Mechanism Of Phosphorylation Research Articles

Phosphorylation of GntR reduces Streptococcus suis oxidative stress resistance and virulence by inhibiting NADH oxidase transcription.

GntR transcription factor of Streptococcus suis serotype 2 (SS2) is a potential substrate protein of STK, but the regulation mechanisms of GntR phosphorylation are still unclear. This study confirmed that STK phosphorylated GntR in vivo, and in vitro phosphorylation experiments showed that STK phosphorylated GntR at Ser-41. The phosphomimetic strain (GntR-S41E) had significantly reduced lethality in mice and reduced bacterial load in the blood, lung, liver, spleen, and brain of infected mice compared to wild-type (WT) SS2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that the promoter of nox was bound by GntR. The phosphomimetic protein GntR-S41E cannot bind to the promoter of nox, and the nox transcription levels were significantly reduced in the GntR-S41E mutant compared to WT SS2. The virulence in mice and the ability to resist oxidative stress of the GntR-S41E strain were restored by complementing transcript levels of nox. NOX is an NADH oxidase that catalyzes the oxidation of NADH to NAD+ with the reduction of oxygen to water. We found that NADH is likely accumulated under oxidative stress in the GntR-S41E strain, and higher NADH levels resulted in increased amplified ROS killing. In total, we report GntR phosphorylation could inhibit the transcription of nox, which impaired the ability of SS2 to resist oxidative stress and virulence.

Unravel the regulatory mechanism of Yrr1p phosphorylation in response to vanillin stress in Saccharomyces cerevisiae

Improving the resistance of Saccharomyces cerevisiae to vanillin, derived from lignin, will benefit the design of robust cell factories for lignocellulosic biorefining. The transcription factor Yrr1p mediates S. cerevisiae resistance to various compounds. In this study, eleven predicted phosphorylation sites were mutated, among which 4 mutants of Yrr1p, Y134A/E and T185A/E could improve vanillin resistance. Both dephosphorylated and phosphorylated mutations at Yrr1p 134 and 185 gathered in the nucleus regardless of the presence or absence of vanillin. However, the phosphorylated mutant Yrr1p inhibited target gene expression, while dephosphorylated mutants promoted expression. Transcriptomic analysis showed that the dephosphorylated Yrr1p T185 mutant, under vanillin stress, upregulated ribosome biogenesis and rRNA processing. These results demonstrate the mechanism by which Yrr1p phosphorylation regulates the expression of target genes. The identification of key phosphorylation sites in Yrr1p offers novel targets for the rational construction of Yrr1p mutants to improve resistance to other compounds.

Phosphorylation-Competent Metastable State of Escherichia coli Toxin HipA.

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

Towards cloud-enabled fully automated end-to-end single molecule imaging.

Single Molecule Super-resolution imaging has revolutionised understanding biological processes on the sub-100 nm scale. Our group developed Fluorophore Localisation Imaging with Photobleaching (FLImP), a super-resolution imaging method that can achieve <4 nm resolution. In concert with molecular dynamics simulations, FLImP was used to reveal the in-situ atomic resolution structures of ligand-bound EGFR oligomers at the plasma membrane of cells (Needham et al., Nat Comms 2016), and those of ligand-unbound dimers and oligomers on cells (Zanetti-Domingues et al., Nat Comms 2018), shedding new light on the mechanisms of EGFR auto-inhibition, phosphorylation and signalling.

Modulation of potassium channels preserves temporal fidelity in auditory network models.

Potassium channels in auditory neurons are rapidly modified by changes in the auditory environment. In response to elevated auditory stimulation, short-term mechanisms such as protein phosphorylation and long-term mechanisms linked to channel synthesis increase the activity of channels that promote high frequency firing. We have now used simple simulations of cochlear hair cells and postsynaptic neurons to demonstrate that the amplitudes of potassium currents in neurons required for optimal encoding of a low-level auditory signal differs substantially from that for louder sounds. Specifically, the cross correlation of the output of a neuron with an auditory stimulus is increased by increasing potassium currents as sound amplitude increases. Although it has been suggested that channel modulation allows neurons to fire at high rates in response to high sound levels, we found that the cross correlation is entirely independent of firing rate and that combinations of currents that maximize firing to a stimulus provide very poor temporal fidelity. We also found that levels of potassium currents that maximize the temporal fidelity of the output of an ensemble of thirty neurons differ from those that maximize temporal fidelity for a single neuron. This suggests that, to maximize preservation of temporal information, modulatory mechanisms must coordinate channel activity in groups of neurons receiving similar synaptic inputs. The simulations provide an explanation for the modulation of the intrinsic excitability of auditory brainstem neurons by changes in environmental sound levels, and the results may extend to information processing in other neural systems.

Review Artikel: Pengaruh Diet Semangka Merah (Citrullus lanatus) terhadap VO2Max

Technological rapidly developments influence in lifestyle changes of people who do sedentary lifestyles. Lack of physical activity can reduce the value of VO2max, so that will impact on lowering physical fitness index. Value of VO2max can be increased through consumption of red watermelon (Citrullus lanatus) that is contained citrulline. Red watermelon is the richest source of citrulline compared to other fruits or vegetable on cucurbitaceae plants. Substance of citrulline that enters the body is converted to nitric oxide (NO) which can increase VO2max through the mechanism of oxidative phosphorylation in mitochondria. This study is to know the effect of red watermelon (Citrullus lanatus) to VO2max. This paper is a qualitative study of literature. Bibliography like articles and literature books were obtained through the Elsevier, PubMed, Cochrane Library, and Google Scholar search engines. The result of analysis shows that the consumption of red watermelon (Citrullus lanatus) affects of VO2max enhancement. Nutrients of citrulline, which is contained in red watermelon, increases NO bioavailability and has an effect on increasing VO2max. In conclusion consumption of red watermelon juice (Citrullus lanatus) affects to value of VO2max.

Phosphorylation Regulation Mechanism of β2 Integrin for the Binding of Filamin Revealed by Markov State Model.

Leukocyte adhesion deficiency-1 (LAD-1) disorder is a severe immunodeficiency syndrome caused by deficiency or mutation of β2 integrin. The phosphorylation on threonine 758 of β2 integrin acts as a molecular switch inhibiting the binding of filamin. However, the switch mechanism of site-specific phosphorylation at the atom level is still poorly understood. To resolve the regulation mechanism, all-atom molecular dynamics simulation and Markov state model were used to study the dynamic regulation pathway of phosphorylation. Wild type system possessed lower binding free energy and fewer number of states than the phosphorylated system. Both systems underwent local disorder-to-order conformation conversion when achieving steady states. To reach steady states, wild type adopted less number of transition paths/shortest path according to the transition path theory than the phosphorylated system. The underlying phosphorylated regulation pathway was from P1 to P0 and then P4 state, and the main driving force should be hydrogen bond and hydrophobic interaction disturbing the secondary structure of phosphorylated states. These studies will shed light on the pathogenesis of LAD-1 disease and lay a foundation for drug development.

Atomistic Insights into the Inhibitory Mechanism of Tyrosine Phosphorylation against the Aggregation of Human Tau Fragment PHF6.

Abnormal aggregation of the microtubule-associated protein tau into intracellular fibrillary inclusions is characterized as the hallmark of tauopathies, including Alzheimer's disease and chronic traumatic encephalopathy. The hexapeptide 306VQIVYK311 (PHF6) of R3 plays an important role in the aggregation of tau. Recent experimental studies reported that phosphorylation of residue tyrosine 310 (Y310) could decrease the propensity of PHF6 to form fibrils and inhibit tau aggregation. However, the underlying inhibitory mechanism is not well understood. In this work, we systematically investigated the influences of phosphorylation on the conformational ensembles and oligomerization dynamics of PHF6 by performing extensive all-atom molecular dynamics (MD) simulations. Our replica exchange MD simulations demonstrate that Y310 phosphorylation could effectively suppress the formation of β-structure and shift PHF6 oligomers toward coil-rich aggregates. The interaction analyses show that hydrogen bonding and hydrophobic interactions among PHF6 peptides, as well as Y310-Y310 π-π stacking and I308-Y310 CH-π interactions, are weakened by phosphorylation. Additional microsecond MD simulations show that Y310 phosphorylation could inhibit the oligomerization of PHF6 by preventing the formation of large β-sheet oligomers and multi-layer β-sheet aggregates. This study provides mechanistic insights into the phosphorylation-inhibited tau aggregation, which may be helpful for the in-depth understanding of the pathogenesis of tauopathies.

The role of domain alterations in F1Fo-ATPase dysfunction associated to neurodegenerative diseases

Mitochondrial dysfunction can lead to degeneration in the central nervous system. F1Fo-ATPase catalyzes most of the intracellular ATP synthesis which plays an essential role in cellular energy supply. The dimerized assembly of F1Fo-ATPase underlies the rotational catalytic function and regulates the mechanisms of oxidative phosphorylation. F1Fo-ATPase dysfunction is involved in a variety of neurological diseases, including epilepsy, Alzheimer's disease, and Parkinson’s disease. Dysregulated expression, activity, and localization of F1Fo-ATPase subunits and the interactions with pathogenic proteins result in decreased F1Fo-ATPase activity and ATP production, and aggravated oxidative stress.

The molecular mechanism of Y473 phosphorylation of UGDH relieves the inhibition effect of UDP-glucose on HuR.

Uridine diphosphate glucose (UDP-Glc) is able to accelerate the decay of snail family transcriptional repressor 1 (SNAI1) mRNA by inhibiting Hu antigen R (HuR, an RNA-binding protein), thereby preventing cancer invasiveness and drug resistance. Nevertheless, the phosphorylation of tyrosine 473 (Y473) of UDP-glucose dehydrogenase (UGDH is capable of converting UDP-Glc to uridine diphosphate glucuronic acid (UDP-GlcUA)) weakens the inhibition of UDP-Glc to HuR, thus initiating the epithelial-mesenchymal transformation of tumor cells and promoting tumor cell migration and metastasis. To address the mechanism, we performed molecular dynamics simulations combined with molecular mechanics generalized Born surface area (MM/GBSA) analysis on wild-type and Y473 phosphorylated UGDH and HuR, UDP-Glc, UDP-GlcUA complexes. We demonstrated that Y473 phosphorylation was able to enhance the binding between UGDH and the HuR/UDP-Glc complex. Compared with HuR, UGDH has a stronger binding ability with UDP-Glc; therefore, UDP-Glc was inclined to bind to UGDH and then was catalyzed to UDP-GlcUA by UGDH, which relieved the inhibition of UDP-Glc to HuR. In addition, the binding ability of HuR for UDP-GlcUA was lower than its affinity for UDP-Glc, significantly reducing the inhibition of HuR. Hence, HuR bound to SNAI1 mRNA more easily to increase the stability of mRNA. Our results revealed the micromolecular mechanism of Y473 phosphorylation of UGDH regulating the interaction between UGDH and HuR as well as relieving the inhibition of UDP-Glc on HuR, which contributed to understanding the role of UGDH and HuR in tumor metastasis and developing small molecule drugs targeting the interaction between UGDH and HuR.

Functional properties of glutelin from Camellia oleifera seed cake: Improvement by alkali-assisted phosphorylation through changes in protein structure

To explore the effect and its mechanism of alkali-assisted phosphorylation on the functional properties of Camellia Oleifera seeds cake glutelin (CSCG), CSCG was treated with different concentration of sodium trimetaphosphate (STMP, 1.0, 2.0, 3.0, 4.0, and 5%, w/v) in different pH environment (3.0, 5.0, 7.0, 9.0, and 11.0). The results showed that alkali assist improved the phosphorylation degree of CSCG, and the optimum pH value is 9.0. FT-IR and XPS confirmed the successful modification of phosphate groups on CSCG through covalent interaction. Alkali-assisted phosphorylation decreased the particle size and increased electronegativity of CSCG, as well as changed in its surface hydrophobicity, crystallinity, and intrinsic fluorescence. All these changes of protein structure triggered by alkali-assisted phosphorylation led to the improvement of water solubility, water/oil absorption capacity, emulsifying ability, foamability, and in vitro digestibility of CSCG. This work could provide a theoretical basis for industrial production of CSCG with excellent functional properties.

CRISPR-dCas9 system for epigenetic editing towards therapeutic applications.

In the past few decades, epigenetics has emerged as an important area of study to enable a better understanding of gene expression and its regulation. Due to epigenetics, stable phenotypic changes have been possible without alterations in DNA sequences. Epigenetic changes may occur due to DNA methylation, acetylation, phosphorylation and other such mechanisms which alter the level of gene expression without making any difference to DNA sequences. In this chapter, CRISPR-dCas9 used to bring about epigenome modifications for regulating gene expression towards a therapeutic approaches for treating human diseases have been discussed.

A single phosphorylation mechanism in early metabolism – the case of phosphoenolpyruvate

A nonenzymatic reaction converting pyruvate to phosphoenolpyruvate (PEP) is shown to proceed through carboxylate phosphorylation, suggesting that only a single phosphorylation mechanism was needed to initiate metabolic networks.

Clinical case report of acute dinitrophenol poisoning with a fatal outcome of Udmurt Republic

Was to conduct the analysis of patient's clinical observation with acute dinitrophenol poisoning, admitted to a toxicological department of CCH №6 of Izhevsk, Udmurt Republic in 2021 yr. In this clinical case report, a 19 years old girl, who took 20 tablets of dinitrophenol, illegally obtained in online-shop, died. The fatal outcome was realized by the uncoupling of oxidative phosphorylation mechanism and cellular respiration, which in its turn led to serious dystrophic changes in all organs and tissues. Disorders of hemodynamics and blood rheological properties dominated in poisoning pathogenesis, led to congestion, stasis in microcirculatory vessels, hyperpermeability with multiple perivascular hemorrhages in organs, occurrence of piecemeal necrosis in kidneys and liver, nephrosis and nonspecific reactive hepatitis. Production ATP from ADP becomes impossible in these conditions, and respiratory energy chain completely disappears as heat, that explains the heat-increasing and fat-burning effects of dinitrophenol.

CSF P-Tau181 and Other Biomarkers in Patients With Neuronal Intranuclear Inclusion Disease.

CSF tau phosphorylated at threonine 181 (p-tau181) is a widely used biomarker for Alzheimer disease (AD) and has recently been regarded to reflect β-amyloid and/or p-tau deposition in the AD brain. Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by intranuclear inclusions in neurons, glial cells, and other somatic cells. Symptoms include dementia, neuropathy, and others. CSF biomarkers were not reported. The objective of this study was to investigate whether CSF biomarkers including p-tau181 are altered in patients with NIID. This was a retrospective observational study. CSF concentrations of p-tau181, total tau, amyloid-beta 1-42 (Aβ42), monoamine metabolites homovanillic acid (HVA), and 5-hydroxyindole acetic acid (5-HIAA) were compared between 12 patients with NIID, 120 patients with Alzheimer clinical syndrome biologically confirmed based on CSF biomarker profiles, and patients clinically diagnosed with other neurocognitive disorders (dementia with Lewy bodies [DLB], 24; frontotemporal dementia [FTD], 13; progressive supranuclear palsy [PSP], 21; and corticobasal syndrome [CBS], 13). Amyloid PET using Pittsburgh compound B (PiB) was performed in 6 patients with NIID. The mean age of patients with NIID, AD, DLB, FTD, PSP, and CBS was 71.3, 74.6, 76.8, 70.2, 75.5, and 71.9 years, respectively. CSF p-tau181 was significantly higher in NIID (72.7 ± 24.8 pg/mL) compared with DLB, PSP, and CBS and was comparable between NIID and AD. CSF p-tau181 was above the cutoff value (50.0 pg/mL) in 11 of 12 patients with NIID (91.7%). Within these patients, only 2 patients showed decreased CSF Aβ42, and these patients showed negative or mild local accumulation in PiB PET, respectively. PiB PET scans were negative in the remaining 4 patients tested. The proportion of patients with increased CSF p-tau181 and normal Aβ42 (A-T+) was significantly higher in NIID (75%) compared with DLB, PSP, and CBS (4.2%, 4.8%, and 7.7%, respectively). CSF HVA and 5-HIAA concentrations were significantly higher in patients with NIID compared with disease controls. CSF p-tau181 was increased in patients with NIID without amyloid accumulation. Although the deposition of p-tau has not been reported in NIID brains, the molecular mechanism of tau phosphorylation or secretion of p-tau may be altered in NIID.

Fluorinated triphenylphosphonium analogs improve cell selectivity and invivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancercells by exploiting their hyperpolarized mitochondrial membrane potential. Todate, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects ofdirect modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining thesame signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR invitro and invivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity invivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.

Customized synthesis of phosphoprotein bearing phosphoserine or its nonhydrolyzable analog

Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs. Here we address this limitation by customizing the cell-free protein synthesis (CFPS) machinery via chassis strain selection and orthogonal translation system (OTS) reconfiguration screening. The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis. Specifically, we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine (O-pSer) or its nonhydrolyzable analog, 2-amino-4-phosphonobutyric acid (C-pSer). This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein. Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins, which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.

Mechanisms of Mitochondrial Apoptosis-Mediated Meat Tenderization Based on Quantitative Phosphoproteomic Analysis.

This study investigates the mechanism of phosphorylation in the regulation of apoptosis-mediated meat tenderization during postmortem aging. The results found that the pork muscle exhibited apoptotic potential at early postmortem (48 h) and showed more tenderness at late postmortem, as evidenced by the increase in mitochondrial membrane permeability (MMP), Ca2+ level, reactive oxygen species (ROS) content, and caspases activity at 0 h to 48 h, and decreases in ATP level at 0 h to 24 h and shear force at 12 h to 120 h (p < 0.05). Phosphoproteomic analysis revealed that phosphorylation regulated apoptosis by modulating ATP and calcium bindings as well as apoptotic signaling, which occurred within early 12 h and mainly occurred at 12 h to 48 h postmortem. Moreover, differential expression of phosphoproteins demonstrated that phosphorylation regulated oxidative stress-induced apoptosis and rigor mortis, thereby promoting the development of meat tenderness. Our results provide insights into the roles of phosphorylation in various physiological processes that affect meat tenderness.

DPP-4 Inhibitor Improved the Cognitive Function in Diabetic Rats.

Diabetes-associated cognitive dysfunction is a major problem of the international community. Dipeptidyl peptidase-4 (DPP-4) inhibitors are drugs with hypoglycemic effect widely used in diabetic treatment in clinic. In this article, we studied the effect of the DPP-4 inhibitor saxagliptin on cognitive function in diabetic rats. Firstly, to observe cognitive dysfunction caused by diabetes, we built the diabetic rat model. Subsequently, the effect of diabetes on cognitive function was evaluated by Morris Water Maze Task. Thirdly, the mechanism of the alleviation effect of DPP-4 inhibitor on cognitive dysfunction was investigated. Specifically, (1) the anti-inflammation mechanism was revealed by quantifying the accumulation of the inflammatory factor interleukin-1β (IL-1β) in the hippocampus area by western blotting and the glial fibrillary acidic protein (GFAP) by immunohistochemistry; (2) the anti-tau phosphorylation mechanism was revealed by quantifying phosphorylated tau by western blotting. This work represents the first study demonstrating the alleviation effect of DPP-4 inhibitor on cognitive dysfunction caused by diabetes. Results obtained here could be useful to seeking for a medical solution with high efficacy to the diabetes-associated cognitive dysfunction.

Nonreceptor Tyrosine Kinase c-Abl-Mediated PHB2 Phosphorylation Aggravates Mitophagy Disorder in Parkinson's Disease Model.

Mitophagy and oxidative stress play important roles in Parkinson's disease (PD). Dysregulated mitophagy exacerbates mitochondrial oxidative damage; however, the regulatory mechanism of mitophagy is unclear. Here, we provide a potential mechanistic link between c-Abl, a nonreceptor tyrosine kinase, and mitophagy in PD progression. We found that c-Abl activation reduces the interaction of prohibitin 2 (PHB2) and microtubule-associated protein 1 light chain 3 (LC3) and decreases the expressive level of antioxidative stress proteins, including nuclear factor erythroid 2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO-1), and the antioxidant enzyme heme oxygenase-1 (HO-1) in 1-methyl-4-phenylpyridinium- (MPP+-) lesioned SH-SY5Y cells. Importantly, we found that MPP+ can increase the expression of phosphorylated proteins at the tyrosine site of PHB2 and the interaction of c-Abl with PHB2. We showed for the first time that PHB2 by changing tyrosine (Y) to aspartate (D) at site 121 resulted in impaired binding of PHB2 and LC3 in vitro. Moreover, silencing of PHB2 can decrease the interaction of PHB2 and LC3 and exacerbate the loss of dopaminergic neurons. We also found that STI 571, a c-Abl family kinase inhibitor, can decrease dopaminergic neuron damage and ameliorate MPTP-induced behavioral deficits in PD mice. Taken together, our findings highlight a novel molecular mechanism for aberrant PHB2 phosphorylation as an inhibitor of c-Abl activity and suggest that c-Abl and PHB2 are potential therapeutic targets for the treatment of individuals with PD. However, these results need to be further validated in PHB2 Y121D mice.