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Bioanalysis Jul 2022Relative quantification techniques have dominated the field of proteomics. However, biomarker discovery, mathematical model development and studies on... (Review)
Review
Relative quantification techniques have dominated the field of proteomics. However, biomarker discovery, mathematical model development and studies on transporter-mediated drug disposition still need absolute quantification of proteins. The quality of data of trace-level protein quantification is solely dependent on the specific selection of surrogate peptides. Selection of surrogate peptides has a major impact on the accuracy of the method. In this article, the advanced approaches for selection of surrogate peptides, which can provide absolute quantification of the proteins are discussed. In addition, internal standardization, which accounts for variations in the quantitation process to achieve absolute protein quantification is discussed.
Topics: Membrane Transport Proteins; Peptides; Proteomics; Reference Standards
PubMed: 36017716
DOI: 10.4155/bio-2022-0071 -
The EMBO Journal Sep 2022The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial...
The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.
Topics: Apoptosis Inducing Factor; Disulfides; Electron Transport Complex I; HEK293 Cells; Humans; Mitochondrial Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Oxidation-Reduction; Protein Transport
PubMed: 35859387
DOI: 10.15252/embj.2022110784 -
Communications Biology Apr 2024Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other...
Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.
Topics: Rats; Animals; Humans; Streptococcus pneumoniae; Klebsiella pneumoniae; Membrane Transport Proteins; Nasopharynx; Pneumococcal Infections; Peptides
PubMed: 38589539
DOI: 10.1038/s42003-024-06113-9 -
Planta Mar 2022A critical investigation into arsenic uptake and transportation, its phytotoxic effects, and defense strategies including complex signaling cascades and regulatory... (Review)
Review
A critical investigation into arsenic uptake and transportation, its phytotoxic effects, and defense strategies including complex signaling cascades and regulatory networks in plants. The metalloid arsenic (As) is a leading pollutant of soil and water. It easily finds its way into the food chain through plants, more precisely crops, a common diet source for humans resulting in serious health risks. Prolonged As exposure causes detrimental effects in plants and is diaphanously observed through numerous physiological, biochemical, and molecular attributes. Different inorganic and organic As species enter into the plant system via a variety of transporters e.g., phosphate transporters, aquaporins, etc. Therefore, plants tend to accumulate elevated levels of As which leads to severe phytotoxic damages including anomalies in biomolecules like protein, lipid, and DNA. To combat this, plants employ quite a few mitigation strategies such as efficient As efflux from the cell, iron plaque formation, regulation of As transporters, and intracellular chelation with an array of thiol-rich molecules such as phytochelatin, glutathione, and metallothionein followed by vacuolar compartmentalization of As through various vacuolar transporters. Moreover, the antioxidant machinery is also implicated to nullify the perilous outcomes of the metalloid. The stress ascribed by the metalloid also marks the commencement of multiple signaling cascades. This whole complicated system is indeed controlled by several transcription factors and microRNAs. This review aims to understand, in general, the plant-soil-arsenic interaction, effects of As in plants, As uptake mechanisms and its dynamics, and multifarious As detoxification mechanisms in plants. A major portion of this article is also devoted to understanding and deciphering the nexus between As stress-responsive mechanisms and its underlying complex interconnected regulatory networks.
Topics: Arsenic; Biological Transport; Crops, Agricultural; Membrane Transport Proteins; Phytochelatins
PubMed: 35303194
DOI: 10.1007/s00425-022-03869-4 -
Science Signaling Sep 2019Intracellular calcium (Ca) signals are of prime importance for cellular function and behavior and are underpinned by a plethora of Ca channels, pumps, transporters, and...
Intracellular calcium (Ca) signals are of prime importance for cellular function and behavior and are underpinned by a plethora of Ca channels, pumps, transporters, and binding proteins that are regulated in complex ways. A series of biennial meetings, the International Meetings of the European Calcium Society (ECS), focuses on a better understanding of these complex mechanisms in the framework of cellular and organismal (patho)physiology.
Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Endoplasmic Reticulum; Humans; Intracellular Calcium-Sensing Proteins; Lysosomes; Membrane Proteins; Membrane Transport Proteins; Mitochondria; Models, Biological
PubMed: 31530730
DOI: 10.1126/scisignal.aaz0961 -
Food Research International (Ottawa,... Feb 2023Two novel hypoglycemic peptides VY and SFLLR were identified from douchi as the major peptides responsible for the glucose uptake activity. The present work aimed to...
Digestion, absorption, and transport properties of soy-fermented douchi hypoglycemic peptides VY and SFLLR under simulated gastrointestinal digestion and Caco-2 cell monolayers.
Two novel hypoglycemic peptides VY and SFLLR were identified from douchi as the major peptides responsible for the glucose uptake activity. The present work aimed to elucidate their digestion, absorption and transport properties using simulated digestion and Caco-2 cell monolayers transport models. Besides, the effects of digestion and absorption on the structure and activity were also studied. The results showed that VY was resistant to gastrointestinal tract digestion and could cross Caco-2 cell monolayers intactly via both TJs-mediated passive paracellular pathway and PepT1-mediated active route. In comparison, SFLLR was partially degraded into small fragments of SFLL, SFL, and SF by the digestive system, leading to increased glucose uptake activity. Notably, SFLLR, SFLL, and SFL were partly hydrolyzed by aminopeptidase N or dipeptidyl peptidase IV during transport, but they were transported intact. SFL was transported via both paracellular diffusion and PepT1-mediated routes, while SFLLR and SFLL were via paracellular route only.
Topics: Humans; Caco-2 Cells; Peptides; Biological Transport; Digestion; Glucose
PubMed: 36737933
DOI: 10.1016/j.foodres.2022.112340 -
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