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The Journal of Biological Chemistry Mar 2022Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens...
Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts.
Topics: Cataract; Connexins; Eye Proteins; Gap Junctions; HeLa Cells; Humans; Lens, Crystalline; Lysosomes; Mutation; Serine
PubMed: 35120923
DOI: 10.1016/j.jbc.2022.101673 -
Journal of Dairy Science Aug 2019Selenium is included in selenoprotein sequences, which participate in enzymatic processes necessary to preserve optimal health. Some lactic acid bacteria carry out the...
Selenium is included in selenoprotein sequences, which participate in enzymatic processes necessary to preserve optimal health. Some lactic acid bacteria carry out the biotransformation of inorganic selenium in their metabolism. The complete biochemical mechanism of selenium biotransformation is still unknown; however, it is known that both the selenocysteine synthesis process and its subsequent incorporation into selenoproteins include serine as part of the action of seryl-RNAt synthetase. Therefore, the aim of this work was to determine the effect of serine during the biotransformation of selenium and the subsequence growth of Streptococcus thermophilus in a minimal medium. Two culture media were prepared, one enriched with the minimum inhibitory concentration of selenite (as NaSeO) and the other as a mixture of the minimum inhibitory concentration of selenite and serine. The absorbed selenium concentration was measured by inductively coupled plasma, and the selenocysteine identification was performed by reverse-phase HPLC. In the second culture medium, decreases in both times, the adaptation and the logarithmic phase, were observed. According to the results, it was possible to establish that the presence of serine allowed the biotransformation of selenite into selenocysteine by Strep. thermophilus.
Topics: Animals; Chromatography, High Pressure Liquid; Culture Media; Selenium; Selenocysteine; Selenoproteins; Serine; Streptococcus thermophilus
PubMed: 31155253
DOI: 10.3168/jds.2019-16365 -
Physiology & Behavior Jun 2022Male song in songbirds is a critical and elaborate signal for mate attraction. In many species female listeners respond to male song both behaviorally and...
Male song in songbirds is a critical and elaborate signal for mate attraction. In many species female listeners respond to male song both behaviorally and physiologically (e.g. copulation solicitation displays and production of the immediate early gene ZENK in auditory regions). It is becoming increasingly well known that females in many species also sing. However, in common lab species, such as canaries (Serinus canaria), female song is limited and has been primarily studied in the context of administering of exogenous testosterone (T) to increase song rate and length. In this study we addressed to what extent female canary songs are masculinized by the administration of exogenous T based on the behavioral and physiological responses of avian receivers. Specifically, are T induced female songs sufficient to elicit courtship behaviors and auditory ZENK expression in female listeners? We found that female songs after 3 weeks of exogenous T were significantly longer and more complex than female songs after 12 weeks of exogenous T. Additionally, we found that playback of 3-week T song significantly increased sexual response behaviors and the expression of ZENK in the auditory brain regions of female listeners. Finally, we conclude that extended periods of T do not necessarily maintain the masculinization of female song.
Topics: Animals; Canaries; Female; Genes, Immediate-Early; Male; Songbirds; Testosterone; Vocalization, Animal
PubMed: 35314175
DOI: 10.1016/j.physbeh.2022.113782 -
Neurochemical Research Feb 2016Deficits in N-methyl-D-aspartate receptor (NMDAR) function are increasingly linked to persistent negative symptoms and cognitive deficits in schizophrenia. Accordingly,...
Deficits in N-methyl-D-aspartate receptor (NMDAR) function are increasingly linked to persistent negative symptoms and cognitive deficits in schizophrenia. Accordingly, clinical studies have been targeting the modulatory site of the NMDA receptor, based on the decreased function of NMDA receptor, to see whether increasing NMDA function can potentially help treat the negative and cognitive deficits seen in the disease. Glycine and D-serine are endogenous ligands to the NMDA modulatory site, but since high doses are needed to affect brain levels, related compounds are being developed, for example glycine transport (GlyT) inhibitors to potentially elevate brain glycine or targeting enzymes, such as D-amino acid oxidase (DAAO) to slow the breakdown and increase the brain level of D-serine. In the present study we further evaluated the effect of DAAO inhibitors 5-chloro-benzo[d]isoxazol-3-ol (CBIO) and sodium benzoate (NaB) in a phencyclidine (PCP) rodent mouse model to see if the inhibitors affect PCP-induced locomotor activity, alter brain D-serine level, and thereby potentially enhance D-serine responses. D-Serine dose-dependently reduced the PCP-induced locomotor activity at doses above 1000 mg/kg. Acute CBIO (30 mg/kg) did not affect PCP-induced locomotor activity, but appeared to reduce locomotor activity when given with D-serine (600 mg/kg); a dose that by itself did not have an effect. However, the effect was also present when the vehicle (Trappsol(®)) was tested with D-serine, suggesting that the reduction in locomotor activity was not related to DAAO inhibition, but possibly reflected enhanced bioavailability of D-serine across the blood brain barrier related to the vehicle. With this acute dose of CBIO, D-serine level in brain and plasma were not increased. Another weaker DAAO inhibitor NaB (400 mg/kg), and NaB plus D-serine also significantly reduced PCP-induced locomotor activity, but without affecting plasma or brain D-serine level, arguing against a DAAO-mediated effect. However, NaB reduced plasma L-serine and based on reports that NaB also elevates various plasma metabolites, for example aminoisobutyric acid (AIB), a potential effect via the System A amino acid carrier may be involved in the regulation of synaptic glycine level to modulate NMDAR function needs to be investigated. Acute ascorbic acid (300 mg/kg) also inhibited PCP-induced locomotor activity, which was further attenuated in the presence of D-serine (600 mg/kg). Ascorbic acid may have an action at the dopamine membrane carrier and/or altering redox mechanisms that modulate NMDARs, but this needs to be further investigated. The findings support an effect of D-serine on PCP-induced hyperactivity. They also offer suggestions on an interaction of NaB via an unknown mechanism, other than DAAO inhibition, perhaps through metabolomic changes, and find unexpected synergy between D-serine and ascorbic acid that supports combined NMDA glycine- and redox-site intervention. Although mechanisms of these specific agents need to be determined, overall it supports continued glutamatergic drug development.
Topics: Animals; Corpus Striatum; D-Amino-Acid Oxidase; Enzyme Inhibitors; Locomotion; Male; Mice; Mice, Inbred C57BL; Models, Animal; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Serine
PubMed: 26857796
DOI: 10.1007/s11064-016-1838-8 -
Nature Communications Oct 2020Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults, with an unknown etiology. A hallmark of TLE is the characteristic loss of...
Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults, with an unknown etiology. A hallmark of TLE is the characteristic loss of layer 3 neurons in the medial entorhinal area (MEA) that underlies seizure development. One approach to intervention is preventing loss of these neurons through better understanding of underlying pathophysiological mechanisms. Here, we show that both neurons and glia together give rise to the pathology that is mitigated by the amino acid D-serine whose levels are potentially diminished under epileptic conditions. Focal administration of D-serine to the MEA attenuates neuronal loss in this region thereby preventing epileptogenesis in an animal model of TLE. Additionally, treatment with D-serine reduces astrocyte counts in the MEA, alters their reactive status, and attenuates proliferation and/or infiltration of microglia to the region thereby curtailing the deleterious consequences of neuroinflammation. Given the paucity of compounds that reduce hyperexcitability and neuron loss, have anti-inflammatory properties, and are well tolerated by the brain, D-serine, an endogenous amino acid, offers new hope as a therapeutic agent for refractory TLE.
Topics: Animals; Astrocytes; Behavior, Animal; Brain; Entorhinal Cortex; Epilepsy, Temporal Lobe; Gliosis; Inflammation; Microglia; Neurons; Rats, Sprague-Dawley; Serine
PubMed: 33009404
DOI: 10.1038/s41467-020-18757-2 -
The FEBS Journal May 2023Lactate dehydrogenase A (LDHA) is a glycolytic enzyme catalysing the reversible conversion of pyruvate to lactate. It has been implicated as a substrate for PIM kinases,...
Lactate dehydrogenase A (LDHA) is a glycolytic enzyme catalysing the reversible conversion of pyruvate to lactate. It has been implicated as a substrate for PIM kinases, yet the relevant target sites and functional consequences of phosphorylation have remained unknown. Here, we show that all three PIM family members can phosphorylate LDHA at serine 161. When we investigated the physiological consequences of this phosphorylation in PC3 prostate cancer and MCF7 breast cancer cells, we noticed that it suppressed ubiquitin-mediated degradation of nuclear LDHA and promoted interactions between LDHA and 14-3-3 proteins. By contrast, in CRISPR/Cas9-edited knock-out cells lacking all three PIM family members, ubiquitination of nuclear LDHA was dramatically increased followed by its decreased expression. Our data suggest that PIM kinases support nuclear LDHA expression and activities by promoting phosphorylation-dependent interactions of LDHA with 14-3-3ε, which shields nuclear LDHA from ubiquitin-mediated degradation.
Topics: Humans; Cell Line, Tumor; Lactate Dehydrogenase 5; Serine; Ubiquitination; Ubiquitins; Protein Serine-Threonine Kinases
PubMed: 36239424
DOI: 10.1111/febs.16653 -
Nature Communications May 2017The ability to generate variable movements is essential for learning and adjusting complex behaviours. This variability has been linked to the temporal irregularity of...
The ability to generate variable movements is essential for learning and adjusting complex behaviours. This variability has been linked to the temporal irregularity of neuronal activity in the central nervous system. However, how neuronal irregularity actually translates into behavioural variability is unclear. Here we combine modelling, electrophysiological and behavioural studies to address this issue. We demonstrate that a model circuit comprising topographically organized and strongly recurrent neural networks can autonomously generate irregular motor behaviours. Simultaneous recordings of neurons in singing finches reveal that neural correlations increase across the circuit driving song variability, in agreement with the model predictions. Analysing behavioural data, we find remarkable similarities in the babbling statistics of 5-6-month-old human infants and juveniles from three songbird species and show that our model naturally accounts for these 'universal' statistics.
Topics: Animals; Canaries; Central Nervous System; Female; Finches; Humans; Infant; Learning; Male; Models, Neurological; Motor Skills; Nerve Net; Neural Pathways; Neurons; Sparrows; Verbal Behavior; Vocalization, Animal
PubMed: 28530225
DOI: 10.1038/ncomms15415 -
Journal of Virology Jul 2017The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyperphosphorylation. Genetic mutation...
The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase Iα, which requires a priming phosphorylation at S235. It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypo- and hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.
Topics: Amino Acid Substitution; Cell Line; DNA Mutational Analysis; Hepacivirus; Hepatocytes; Humans; Protein Processing, Post-Translational; Serine; Viral Nonstructural Proteins; Virus Replication
PubMed: 28446668
DOI: 10.1128/JVI.00194-17 -
Cancer Letters Apr 2020Upregulation of serine biosynthesis pathway activity is an increasingly apparent feature of many cancers. Most notably, the first rate-limiting enzyme of the pathway,...
Upregulation of serine biosynthesis pathway activity is an increasingly apparent feature of many cancers. Most notably, the first rate-limiting enzyme of the pathway, phosphoglycerate dehydrogenase (PHGDH), is genomically amplified in some melanomas and breast cancers and can be transcriptionally regulated by various tumor suppressors and oncogenes. Yet emerging evidence suggests that serine-in particular, serine biosynthetic pathway activity-may promote cancer in ways beyond providing the building blocks to support cell proliferation. Here, we summarize how mammalian cells tightly control serine synthesis before discussing alternate ways in which increased serine synthetic flux through PHGDH may benefit cancer cells, such as maintenance of TCA cycle flux through alpha-ketoglutarate (αKG) and modulation of cellular redox balance. We will also provide an overview of the current landscape of therapeutics targeting serine synthesis and offer a perspective on future strategies.
Topics: Animals; Cell Proliferation; Humans; Neoplasms; Oxidation-Reduction; Phosphoglycerate Dehydrogenase; Serine
PubMed: 32032680
DOI: 10.1016/j.canlet.2020.01.036 -
Laboratory Investigation; a Journal of... Oct 2021Stable isotope labeling techniques have been widely applied in the field of metabolomics and proteomics. Before the measured mass spectral data can be used for...
Stable isotope labeling techniques have been widely applied in the field of metabolomics and proteomics. Before the measured mass spectral data can be used for quantitative analysis, it must be accurately corrected for isotope natural abundance and tracer isotopic impurity. Despite the increasing popularity of dual-isotope tracing strategy such as C-N or C-H, there are no accurate tools for correcting isotope natural abundance for such experiments in a resolution-dependent manner. Here, we present AccuCor2 as an R-based tool to perform the correction for C-N or C-H labeling experiments. Our method uses a newly designed algorithm to construct the correction matrices that link labeling pattern and measured mass fractions, then use non-negative least-squares to solve the labeling patterns. Our results show that the dual-isotope experiments often require a mass resolution that is high enough to resolve C and N or C and H. Otherwise, the labeling pattern is not solvable. However, this mass resolution may not be sufficiently high to resolve other non-tracer elements such as oxygen or sulfur from the tracer elements. Therefore, we design AccuCor2 to perform the correction based on the actual mass resolution of the measurements. Using both simulated and experimental data, we show that AccuCor2 performs accurate and resolution-dependent correction for dual-isotope tracer data.
Topics: Algorithms; Isotope Labeling; Isotopes; Mass Spectrometry; Metabolomics; Serine; Software
PubMed: 34193963
DOI: 10.1038/s41374-021-00631-4