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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 -
Fluids and Barriers of the CNS Aug 2023The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such...
BACKGROUND
The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency.
METHODS
A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1 and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology.
RESULTS
The most effective histidine mutant, M1-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30-60 min, biotinylated M1-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2-16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect.
CONCLUSION
The pH-sensitive mouse transferrin receptor binding nanobody M1 may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.
Topics: Animals; Mice; Blood-Brain Barrier; Histidine; Hypothermia; Neurotensin; Transcytosis; Hydrogen-Ion Concentration
PubMed: 37620930
DOI: 10.1186/s12987-023-00462-z -
PLoS Biology Aug 2023Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins,...
Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.
Topics: Mice; Animals; Conotoxins; Calcium Channels; Peptides; Sensory Receptor Cells; Snails
PubMed: 37535677
DOI: 10.1371/journal.pbio.3002217 -
Analytical Chemistry Aug 2023De novo design of peptides that bind specifically to functional proteins is beneficial for diagnostics and therapeutics. However, complex permutations and combinations...
De novo design of peptides that bind specifically to functional proteins is beneficial for diagnostics and therapeutics. However, complex permutations and combinations of amino acids pose significant challenges to the rational design of peptides with desirable stability and affinity. Herein, we develop a computational-based evolution method, namely, peptidomimetics-driven recognition elements design (PepDRED), to derive hemoglobin-inspired peptidomimetics. PepDRED mimics the natural evolutionism pipeline to generate stable apovariant (AVs) structures for wild-type counterparts via automated point mutations and validates their efficiency through free binding energy analysis and per residue energy decomposition analysis. For application demonstration, we applied PepDRED to design de novo peptides to bind FhuA, a typical TonB-dependent transporter (TBDT). TBDTs are Gram-negative bacterial outer membrane proteins responsible for iron transport and vital for bacterial resistance. PepDRED generated a pool of AVs and proceeded to reach an optimized peptide, AV440, with a remarkable binding affinity of -21 kcal/mol. AV440 is ∼2.5-fold stronger than the existing FhuA inhibitor Microcin J25. Network energy analysis further unveils that incorporating methionine (M42) in the N-terminal region significantly enhances inter-residue contacts and binding affinity. PepDRED offers a prompt and efficient in silico approach to develop potent peptide candidates for target proteins.
Topics: Escherichia coli Proteins; Peptidomimetics; Escherichia coli; Bacterial Outer Membrane Proteins; Peptides; Protein Binding; Bacterial Proteins
PubMed: 37553082
DOI: 10.1021/acs.analchem.3c01057 -
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 -
Handbook of Experimental Pharmacology 2024Blood-brain barrier (BBB) is a special biological property of the brain neurovascular unit (including brain microvessels and capillaries), which facilitates the...
Blood-brain barrier (BBB) is a special biological property of the brain neurovascular unit (including brain microvessels and capillaries), which facilitates the transport of nutrients into the central nervous system (CNS) and exchanges metabolites but restricts passage of blood-borne neurotoxic substances and drugs/xenobiotics into CNS. BBB plays a crucial role in maintaining the homeostasis and normal physiological functions of CNS but severely impedes the delivery of drugs and biotherapeutics into CNS for treatment of neurological disorders. A variety of technologies have been developed in the past decade for brain drug delivery. Most of these technologies are still in preclinical stage and some are undergoing clinical studies. Only a few have been approved by regulatory agencies for clinical applications. This chapter will overview the strategies and technologies/approaches for brain drug delivery and discuss some of the recent advances in the field.
Topics: Humans; Blood-Brain Barrier; Brain; Biological Transport; Central Nervous System; Drug Delivery Systems; Pharmaceutical Preparations
PubMed: 37528323
DOI: 10.1007/164_2023_689 -
Current Opinion in Lipidology Apr 2024The angiopoietin-like (ANGPTL) proteins ANGPTL3 and ANGPTL4 are critical lipoprotein lipase (LPL) inhibitors. This review discusses the unique ability of the... (Review)
Review
PURPOSE OF REVIEW
The angiopoietin-like (ANGPTL) proteins ANGPTL3 and ANGPTL4 are critical lipoprotein lipase (LPL) inhibitors. This review discusses the unique ability of the insulin-responsive protein ANGPTL8 to regulate triglyceride (TG) metabolism by forming ANGPTL3/8 and ANGPTL4/8 complexes that control tissue-specific LPL activities.
RECENT FINDINGS
After feeding, ANGPTL4/8 acts locally in adipose tissue, has decreased LPL-inhibitory activity compared to ANGPTL4, and binds tissue plasminogen activator (tPA) and plasminogen to generate plasmin, which cleaves ANGPTL4/8 and other LPL inhibitors. This enables LPL to be fully active postprandially to promote efficient fatty acid (FA) uptake and minimize ectopic fat deposition. In contrast, liver-derived ANGPTL3/8 acts in an endocrine manner, has markedly increased LPL-inhibitory activity compared to ANGPTL3, and potently inhibits LPL in oxidative tissues to direct TG toward adipose tissue for storage. Circulating ANGPTL3/8 levels are strongly correlated with serum TG, and the ANGPTL3/8 LPL-inhibitory epitope is blocked by the TG-lowering protein apolipoprotein A5 (ApoA5).
SUMMARY
ANGPTL8 plays a crucial role in TG metabolism by forming ANGPTL3/8 and ANGPTL4/8 complexes that differentially modulate LPL activities in oxidative and adipose tissues respectively. Selective ANGPTL8 inhibition in the context of the ANGPTL3/8 complex has the potential to be a promising strategy for treating dyslipidemia.
Topics: Humans; Angiopoietin-Like Protein 8; Angiopoietin-like Proteins; Tissue Plasminogen Activator; Biological Transport; Lipoprotein Lipase; Triglycerides; Angiopoietin-Like Protein 3; Peptide Hormones
PubMed: 37962908
DOI: 10.1097/MOL.0000000000000910 -
Biochemical and Biophysical Research... Oct 2023Podocytes are sensitive to insulin, which governs the functional and structural integrity of podocytes that are essential for proper function of the glomerular...
Podocytes are sensitive to insulin, which governs the functional and structural integrity of podocytes that are essential for proper function of the glomerular filtration barrier. Lysosomes are acidic organelles that are implicated in regulation of the insulin signaling pathway. Cathepsin D (CTPD) and lysosome-associated membrane protein 1 (LAMP1) are major lysosomal proteins that reflect the functional state of lysosomes. However, the effect of insulin on lysosome activity and role of lysosomes in the regulation of insulin-dependent glucose uptake in podocytes are unknown. Our studies showed that the short-term incubation of podocytes with insulin decreased LAMP1 and CTPD mRNA levels. Insulin and bafilomycin A1 reduced both the amounts of LAMP1 and CTPD proteins and activity of CTPD, which were associated with a decrease in the fluorescence intensity of lysosomes that were labeled with LysoTracker. Bafilomycin A1 inhibited insulin-dependent endocytosis of the insulin receptor and increased the amounts of the insulin receptor and glucose transporter 4 on the cell surface of podocytes. Bafilomycin A1 also inhibited insulin-dependent glucose uptake despite an increase in the amount of glucose transporter 4 in the plasma membrane of podocytes. These results suggest that lysosomes are signaling hubs that may be involved in the coupling of insulin signaling with the regulation of glucose uptake in podocytes. The dysregulation of this mechanism can lead to the dysfunction of podocytes and development of insulin resistance.
Topics: Rats; Animals; Podocytes; Insulin; Receptor, Insulin; Transcription Factors; Lysosomes; Signal Transduction; Glucose; Glucose Transport Proteins, Facilitative
PubMed: 37696068
DOI: 10.1016/j.bbrc.2023.09.012 -
Early Human Development Nov 2023Stress exposure during Neonatal Intensive Care Unit (NICU) stay may have long-lasting effects on neurodevelopmental outcomes in extremely preterm infants. Altered DNA...
BACKGROUND
Stress exposure during Neonatal Intensive Care Unit (NICU) stay may have long-lasting effects on neurodevelopmental outcomes in extremely preterm infants. Altered DNA methylation of stress-related and neurodevelopmentally relevant genes may be an underlying mechanism.
AIMS
This exploratory study aimed to investigate the association between neonatal stress exposure and DNA methylation in these genes at two different time points: early during the NICU stay (7-14 days after birth) and later, at discharge from the NICU.
SUBJECTS
We included 45 extremely preterm infants in this prospective cohort study, gestational age 24-30 weeks.
OUTCOME MEASURES
We collected fecal samples at days 7-14 (n = 44) and discharge (n = 28) and determined DNA methylation status in predefined regions of NR3C1, SLC6A4, HSD11B2, OPRM1, SLC7A5, SLC1A2, IGF2, NNAT, BDNF and GABRA6 using pyrosequencing. Because of low DNA concentrations in some fecal samples, we could do so in 25-50 % of collected samples. We prospectively quantified daily neonatal stress exposure using the Neonatal Infant Stressor Scale (NISS) and explored associations between cumulative NISS scores and average DNA methylation status.
RESULTS
Rates of methylation of most genes were not statistically different between day 7-14 and discharge, except for OPRM1. We found moderately high and mostly negative correlation coefficients upon discharge with the cumulative NISS for the NR3C1, SLC6A4, SLC1A2, IGF2, BDNF and OPRM1 genes, albeit not statistically significant.
CONCLUSIONS
Findings suggest that expression of stress-related and neurodevelopmentally relevant genes may be differently regulated following higher neonatal stress exposure. Larger studies should challenge the findings of this study and ideally test the effects on gene expression.
Topics: Infant; Infant, Newborn; Humans; DNA Methylation; Prospective Studies; Brain-Derived Neurotrophic Factor; Infant, Extremely Premature; Gestational Age; Intensive Care Units, Neonatal; Serotonin Plasma Membrane Transport Proteins
PubMed: 37797474
DOI: 10.1016/j.earlhumdev.2023.105868 -
Scientific Reports May 2024Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins...
Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins of the ABC transporter Ami-AliA/AliB oligopeptide permease. Exposure to these peptides alters pneumococcal phenotypes such as growth. Using a mutant in which a permease domain of the transporter was disrupted, by growth analysis and epifluorescence microscopy, we confirmed peptide uptake via the Ami permease and intracellular location in the pneumococcus. By RNA-sequencing we found that the peptides modulated expression of genes involved in metabolism, as pathways affected were mostly associated with energy or synthesis and transport of amino acids. Both peptides downregulated expression of genes involved in branched-chain amino acid metabolism and the Ami permease; and upregulated fatty acid biosynthesis genes but differed in their regulation of genes involved in purine and pyrimidine biosynthesis. The transcriptomic changes are consistent with growth suppression by peptide treatment. The peptides inhibited growth of pneumococcal isolates of serotypes 3, 8, 9N, 12F and 19A, currently prevalent in Switzerland, and caused no detectable toxic effect to primary human airway epithelial cells. We conclude that pneumococci take up K. pneumoniae peptides from the environment via binding and transport through the Ami permease. This changes gene expression resulting in altered phenotypes, particularly reduced growth.
Topics: Klebsiella pneumoniae; Bacterial Proteins; Streptococcus pneumoniae; Transcriptome; Gene Expression Regulation, Bacterial; Humans; Ligands; Membrane Transport Proteins; Peptides
PubMed: 38816440
DOI: 10.1038/s41598-024-63217-2