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Biomolecules Jun 2024Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead...
Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead sulfoxide-substituted analogs showing characteristics of atypical inhibition (e.g., JJC8-091). Interestingly, the only distinction between sulfoxide and sulfide substitution is the presence of one additional oxygen atom. To elucidate why such a subtle difference in ligand structure can result in different typical or atypical profiles, we investigated two pairs of analogs. Our quantum mechanical calculations revealed a more negatively charged distribution of the electrostatic potential surface of the sulfoxide substitution. Using molecular dynamics simulations, we demonstrated that sulfoxide-substituted modafinil analogs have a propensity to attract more water into the binding pocket. They also exhibited a tendency to dissociate from Asp79 and form a new interaction with Asp421, consequently promoting an inward-facing conformation of hDAT. In contrast, sulfide-substituted analogs did not display these effects. These findings elucidate the structural basis of the activity cliff observed with modafinil analogs and also enhance our understanding of the functionally relevant conformational spectrum of hDAT.
Topics: Modafinil; Dopamine Plasma Membrane Transport Proteins; Humans; Molecular Dynamics Simulation; Binding Sites; Dopamine Uptake Inhibitors; Structure-Activity Relationship; Protein Binding
PubMed: 38927116
DOI: 10.3390/biom14060713 -
Biomolecules Jun 2024This retrospective begins with Galvani's experiments on frogs at the end of the 18th century and his discovery of 'animal electricity'. It goes on to illustrate the... (Review)
Review
This retrospective begins with Galvani's experiments on frogs at the end of the 18th century and his discovery of 'animal electricity'. It goes on to illustrate the numerous contributions to the field of physical chemistry in the second half of the 19th century (Nernst's equilibrium potential, based on the work of Wilhelm Ostwald, Max Planck's ion electrodiffusion, Einstein's studies of Brownian motion) which led Bernstein to propose his membrane theory in the early 1900s as an explanation of Galvani's findings and cell excitability. These processes were fully elucidated by Hodgkin and Huxley in 1952 who detailed the ionic basis of resting and action potentials, but without addressing the question of where these ions passed. The emerging question of the existence of ion channels, widely debated over the next two decades, was finally accepted and, a decade later, many of them began to be cloned. This led to the possibility of modelling the activity of individual neurons in the brain and then that of simple circuits. Taking advantage of the remarkable advances in computer science in the new millennium, together with a much deeper understanding of brain architecture, more ambitious scientific goals were dreamed of to understand the brain and how it works. The retrospective concludes by reviewing the main efforts in this direction, namely the construction of a digital brain, an in silico copy of the brain that would run on supercomputers and behave just like a real brain.
Topics: Animals; Humans; Brain; Ion Channels; History, 20th Century; History, 19th Century; Electricity; History, 18th Century; Models, Neurological
PubMed: 38927086
DOI: 10.3390/biom14060684 -
Biomolecules Jun 2024Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in...
Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized mice and have been implicated in cardiac dysfunction. Using a mouse model of menopause in which ovarian failure occurs over 120 days, we sought to determine if perimenopause impacted calcium removal mechanisms in the heart and identify the molecular mechanisms. Mice were injected with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure over 120 days, mimicking perimenopause. Hearts were removed at 60 and 120 days after VCD injections, representing the middle and end of perimenopause. SERCA2a function was significantly diminished at the end of perimenopause. Neither SERCA2a nor phospholamban expression changed at either time point, but phospholamban phosphorylation at S16 and T17 was dynamically altered. Intrinsic SERCA inhibitors sarcolipin and myoregulin increased >4-fold at day 60, as did the native activator DWORF. At the end of perimenopause, sarcolipin and myoregulin returned to baseline levels while DWORF was significantly reduced below controls. Sodium-calcium exchanger expression was significantly increased at the end of perimenopause. These results show that the foundation for increased cardiovascular disease mortality develops in the heart during perimenopause and that regulators of calcium handling exhibit significant fluctuations over time. Understanding the temporal development of cardiovascular risk associated with menopause and the underlying mechanisms is critical to developing interventions that mitigate the rise in cardiovascular mortality that arises after menopause.
Topics: Animals; Female; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Mice; Perimenopause; Disease Models, Animal; Vinyl Compounds; Myocardium; Calcium; Calcium-Binding Proteins; Primary Ovarian Insufficiency; Cyclohexenes; Mice, Inbred C57BL; Phosphorylation
PubMed: 38927078
DOI: 10.3390/biom14060675 -
Biomolecules Jun 2024Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that (Nb) infection or its derived products could...
Anti-Inflammatory Responses Produced with -Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model.
Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that (Nb) infection or its derived products could inhibit AS development by inducing an anti-inflammatory response. We performed a metabolic analysis to screen Nb-derived metabolites with anti-inflammation activity and evaluated the AS-prevention effect. We observed that the metabolite uridine had higher expression levels in mice infected with the Nb and ES (excretory-secretory) products and could be selected as a key metabolite. ES and uridine interventions could reduce the pro-inflammatory responses and increase the anti-inflammatory responses in vitro and in vivo. The apolipoprotein E gene knockout (ApoE) mice were fed with a high-fat diet for the AS modeling. Following the in vivo intervention, ES products or uridine significantly reduced serum and liver lipid levels, alleviated the formation of atherosclerosis, and reduced the pro-inflammatory responses in serum or plaques, while the anti-inflammatory responses showed opposite trends. After blocking with 5-HD (5-hydroxydecanoate sodium) in vitro, the mRNA levels of M2 markers were significantly reduced. When blocked with 5-HD in vivo, the degree of atherosclerosis was worsened, the pro-inflammatory responses were increased compared to the uridine group, while the anti-inflammatory responses decreased accordingly. Uridine, a key metabolite from , showed anti-inflammatory and anti-atherosclerotic effects in vitro and in vivo, which depend on the activation of the mitochondrial ATP-sensitive potassium channel.
Topics: Animals; Mice; Atherosclerosis; Uridine; Anti-Inflammatory Agents; Nippostrongylus; Mice, Knockout; Apolipoproteins E; Disease Models, Animal; KATP Channels; Male; Mitochondria
PubMed: 38927075
DOI: 10.3390/biom14060672 -
Biomolecules Jun 2024The retina, a tissue of the central nervous system, is vital for vision as its photoreceptors capture light and transform it into electrical signals, which are further... (Review)
Review
The retina, a tissue of the central nervous system, is vital for vision as its photoreceptors capture light and transform it into electrical signals, which are further processed before they are sent to the brain to be interpreted as images. The retina is unique in that it is continuously exposed to light and has the highest metabolic rate and demand for energy amongst all the tissues in the body. Consequently, the retina is very susceptible to oxidative stress. VDAC, a pore in the outer membrane of mitochondria, shuttles metabolites between mitochondria and the cytosol and normally protects cells from oxidative damage, but when a cell's integrity is greatly compromised it initiates cell death. There are three isoforms of VDAC, and existing evidence indicates that all three are expressed in the retina. However, their precise localization and function in each cell type is unknown. It appears that most retinal cells express substantial amounts of VDAC2 and VDAC3, presumably to protect them from oxidative stress. Photoreceptors express VDAC2, HK2, and PKM2-key proteins in the Warburg pathway that also protect these cells. Consistent with its role in initiating cell death, VDAC is overexpressed in the retinal degenerative diseases retinitis pigmentosa, age related macular degeneration (AMD), and glaucoma. Treatment with antioxidants or inhibiting VDAC oligomerization reduced its expression and improved cell survival. Thus, VDAC may be a promising therapeutic candidate for the treatment of these diseases.
Topics: Humans; Voltage-Dependent Anion Channels; Retina; Animals; Oxidative Stress; Retinal Diseases; Mitochondria; Retinitis Pigmentosa
PubMed: 38927058
DOI: 10.3390/biom14060654 -
Modulation of Adverse Health Effects of Environmental Cadmium Exposure by Zinc and Its Transporters.Biomolecules May 2024Zinc (Zn) is the second most abundant metal in the human body and is essential for the function of 10% of all proteins. As metals cannot be synthesized or degraded, they... (Review)
Review
Zinc (Zn) is the second most abundant metal in the human body and is essential for the function of 10% of all proteins. As metals cannot be synthesized or degraded, they must be assimilated from the diet by specialized transport proteins, which unfortunately also provide an entry route for the toxic metal pollutant cadmium (Cd). The intestinal absorption of Zn depends on the composition of food that is consumed, firstly the amount of Zn itself and then the quantity of other food constituents such as phytate, protein, and calcium (Ca). In cells, Zn is involved in the regulation of intermediary metabolism, gene expression, cell growth, differentiation, apoptosis, and antioxidant defense mechanisms. The cellular influx, efflux, subcellular compartmentalization, and trafficking of Zn are coordinated by transporter proteins, solute-linked carriers 30A and 39A (SLC30A and SLC39A), known as the ZnT and Zrt/Irt-like protein (ZIP). Because of its chemical similarity with Zn and Ca, Cd disrupts the physiological functions of both. The concurrent induction of a Zn efflux transporter ZnT1 (SLC30A1) and metallothionein by Cd disrupts the homeostasis and reduces the bioavailability of Zn. The present review highlights the increased mortality and the severity of various diseases among Cd-exposed persons and the roles of Zn and other transport proteins in the manifestation of Cd cytotoxicity. Special emphasis is given to Zn intake levels that may lower the risk of vision loss and bone fracture associated with Cd exposure. The difficult challenge of determining a permissible intake level of Cd is discussed in relation to the recommended dietary Zn intake levels.
Topics: Humans; Cadmium; Zinc; Environmental Exposure; Animals; Cation Transport Proteins; Metallothionein
PubMed: 38927054
DOI: 10.3390/biom14060650 -
Biomolecules May 2024Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel involved in sensitivity to a plethora of irritating agents and endogenous mediators of...
Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel involved in sensitivity to a plethora of irritating agents and endogenous mediators of oxidative stress. TRPA1 influences neuroinflammation and macrophage and lymphocyte functions, but its role is controversial in immune cells. We reported earlier a detectable, but orders-of-magnitude-lower level of mRNA in monocytes and lymphocytes than in sensory neurons by qRT-PCR analyses of cells from lymphoid organs of mice. Our present goals were to (a) further elucidate the expression of mRNA in immune cells by RNAscope in situ hybridization (ISH) and (b) test the role of TRPA1 in lymphocyte activation. RNAscope ISH confirmed that transcripts were detectable in CD14 and CD4 cells from the peritoneal cavity of mice. A selective TRPA1 agonist JT010 elevated Ca levels in these cells only at high concentrations. However, a concentration-dependent inhibitory effect of JT010 was observed on T-cell receptor (TcR)-induced Ca signals in CD4 T lymphocytes, while JT010 neither modified B cell activation nor ionomycin-stimulated Ca level. Based on our present and past findings, TRPA1 activation negatively modulates T lymphocyte activation, but it does not appear to be a key regulator of TcR-stimulated calcium signaling.
Topics: TRPA1 Cation Channel; Animals; Mice; Lymphocyte Activation; T-Lymphocytes; Ligands; CD4-Positive T-Lymphocytes; Acetanilides; Mice, Inbred C57BL; Calcium; Receptors, Antigen, T-Cell; RNA, Messenger; Male; Calcium Signaling
PubMed: 38927036
DOI: 10.3390/biom14060632 -
Biomolecules May 2024Recent studies increasingly suggest that targeting brown/beige adipose tissues to enhance energy expenditure offers a novel therapeutic approach for treating metabolic...
Recent studies increasingly suggest that targeting brown/beige adipose tissues to enhance energy expenditure offers a novel therapeutic approach for treating metabolic diseases. Brown/beige adipocytes exhibit elevated expression of uncoupling protein 1 (UCP1), which is a thermogenic protein that efficiently converts energy into heat, particularly in response to cold stimulation. Polyphenols possess potential anti-obesity properties, but their pharmacological effects are limited by their bioavailability and distribution within tissue. This study discovered , a polyphenol compound with a favorable distribution within adipose tissues, which transcriptionally activates UCP1, thereby promoting thermogenesis and enhancing mitochondrial respiration in brown adipocytes. Furthermore, in vivo studies demonstrated that prevents high-fat-diet-induced weight gain and improves insulin sensitivity. Our research provides strong mechanistic evidence that UCP1 is a complex mediator of -induced thermogenesis, which is a critical process in obesity mitigation. Brown adipose thermogenesis is triggered by via the AMPK-PGC-1α pathway. As a result, our research highlights a thermogenic controlled polyphenol compound and clarifies its underlying mechanisms, thus offering a potential strategy for the thermogenic targeting of adipose tissue to reduce the incidence of obesity and its related metabolic problems.
Topics: Uncoupling Protein 1; Thermogenesis; Animals; Obesity; Polyphenols; Mice; Diet, High-Fat; Adipose Tissue, Brown; Male; Mice, Inbred C57BL; Humans; Energy Metabolism
PubMed: 38927022
DOI: 10.3390/biom14060618 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
BMC Biology Jun 2024The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans....
BACKGROUND
The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown.
RESULTS
To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments.
CONCLUSIONS
We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.
Topics: Animals; Mice; Mice, Knockout; Synaptic Vesicles; Vacuolar Proton-Translocating ATPases; Synaptic Transmission; Brain; Behavior, Animal; Synapses; Neurons; Vesicular Transport Proteins
PubMed: 38926759
DOI: 10.1186/s12915-024-01940-y