cell Membrane; Bacteria
PubMed: 37948297
DOI: 10.1099/mic.0.001412 -
PloS One 2021P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the...
P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer's associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphate; Alzheimer Disease; Amyloid beta-Peptides; Biocatalysis; Blood-Brain Barrier; Cell Line, Tumor; Disease Progression; Humans; Hydrolysis; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Peptide Fragments; Protein Binding; Protein Conformation, beta-Strand; Protein Domains; Protein Transport; Quinolines; Signal Transduction; Substrate Specificity
PubMed: 33901197
DOI: 10.1371/journal.pone.0250371 -
MBio Apr 2023The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is a leading cause of infections and mortality in immunocompromised patients. This organism can overcome...
The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is a leading cause of infections and mortality in immunocompromised patients. This organism can overcome iron deprivation during infection via the synthesis of two iron-chelating siderophores, pyoverdine and pyochelin, which scavenge iron from host proteins. P. aeruginosa can also uptake xenosiderophores produced by other bacteria or fungi using dedicated transporter systems. The precise substrate specificity of these siderophore transporters remains to be determined. The thiopeptide antibiotic thiostrepton exploits the pyoverdine transporters FpvA and FpvB to cross the outer membrane and reach intracellular targets. Using a series of intricate biochemical experiments, a recent study by Chan and Burrows capitalized on the specificity of thiostrepton to uncover that FpvB transports the xenosiderophores ferrichrome and ferrioxamine B with higher affinity than pyoverdine. This surprising result highlights an alternative uptake pathway for these siderophores and has significant implications for our understanding of iron acquisition in this organism.
Topics: Humans; Siderophores; Anti-Bacterial Agents; Thiostrepton; Bacterial Outer Membrane Proteins; Iron; Membrane Transport Proteins; Pseudomonas aeruginosa
PubMed: 36946760
DOI: 10.1128/mbio.03326-22 -
Cellular and Molecular Life Sciences :... Jul 2022The blood-brain barrier (BBB) provides essential neuroprotection from environmental toxins and xenobiotics, through high expression of drug efflux transporters in... (Review)
Review
The blood-brain barrier (BBB) provides essential neuroprotection from environmental toxins and xenobiotics, through high expression of drug efflux transporters in endothelial cells of the cerebral capillaries. However, xenobiotic exposure, stress, and inflammatory stimuli have the potential to disrupt BBB permeability in fetal and post-natal life. Understanding the role and ability of the BBB in protecting the developing brain, particularly with respect to drug/toxin transport, is key to promoting long-term brain health. Drug transporters, particularly P-gp and BCRP are expressed in early gestation at the developing BBB and have a crucial role in developmental homeostasis and fetal brain protection. We have highlighted several factors that modulate drug transporters at the developing BBB, including synthetic glucocorticoid (sGC), cytokines, maternal infection, and growth factors. Some factors have the potential to increase expression and function of drug transporters and increase brain protection (e.g., sGC, transforming growth factor [TGF]-β). However, others inhibit drug transporters expression and function at the BBB, increasing brain exposure to xenobiotics (e.g., tumor necrosis factor [TNF], interleukin [IL]-6), negatively impacting brain development. This has implications for pregnant women and neonates, who represent a vulnerable population and may be exposed to drugs and environmental toxins, many of which are P-gp and BCRP substrates. Thus, alterations in regulated transport across the developing BBB may induce long-term changes in brain health and compromise pregnancy outcome. Furthermore, a large portion of neonatal adverse drug reactions are attributed to agents that target or access the nervous system, such as stimulants (e.g., caffeine), anesthetics (e.g., midazolam), analgesics (e.g., morphine) and antiretrovirals (e.g., Zidovudine); thus, understanding brain protection is key for the development of strategies to protect the fetal and neonatal brain.
Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Adenosine Triphosphate; Blood-Brain Barrier; Brain; Endothelial Cells; Female; Humans; Infant, Newborn; Interleukin-6; Membrane Transport Proteins; Neoplasm Proteins; Pregnancy; Xenobiotics
PubMed: 35821142
DOI: 10.1007/s00018-022-04432-w -
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 -
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 -
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