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Nature Communications Oct 2022During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The contributions of specific lipids, such as cholesterol, to the...
During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The contributions of specific lipids, such as cholesterol, to the membranes that form the autophagosome, remain to be fully characterized. Here, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and a corresponding increase in autophagy initiation events. We further show that the ER-localized cholesterol transport protein GRAMD1C functions as a negative regulator of starvation-induced autophagy and that both its cholesterol transport VASt domain and membrane binding GRAM domain are required for GRAMD1C-mediated suppression of autophagy initiation. Similar to its yeast orthologue, GRAMD1C associates with mitochondria through its GRAM domain. Cells lacking GRAMD1C or its VASt domain show increased mitochondrial cholesterol levels and mitochondrial oxidative phosphorylation, suggesting that GRAMD1C may facilitate cholesterol transfer at ER-mitochondria contact sites. Finally, we demonstrate that expression of GRAMD family proteins is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.
Topics: Carrier Proteins; Autophagy; Mitochondria; Saccharomyces cerevisiae; Cholesterol; Energy Metabolism; Protein Transport
PubMed: 36270994
DOI: 10.1038/s41467-022-33933-2 -
Molecules (Basel, Switzerland) May 2022Bile acids (BAs) are important steroidal molecules with a rapidly growing span of applications across a variety of fields such as supramolecular chemistry, pharmacy, and... (Review)
Review
Bile acids (BAs) are important steroidal molecules with a rapidly growing span of applications across a variety of fields such as supramolecular chemistry, pharmacy, and biomedicine. This work provides a systematic review on their transport processes within the enterohepatic circulation and related processes. The focus is laid on the description of specific or less-specific BA transport proteins and their localization. Initially, the reader is provided with essential information about BAs' properties, their systemic flow, metabolism, and functions. Later, the transport processes are described in detail and schematically illustrated, moving step by step from the liver via bile ducts to the gallbladder, small intestine, and colon; this description is accompanied by descriptions of major proteins known to be involved in BA transport. Spillage of BAs into systemic circulation and urine excretion are also discussed. Finally, the review also points out some of the less-studied areas of the enterohepatic circulation, which can be crucial for the development of BA-related drugs, prodrugs, and drug carrier systems.
Topics: Bile Acids and Salts; Bile Ducts; Carrier Proteins; Enterohepatic Circulation; Liver
PubMed: 35566302
DOI: 10.3390/molecules27092961 -
MSphere Jun 2023Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii forms a large, acidic -containing vacuole (CCV) and uses a type 4B...
Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii forms a large, acidic -containing vacuole (CCV) and uses a type 4B secretion system to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport and metabolism is critical for successful infection. The mammalian lipid transport protein ORP1L (oxysterol binding protein-like protein 1 Long) localizes to the CCV membrane and mediates CCV-endoplasmic reticulum (ER) membrane contact sites. ORP1L functions in lipid sensing and transport, including cholesterol efflux from late endosomes and lysosomes (LELs), and the ER. Its sister isoform, ORP1S (oxysterol binding protein-like protein 1 Short) also binds cholesterol but has cytoplasmic and nuclear localization. In ORP1-null cells, we found that CCVs were smaller than in wild-type cells, highlighting the importance of ORP1 in CCV development. This effect was consistent between HeLa cells and murine alveolar macrophages (MH-S cells). CCVs in ORP1-null cells had higher cholesterol content than CCVs in wild-type cells at 4 days of infection, suggesting ORP1 functions in cholesterol efflux from the CCV. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrated that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol. Coxiella burnetii is an emerging zoonotic pathogen and bioterrorism threat. No licensed vaccine exists in the United States, and the chronic form of the disease is difficult to treat and potentially lethal. Postinfectious sequelae of C. burnetii infection, including debilitating fatigue, place a significant burden on individuals and communities recovering from an outbreak. C. burnetii must manipulate host cell processes in order to promote infection. Our results establish a link between host cell lipid transport processes and C. burnetii's avoidance of cholesterol toxicity during infection of alveolar macrophages. Elucidating the mechanisms behind bacterial manipulation of the host will yield insight for new strategies to combat this intracellular pathogen.
Topics: Humans; Animals; Mice; Coxiella burnetii; Vacuoles; HeLa Cells; Macrophages; Carrier Proteins; Cholesterol; Lipids; Mammals
PubMed: 37017523
DOI: 10.1128/msphere.00104-23 -
Genetics Research Dec 2019Regulated transport through the secretory pathway is essential for embryonic development and homeostasis. Disruptions in this process impact cell fate, differentiation... (Review)
Review
Regulated transport through the secretory pathway is essential for embryonic development and homeostasis. Disruptions in this process impact cell fate, differentiation and survival, often resulting in abnormalities in morphogenesis and in disease. Several congenital malformations are caused by mutations in genes coding for proteins that regulate cargo protein transport in the secretory pathway. The severity of mutant phenotypes and the unclear aetiology of transport protein-associated pathologies have motivated research on the regulation and mechanisms through which these proteins contribute to morphogenesis. This review focuses on the role of the p24/transmembrane emp24 domain (TMED) family of cargo receptors in development and disease.
Topics: Animals; Carrier Proteins; Humans; Intracellular Membranes; Membrane Proteins; Protein Transport; Transport Vesicles; Vesicular Transport Proteins
PubMed: 31878985
DOI: 10.1017/S0016672319000090 -
Veterinary Parasitology Oct 2021The equine roundworm Parascaris univalens has developed resistance to the three anthelmintic substances most commonly used in horses. The mechanisms responsible for...
The equine roundworm Parascaris univalens has developed resistance to the three anthelmintic substances most commonly used in horses. The mechanisms responsible for resistance are believed to be multi-genic, and transport proteins such as the P-glycoprotein (Pgp) family have been suggested to be involved in resistance in several parasites including P. univlaens. To facilitate further research into the mechanisms behind drug metabolism and resistance development in P. univalens we aimed to develop an in vitro model based on larvae. We developed a fast and easy protocol for hatching P. univalens larvae for in vitro studies, resulting in a hatching rate of 92 %. The expression of transport protein genes pgp-2, pgp-9, pgp-11.1, pgp-16.1 and major facilitator superfamily (MFS) genes PgR006_g137 and PgR015_g078 were studied in hatched larvae exposed to the anthelmintic drugs ivermecin (IVM) 10 M, pyrantel citrate (PYR) 10 M and thiabendazole (TBZ) 10 M for 24 h. In comparison, the expression of these transport protein genes was studied in the anterior end and intestinal tissues of adult worms in vitro exposed to IVM, TBZ and PYR, at the same concentrations as larvae, for 3 h, 10 h and 24 h. Larval exposure to sub-lethal doses of IVM for 24 h did not affect the expression levels of any of the investigated genes, however larvae exposed to PYR and TBZ for 24 h showed significantly increased expression of pgp-9. In vitro drug exposure of adult worms did not result in any significant increases in expression of transport protein genes. Comparisons of constitutive expression between larvae and adult worm tissues showed that pgp-9, pgp-11.1, pgp-16.1 and MFS gene PgR015_g078 were expressed at lower levels in larvae than in adult tissues, while pgp-2 and MFS gene PgR006_g137 had similar expression levels in larvae and adult worms. All investigated transport protein genes were expressed at higher rates in the intestine than in the anterior end of adult worms, except pgp-11.1 where the expression was similar between the two tissues. This high constitutive expression in the intestine suggests that this is an important site for xenobiotic efflux in P. univalens. Despite the fact that the results of this study show differences in expression of transport protein genes between larvae and adult tissues, we believe that the larval assay system described here will be an important tool for further research into the molecular mechanisms behind anthelmintic resistance development and for other in vitro studies.
Topics: Animals; Anthelmintics; Ascaridoidea; Carrier Proteins; Drug Resistance; Gene Expression Regulation; Ivermectin; Larva
PubMed: 34340009
DOI: 10.1016/j.vetpar.2021.109535 -
Autophagy Jul 2023Autophagosomes are crucial components of the cellular recycling machinery that form at endoplasmic reticulum (ER)-associated sites. As the autophagosome membrane is...
Autophagosomes are crucial components of the cellular recycling machinery that form at endoplasmic reticulum (ER)-associated sites. As the autophagosome membrane is largely devoid of transmembrane proteins, autophagosome biogenesis is thought to be largely regulated by lipid transfer and lipid modifications, as well as membrane-associated proteins. While the membrane origin of autophagosomes and their lipid composition are still incompletely understood, previous studies have found the autophagosome membrane to be enriched in unsaturated fatty acids and have little cholesterol, suggesting that cholesterol removal is an integral step during autophagosome biogenesis. In our study, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and identify the ER-localized cholesterol transport protein GRAMD1C as a negative regulator of starvation-induced macroautophagy/autophagy. ATG: autophagy related; ccRCC: clear cell renal cell carcinoma; ER: endoplasmic reticulum; GRAM: glucosyltransferases, RAB-like GTPase activators and myotubularins; GRAMD: GRAM domain containing; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCBD: methyl-cyclodextrin; MTOR: mechanistic target of rapamycin kinase; VASt: VAD1 analog of StAR-related lipid transfer.
Topics: Autophagosomes; Autophagy; Carrier Proteins; Macroautophagy; Membrane Proteins; Cholesterol; Lipids
PubMed: 36469687
DOI: 10.1080/15548627.2022.2155020 -
Plant Signaling & Behavior Aug 2021Plants have evolved elaborate physiological and molecular responses to diverse environmental challenges, including biotic and abiotic stresses. Accumulating evidence...
Plants have evolved elaborate physiological and molecular responses to diverse environmental challenges, including biotic and abiotic stresses. Accumulating evidence suggests that biotic and abiotic stress signaling pathways are intricately intertwined, and factors involved in molecular crosstalk between these pathways have been identified. The R2R3-type MYB96 transcription factor is a key player that mediates plant response to drought and osmotic stresses as well as to microbial pathogens, acting as a molecular signaling integrator. Here, we report that MYB96 is required for the transcriptional regulation of () that lies at the intersection of abscisic acid (ABA) and defense signaling pathways. MYB96 directly binds to the promoter and activates gene expression upon exogenous application of ABA and bacterial flagellin peptide flg22. Our findings indicate that MYB96 integrates biotic and abiotic stress signals and possibly induces sugar uptake to confer tolerance to a wide range of adverse environmental challenges.
Topics: Adaptation, Physiological; Arabidopsis; Arabidopsis Proteins; Biological Transport; Carrier Proteins; Disease Resistance; Gene Expression Regulation, Plant; Monosaccharide Transport Proteins; Signal Transduction; Stress, Physiological; Sugars; Transcription Factors; Transcriptional Activation
PubMed: 33899679
DOI: 10.1080/15592324.2021.1920759 -
The Biochemical Journal Dec 1999The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems... (Review)
Review
The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating beta-alpha-beta topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH(2)- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.
Topics: Amino Acid Sequence; Antiporters; Bacteria; Bacterial Proteins; Biological Transport; Carrier Proteins; Escherichia coli Proteins; Membrane Transport Proteins; Molecular Sequence Data; Periplasmic Binding Proteins; Polyamines; Protein Structure, Secondary; Putrescine; Receptors, Biogenic Amine; Sequence Alignment; Spermidine; Yeasts
PubMed: 10585849
DOI: No ID Found -
The Journals of Gerontology. Series A,... Dec 2012Urine concentrating ability is reduced during normal aging in people and rats. The abundance of many of the key transport proteins that contribute to urine concentrating... (Review)
Review
Urine concentrating ability is reduced during normal aging in people and rats. The abundance of many of the key transport proteins that contribute to urine concentrating ability is reduced in the kidney medulla of aged rats. The reductions in water, sodium, and urea transport protein abundances, and their reduced response to water restriction, contribute to the reduced ability of aged rats to concentrate their urine and conserve water. If similar mechanisms occur in human kidneys, it would provide a molecular explanation for the reduced urine concentrating ability in aging and may provide opportunities for novel therapeutic approaches to improve urine concentrating ability and/or nocturnal polyuria.
Topics: Aging; Animals; Aquaporin 2; Aquaporins; Biological Transport, Active; Carrier Proteins; Humans; Kidney; Kidney Concentrating Ability; Kidney Tubules, Collecting; Loop of Henle; Membrane Transport Proteins; Receptors, Vasopressin; Sodium; Vasopressins; Urea Transporters
PubMed: 22588950
DOI: 10.1093/gerona/gls128 -
Nature Mar 2009The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely...
The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.
Topics: Biological Transport; Carbon Monoxide; Carrier Proteins; Drug Design; Globins; Glutamic Acid; Heme; Histidine; Humans; Kinetics; Ligands; Nerve Tissue Proteins; Neuroglobin; Oxidation-Reduction; Oxygen; Protein Engineering; Protein Structure, Secondary; Rotation; Spectroscopy, Fourier Transform Infrared; Substrate Specificity; Water
PubMed: 19295603
DOI: 10.1038/nature07841