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Microbiology (Reading, England) Jan 2020Bacteria offer resistance to a broad range of antibiotics by activating their export channels of ATP-binding cassette transporters. These transporters perform a central... (Review)
Review
Bacteria offer resistance to a broad range of antibiotics by activating their export channels of ATP-binding cassette transporters. These transporters perform a central role in vital processes of self-immunity, antibiotic transport and resistance. The majority of ATP-binding cassette transporters are capable of detecting the presence of antibiotics in an external vicinity and are tightly regulated by two-component systems. The presence of an extracellular loop and an adjacent location of both the transporter and two-component system offers serious assistance to induce a quick and specific response against antibiotics. Both systems have demonstrated their ability of sensing such agents, however, the exact mechanism is not yet fully established. This review highlighted the three key functions of antibiotic resistance, transport and self-immunity of ATP-binding cassette transporters and an adjacent two-component regulatory system.
Topics: ATP-Binding Cassette Transporters; Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Membrane Transport Proteins; Protein Domains; Signal Transduction; Substrate Specificity
PubMed: 31204967
DOI: 10.1099/mic.0.000823 -
Antioxidants & Redox Signaling Jul 2023Ferroptosis, a type of oxidative cell death driven by unlimited lipid peroxidation, is emerging as a target for cancer therapy. Although mitochondrial dysfunction may...
Ferroptosis, a type of oxidative cell death driven by unlimited lipid peroxidation, is emerging as a target for cancer therapy. Although mitochondrial dysfunction may lead to ferroptosis, the underlying molecular mechanisms and metabolic pathways for ferroptosis are incompletely understood. Here, we identify solute carrier family 25 member 22 (SLC25A22), a mitochondrial glutamate transporter, as a driver of ferroptosis resistance in pancreatic ductal adenocarcinoma (PDAC) cells. The downregulation of SLC25A22 expression was associated with increased sensitivity to ferroptosis, but not to apoptosis. Mechanistically, on the one hand, SLC25A22-dependent NAPDH synthesis blocks ferroptotic cell death in PDAC cells through mediating the production of glutathione (GSH), the most important hydrophilic antioxidant. On the other hand, SLC25A22 promotes the expression of stearoyl-CoA desaturase in PDAC cells in an AMP-activated protein kinase-dependent manner, resulting in the production of antiferroptotic monounsaturated fatty acids (MUFAs). The animal study further confirms that SLC25A22 inhibits ferroptosis-mediated tumor suppression. SLC25A22 is a novel metabolic repressor of ferroptosis by producing GSH and MUFAs. These findings establish a previously unrecognized metabolic defense pathway to limit ferroptotic cell death and . 39, 166-185.
Topics: Animals; Ferroptosis; Fatty Acids, Monounsaturated; Apoptosis; Mitochondrial Membrane Transport Proteins; Glutathione; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal
PubMed: 37051693
DOI: 10.1089/ars.2022.0203 -
Molecular Immunology Feb 2022In the late 1980s and early 1990s, the hunt for a transporter molecule ostensibly responsible for the translocation of peptides across the endoplasmic reticulum (ER)... (Review)
Review
In the late 1980s and early 1990s, the hunt for a transporter molecule ostensibly responsible for the translocation of peptides across the endoplasmic reticulum (ER) membrane yielded the successful discovery of transporter associated with antigen processing (TAP) protein. TAP is a heterodimer complex comprised of TAP1 and TAP2, which utilizes ATP to transport cytosolic peptides into the ER across its membrane. In the ER, together with other components it forms the peptide loading complex (PLC), which directs loading of high affinity peptides onto nascent major histocompatibility complex class I (MHC-I) molecules that are then transported to the cell surface for presentation to CD8 T cells. TAP also plays a crucial role in transporting peptides into phagosomes and endosomes during cross-presentation in dendritic cells (DCs). Because of the critical role that TAP plays in both classical MHC-I presentation and cross-presentation, its expression and function are often compromised by numerous types of cancers and viruses to evade recognition by cytotoxic CD8 T cells. Here we review the discovery and function of TAP with a major focus on its role in cross-presentation in DCs. We discuss a recently described emergency route of noncanonical cross-presentation that is mobilized in DCs upon TAP blockade to restore CD8 T cell cross-priming. We also discuss the various strategies employed by cancer cells and viruses to target TAP expression or function to evade immunosurveillance - along with some strategies by which the repertoire of peptides presented by cells which downregulate TAP can be targeted as a therapeutic strategy to mobilize a TAP-independent CD8 T cell response. Lastly, we discuss TAP polymorphisms and the role of TAP in inherited disorders.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP Binding Cassette Transporter, Subfamily B, Member 3; ATP-Binding Cassette Transporters; Antigen Presentation; Cross-Priming; Dendritic Cells; Endoplasmic Reticulum; Humans; Major Histocompatibility Complex; Neoplasms; Protein Transport; T-Lymphocytes, Cytotoxic; Tumor Escape; Viruses
PubMed: 34973498
DOI: 10.1016/j.molimm.2021.12.013 -
Journal of Molecular Biology Oct 2023ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance...
ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.
Topics: Humans; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cryoelectron Microscopy; Hydrolysis; Membrane Transport Proteins; Neoplasm Proteins; Single-Domain Antibodies
PubMed: 37597690
DOI: 10.1016/j.jmb.2023.168234 -
Accounts of Chemical Research Aug 2022There is a continuous demand to improve our understanding of fundamental processes that underlie human health and disease. Therefore, novel strategies that can assist in...
There is a continuous demand to improve our understanding of fundamental processes that underlie human health and disease. Therefore, novel strategies that can assist in these efforts are required. For example, molecular biology and genetic approaches have revolutionized our understanding of protein-mediated processes by facilitating their direct visualization and analyses in living cells. Despite these developments, genetic manipulation has limitations in controlling events that occur after translation such as posttranslational modifications (PTMs), which are imperative regulatory elements. As a result, developing new methods to study PTMs in live cells is a major bottleneck in deciphering their exact roles in the myriad cellular processes.Synthetic and semisynthetic proteins are prepared by combining solid phase peptide synthesis (SPPS) and chemoselective ligation approaches with synthetic or recombinant peptides. Employing protein synthesis allows chemists to incorporate natural and unnatural modifications with virtually unlimited number of functional groups into the protein's sequence, such as PTMs and their mimics. In addition, synthetic proteins can include additional elements such as fluorescent tags, reactive groups, caged units, and enrichment handles. Therefore, harnessing the power of chemical protein synthesis offers great opportunities to study fundamental biological processes.Unfortunately, the low cell permeability of proteins limits their applications mainly to settings, excluding live cell studies. As a result, chemical biologists have been attempting to overcome these limitations by developing protein delivery methods that would enable the study of custom-made proteins in a biological context. Success with these strategies should enable accurate determination of protein localization, degradation, folding, interactions, and involvement in the assembly of membrane-less organelles formed by liquid-liquid phase separation inside cells. Importantly, protein delivery approaches are complementary to genetic manipulations, and combining these approaches should pave the way to new discoveries.In this Account, we describe recent developments in protein delivery methods, with emphasis on those most compatible with synthetic proteins. We highlight experimental approaches and conceptual adaptations required to design and study synthetic proteins in live cells, with or without genetic manipulation. In addition, we highlight the strength and weakness of these approaches for both the delivery and the subsequent studies. We also describe our endeavors to deliver synthetic proteins to cells via cell penetrating peptides (CPPs) and multiplexed bead loading (MBL), as showcases of the applications of these methods to shed light on biological processes. Lastly, we contemplate other future applications of synthetic proteins to answer questions that are currently unapproachable.
Topics: Cell-Penetrating Peptides; Humans; Protein Processing, Post-Translational; Protein Transport; Proteins; Solid-Phase Synthesis Techniques
PubMed: 35833291
DOI: 10.1021/acs.accounts.2c00236 -
Parasites & Vectors Oct 2021The roundworm Ascaris suum is one of the parasites with the greatest economic impact on pig farming. In this context, lower weight gain is hypothesized to be due to...
BACKGROUND
The roundworm Ascaris suum is one of the parasites with the greatest economic impact on pig farming. In this context, lower weight gain is hypothesized to be due to decreased nutrient absorption. This study aims at characterizing the effects of A. suum infection on intestinal nutrient transport processes and potential molecular mechanisms.
METHODS
Three groups of six piglets each were infected orally (10,000 embryonated A. suum eggs) in a single dose ("single infection"). Another three groups were infected orally (1000 embryonated eggs) for 10 consecutive days ("trickle infection"). Animals were necropsied 21, 35 and 49 days post-infection (dpi). Three groups served as respective controls. The Ussing chamber technique was applied for the functional characterization of small intestinal tissues [short-circuit currents (I) as induced by glucose, alanine and peptides; H-glucose net flux rates; tissue conductance (G)]. Transcription and expression levels of relevant cytokines and nutrient transporters were evaluated (qPCR/western blot).
RESULTS
Peptide- and alanine-induced changes in I were significantly decreased in the jejunum and ileum of the trickle-infected group at 49 dpi and in the ileum of the single-infected group at 49 dpi. No significant differences regarding glucose transport were observed between the Ascaris-infected groups and the control group in Ussing chamber experiments. Transcription levels of the glucose and peptide transporters as well as of selected transcription factors (transcription of signal transducer and activator of transcription 6 [STAT6] and hypoxia-inducible factor 1-alpha [Hif-1α]) were significantly increased in response to both infection types after some periods. The transcription of interleukins 4 and 13 varied between decrease and increase regarding the respective time points, as did the protein expression of glucose transporters. The expression of the peptide transporter PepT1 was significantly decreased in the ileal single-infected group at 35 dpi. Hif-1α was significantly increased in the ileal tissue from the single-infected group at 21 dpi and in the trickle-infected group at 35 dpi. The expression levels of Na/K-ATPase and ASCT1 remained unaffected.
CONCLUSIONS
In contrast to the current hypothesis, these results indicate that the nutrient deprivation induced by A. suum cannot be explained by transcriptional or expression changes alone and requires further studies.
Topics: Alanine; Animals; Ascariasis; Ascaris suum; Biological Transport; Cytokines; Glucose; Intestines; Nutrients; Peptides; Swine; Swine Diseases
PubMed: 34649607
DOI: 10.1186/s13071-021-05029-1 -
Scientific Reports Apr 2023We have previously shown that high expression of prolactin-induced protein (PIP) correlates with the response of breast cancer (BC) patients to standard adjuvant...
We have previously shown that high expression of prolactin-induced protein (PIP) correlates with the response of breast cancer (BC) patients to standard adjuvant chemotherapy (doxorubicin and cyclophosphamide), which suggests that the absence of this glycoprotein is associated with resistance of tumor cells to chemotherapy. Therefore, in the present study, we analyzed the impact of PIP expression on resistance of BC cells to anti-cancer drugs and its biological role in BC progression. Expression of PIP and apoptotic genes in BC cell lines was analyzed using real-time PCR and Western blotting. PIP was detected in BC tissue specimens using immunohistochemistry. The tumorigenicity of cancer cells was analyzed by the in vivo tumor growth assay. Apoptotic cells were detected based on caspase-3 activation, Annexin V binding and TUNEL assay. The interaction of PIP with BC cells was analyzed using flow cytometry. Using two cellular models of BC (i.e. T47D cells with the knockdown of the PIP gene and MDA-MB-231 cells overexpressing PIP), we found that high expression of PIP resulted in (1) increased sensitivity of BC cells to apoptosis induced by doxorubicin (DOX), 4-hydroperoxycyclophosphamide (4-HC), and paclitaxel (PAX), and (2) improved efficacy of anti-cancer therapy with DOX in the xenograft mice model. Accordingly, a clinical study revealed that BC patients with higher PIP expression were characterized by longer 5-year overall survival and disease-free survival. Subsequent studies showed that PIP up-regulated the expression of the following pro-apoptotic genes: CRADD, DAPK1, FASLG, CD40 and BNIP2. This pro-apoptotic activity is mediated by secreted PIP and most probably involves the specific surface receptor. This study demonstrates that a high expression level of PIP sensitizes BC cells to anti-cancer drugs. Increased sensitivity to chemotherapy is the result of pro-apoptotic activity of PIP, which is evidenced by up-regulation of specific pro-apoptotic genes. As high expression of PIP significantly correlated with a better response of patients to anti-cancer drugs, this glycoprotein can be a marker for the prognostic evaluation of adjuvant chemotherapy.
Topics: Animals; Female; Humans; Mice; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Glycoproteins; Membrane Transport Proteins; Prolactin; Paclitaxel
PubMed: 37085653
DOI: 10.1038/s41598-023-33707-w -
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 -
Journal of Hepatology Oct 2022Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and...
BACKGROUND & AIMS
Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular metabolism, most of the SLC25 family members remain uncharacterized. We undertook a comprehensive tissue distribution analysis of all Slc25 family members across metabolic organs and identified SLC25A47 as a liver-specific mitochondrial carrier.
METHODS
We used a murine loss-of-function model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. We performed extensive metabolic phenotyping and molecular characterization of newly generated Slc25a47 and Slc25a47-Fgf21 mice.
RESULTS
Slc25a47 mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration. This mitochondrial phenotype was associated with an activation of the mitochondrial stress response (MSR) in the liver, and the development of fibrosis, which was exacerbated upon feeding a high-fat high-sucrose diet. The MSR induced the secretion of several mitokines, amongst which FGF21 played a preponderant role on systemic physiology. To dissect the FGF21-dependent and -independent physiological changes induced in Slc25a47 mice, we generated a double Slc25a47-Fgf21 mouse model and demonstrated that several aspects of the hypermetabolic state were driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility observed in Slc25a47 mice could not be rescued with the genetic removal of Fgf21.
CONCLUSION
Collectively, our data place the Slc25a47 locus at the center of mitochondrial homeostasis, which upon dysfunction triggers robust liver-specific and systemic adaptive stress responses. The prominent role of the Slc25a47 locus in hepatic fibrosis identifies this carrier, or its transported metabolite, as a potential target for therapeutic intervention.
LAY SUMMARY
Herein, we report the importance of a locus containing a liver-specific gene coding for a mitochondrial transport protein called SLC25A47. Mitochondria are the powerhouses of cells. They are crucial for metabolism and energy generation. We show that mice with genetic disruption of the Slc25a47 locus cannot maintain mitochondrial homeostasis (balance), leading to wide-ranging problems in the liver that have far-reaching physiological consequences.
Topics: Animals; Carrier Proteins; Fibroblast Growth Factors; Liver; Liver Cirrhosis; Mice; Mice, Knockout; Mitochondria; Mitochondrial Membrane Transport Proteins; Sucrose
PubMed: 35714811
DOI: 10.1016/j.jhep.2022.05.040