Phosphorylation Of Serine Residues Research Articles

Phosphorylation of Ser711 residue in the hypervariable region of zoonotic genotype 3 hepatitis E virus is important for virus replication.

Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both in vitro and in vivo. We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.

Tyrosine hydroxylase inhibits HCC progression by downregulating TGFβ/Smad signaling

The alteration of metabolic processes has been found to have significant impacts on the development of hepatocellular carcinoma (HCC). Nevertheless, the effects of dysfunction of tyrosine metabolism on the development of HCC remains to be discovered. This research demonstrated that tyrosine hydroxylase (TH), which responsible for the initial and limiting step in the bio-generation of the neuro-transmitters dopamine and adrenaline, et al. was shown to be reduced in HCC. Increased expression of TH was found facilitates the survival of HCC patients. In addition, decreased TH indicated larger tumor size, much more numbers of tumor, higher level of AFP, and the presence of cirrhosis. TH effectively impairs the growth and metastasis of HCC cells, a process dependent on the phosphorylation of serine residues (S19/S40). TH directly binds to Smad2 and hinders the cascade activation of TGFβ/Smad signaling with the treatment of TGFβ1. In summary, our study uncovered the non-metabolic functions of TH in the development of HCC and proposes that TH might be a promising biomarker for diagnosis as well as an innovative target for metastatic HCC.

Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein)

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP’s influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands.Key points• YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity.• YAP5SA regulates genes involved in proliferation, survival, and mTOR activation.• YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

Protein pyrophosphorylation by inositol phosphates: a novel post-translational modification in plants?

Inositol pyrophosphates (PP-InsPs) are energy-rich molecules harboring one or more diphosphate moieties. PP-InsPs are found in all eukaryotes evaluated and their functional versatility is reflected in the various cellular events in which they take part. These include, among others, insulin signaling and intracellular trafficking in mammals, as well as innate immunity and hormone and phosphate signaling in plants. The molecular mechanisms by which PP-InsPs exert such functions are proposed to rely on the allosteric regulation via direct binding to proteins, by competing with other ligands, or by protein pyrophosphorylation. The latter is the focus of this review, where we outline a historical perspective surrounding the first findings, almost 20 years ago, that certain proteins can be phosphorylated by PP-InsPs in vitro. Strikingly, in vitro phosphorylation occurs by an apparent enzyme-independent but Mg2+-dependent transfer of the β-phosphoryl group of an inositol pyrophosphate to an already phosphorylated serine residue at Glu/Asp-rich protein regions. Ribosome biogenesis, vesicle trafficking and transcription are among the cellular events suggested to be modulated by protein pyrophosphorylation in yeast and mammals. Here we discuss the latest efforts in identifying targets of protein pyrophosphorylation, pointing out the methodological challenges that have hindered the full understanding of this unique post-translational modification, and focusing on the latest advances in mass spectrometry that finally provided convincing evidence that PP-InsP-mediated pyrophosphorylation also occurs in vivo. We also speculate about the relevance of this post-translational modification in plants in a discussion centered around the protein kinase CK2, whose activity is critical for pyrophosphorylation of animal and yeast proteins. This enzyme is widely present in plant species and several of its functions overlap with those of PP-InsPs. Until now, there is virtually no data on pyrophosphorylation of plant proteins, which is an exciting field that remains to be explored.

Phosphorylation Codes in IRS-1 and IRS-2 Are Associated with the Activation/Inhibition of Insulin Canonical Signaling Pathways.

Insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) are signaling adaptor proteins that participate in canonical pathways, where insulin cascade activation occurs, as well as in non-canonical pathways, in which phosphorylation of substrates is carried out by a diverse array of receptors including integrins, cytokines, steroid hormones, and others. IRS proteins are subject to a spectrum of post-translational modifications essential for their activation, encompassing phosphorylation events in distinct tyrosine, serine, and threonine residues. Tyrosine residue phosphorylation is intricately linked to the activation of the insulin receptor cascade and its interaction with SH2 domains within a spectrum of proteins, including PI3K. Conversely, serine residue phosphorylation assumes a different function, serving to attenuate the effects of insulin. In this review, we have identified over 50 serine residues within IRS-1 that have been reported to undergo phosphorylation orchestrated by a spectrum of kinases, thereby engendering the activation or inhibition of different signaling pathways. Furthermore, we delineate the phosphorylation of over 10 distinct tyrosine residues at IRS-1 or IRS-2 in response to insulin, a process essential for signal transduction and the subsequent activation of PI3K.

Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.

A phosphorylation-deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana, rescuing mutant defects from global translation and gene expression to photosynthesis and growth.

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from Arabidopsis thaliana, which indicate that plants expressing only a phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in both rps6a and rps6b genes when expressed at approximately twice the wild-type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl-terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho-enabled version of eS6z, the phospho-deficient seedlings retained a mild pointed-leaf phenotype, root growth was reduced, and certain cell cycle-related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects. There was little or no evidence for gain-of-function defects. As previously published, the phospho-deficient eS6z also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho-deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

Improving insulin resistance by sulforaphane via activating the Bacteroides and Lactobacillus SCFAs-GPR-GLP1 signal axis.

Background: Insulin resistance (IR) is closely associated with non-alcoholic fatty liver disease (NAFLD), and the gut microbiome contributes to the development of NAFLD. Sulforaphane (SFN) is a phytochemical in cruciferous vegetables that could improve lipid metabolism disorder. However, whether SFN can alleviate IR in NAFLD by regulating the intestinal flora remains unclear. Methods: SFN was administered to high fat diet (HFD)-fed Wistar rats for 10 weeks. Gut microbiota was analysed by 16S rRNA sequencing and the short chain fatty acids (SCFAs) by gas chromatography. The expression of tight junction protein and the numbers of Lactobacillus, Bacteroides and Bifidobacterium were determined by qPCR. The expression of G-protein-coupled receptor 41/43 (GPR41/43) was determined by western blot. A randomized controlled trial (RCT) was conducted in NAFLD patients with broccoli seed tablets (rich in SFN, 42 mg d-1) as intervention for 12 weeks. Thirty-six volunteers with abnormal glucose before the broccoli seed tablet treatment were selected in the intervention group to analyze their blood glucose, insulin, homeostasis model assessment-insulin resistance index (HOMA-IRI), homeostasis model assessment-insulin sensitivity index (HOMA-ISI) and glucagon-like peptide (GLP-1). Results: SFN reduced blood glucose and HOMA-IRI while increasing insulin sensitivity in HFD rats. SFN reduced glycogen synthase kinase 3 (GSK-3), phosphoenolpyruvate carboxykinase (PEPCK) activity, and phosphorylation of serine residues of IRS-2 induced by HFD. SFN reshaped the gut microbiota composition of HFD-induced rats and, especially, increased the content of Bacteroidaceae, Lactobacillaceae and Bifidobacteriaceae, which are related to the improvement from SFN of the blood glucose and HOMA-IRI. The increased numbers of Bacteroides and Lactobacillus were the targets of SFN to enhance the expression of tight junction proteins ZO-1 and occludin, thereby lowering lipopolysaccharide content to reduce inflammation, ultimately alleviating IR. Bacteroides and Lactobacillus produced SCFAs, which activated GPR41/43 to secrete GLP1. Moreover, it was also confirmed in RCT that SFN intervention increased the level of GLP1 in NAFLD patients, which was positively correlated with the reduction of blood glucose and HOMA-IR. Conclusions: SFN alleviated IR in NAFLD via the Bacteroides and Lactobacillus SCFAs-GPR41/43-GLP1 axis and protected the intestinal mucosal barrier to decrease inflammation.

RNA Polymerase II evolution and adaptations: Insights from Plasmodium and other parasitic protists

The C-terminal domain (CTD) of RNA polymerase II plays a crucial role in regulating transcription dynamics in eukaryotes. The phosphorylation of serine residues within the CTD controls transcription initiation, elongation, and termination. While the CTD is highly conserved across eukaryotes, lower eukaryotes like protists, including Plasmodium, exhibit some differences. In this study, we performed a comparative analysis of CTD in eukaryotic systems to understand why the parasites evolved in this particular manner. The Plasmodium falciparum RPB1 is exceptionally large and feature a gap between the first and second heptad repeats, resulting in fifteen canonical heptad repeats excluding the initial repeat. Analysis of this intervening sequence revealed sub motifs of heptads where two serine residues occupy the first and fourth positions (S1X2X3S4). These motifs lie in the intrinsically disordered region of RPB1, a characteristic feature of the CTD. Interestingly, the S1X2X3S4 sub-motif was also observed in early-divergingeukaryotes like Leishmania major, which lack canonical heptad repeats. Furthermore, eukaryotes across the phylogenetic tree revealed a sigmoid pattern of increasing serine frequency in the CTD, indicating that serine enrichment is a significant step in the evolution of heptad-rich RPB1. Based on these observations and analysis, we proposed an evolutionary model for RNA Polymerase II CTD, encompassing organisms previously deemed exceptions, notably Plasmodium species. Thus, our study provides novel insights into the evolution of the CTD and will prompt further investigations into the differences exhibited by Plasmodium RNA Pol II and determine if they confer a survival advantage to the parasite.

Abstract P2041: Identification Of The Nuclear Localization Signal In RBM20 And Its Function In The Pathogenesis Of Dilated Cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop an aggressive and arrhythmogenic form of dilated cardiomyopathy (DCM). Five of over 900 genetic mutations in RBM20 have recently been validated in animal models. Four of these five mutations are located in the arginine/serine-rich (RS) domain with the fifth in the RNA recognition motif (RRM). Interestingly, only mutations in the RS domain cause severe DCM, which suggests that disruption of RS domain function is crucial for the pathogenesis of DCM. To test this hypothesis, we generated mice expressing RBM20 with an in-frame deletion of the RS domain ( Rbm20 ΔRS ). We show that Rbm20 ΔRS mice manifest DCM with RBM20 mis-localization and granule formation, similar to that in RS domain mutation knock-in (KI) animals. Conversely, mice expressing RBM20 lacking the RRM do not exhibit RBM20 nucleocytoplasmic transport or granule formation, and, similar to RRM mutation KI mice, do not develop DCM. These data suggest that the critical nuclear localization signal (NLS) in RBM20 is located within the RS domain and that disruption of this sequence leads to RBM20 mis-localization and granule formation. In vitro experiments employing sequence deletion plasmids and immunocytochemical staining revealed that the first nine amino acids in the RS domain constitute the core NLS in RBM20, which is further supported by the fact that all three validated DCM-causing mutations are located in this segment. Analysis of plasmids containing DCM-associated mutations in other regions of RBM20, as well as in the non-NLS RS domain sequence, further confirmed that only mutations in the NLS facilitate re-localization and granule formation. Phosphorylation of residues within the NLS has been shown play an important role in protein nucleocytoplasmic transport. To test this, we mutated all phosphorylatable serine residues in the NLS to unphosphorylatable alanine or phosphomimetic aspartate and provide evidence that phosphorylation is dispensable for nucleocytoplasmic transport. Collectively, our findings identify the critical NLS in RBM20 and demonstrate that mutations in this NLS cause severe DCM through disruption of RS domain-mediated nuclear localization and sarcoplasmic granule formation.

Phosphorylation of E2 Serine Residue 402 Is Required for the Transcription and Replication of the HPV5 Genome.

Cutaneous human papillomavirus type 5 (HPV5) belongs to the supposedly oncogenic β-HPVs associated with specific types of skin and oral cavity cancers. Three viral proteins, namely, helicase E1 and transcription factors E2 and E8^E2, are master regulators of the viral life cycle. HPV5 E2 is a transcriptional activator that also participates in the E1-dependent replication and nuclear retention of the viral genome, whereas E8^E2 counterbalances the activity of E2 and inhibits HPV transcription and replication. In the present study, we demonstrate that the HPV5 E2 protein is extensively phosphorylated by cellular protein kinases, and serine residue 402 (S402) is the highest scoring phosphoacceptor site. This residue is located within a motif conserved among many β-HPVs and in the oncogenic HPV31 α-type. Using the nonphosphorylatable and phosphomimetic mutants, we demonstrate that phosphorylation of the E2 S402 residue is required for the transcription and replication of the HPV5 genome in U2OS cells and human primary keratinocytes. Mechanistically, the E2-S402-phopshodeficient protein is unable to trigger viral gene transcription and has an impaired ability to support E1-dependent replication, but the respective E8^E2-S213 mutant displays no phenotype. However, phosphorylation of the E2 S402 residue has no impact on the E2 stability, subcellular localization, self-assembly, DNA-binding capacity, and affinity to the E1 and BRD4 proteins. Further studies are needed to identify the protein kinase(s) responsible for this phosphorylation. IMPORTANCE Human papillomavirus type 5 (HPV5) may play a role in the development of specific types of cutaneous and head and neck cancers. The persistence of the HPV genome in host cells depends on the activity of its proteins, namely, a helicase E1 and transcription/replication factor E2. The latter also facilitates the attachment of episomal viral genomes to host cell chromosomes. In the present study, we show that the HPV5 E2 protein is extensively phosphorylated by host cell protein kinases, and we identify serine residue 402 as the highest scoring phosphoacceptor site of E2. We demonstrate that the replication of the HPV5 genome may be blocked by a single point mutation that prevents phosphorylation of this serine residue and switches off the transcriptional activity of the E2 protein. The present study contributes to a better understanding of β-HPV5 replication and its regulation by host cell protein kinases.

Molecular Mechanisms of Functional Modulation of Transcriptional Coactivator PC4 via Phosphorylation on Its Intrinsically Disordered Region.

To investigate the effects of phosphorylation on the function of the human positive cofactor 4 (PC4), an enhanced molecular dynamics (MD) simulation was performed. The simulation system consists of the N-terminal intrinsic disordered region (IDR) of PC4 and a complex that comprises the C-terminal acidic activation domain of a herpes simplex virion protein 16 (VP16ad) and a homodimer of the C-terminal structured core domain of PC4 (PC4ctd). An earlier report of an experimental study reported that the PC4-VP16ad interaction is modulated by incremental phosphorylation of the IDR. That report also proposed a dynamic model where phosphorylated serine residues of a segment (SEAC) in the IDR contact positively charged residues (lysin and arginine) of another segment (K-rich) in the IDR. This contact formation induced by the phosphorylation results in variation of PC4-VP16ad interaction. However, this contact formation has not yet been measured directly because it is transiently formed and because the SEAC and K-rich segments are unstructured with high flexibility. We performed two simulations to mimic the incremental phosphorylation: the IDR was not phosphorylated in one simulation and only partially phosphorylated in the other. Our simulation results indicate that the phosphorylation weakens the IDR-VP16ad contact considerably with the induction of a compact structure in the IDR. This structure was stabilized by electrostatic interactions between the phosphorylated serine residues of a segment and lysine or arginine residues of another segment in the IDR, but the conformational fluctuation of this compact structure was considerably large. Consequently, the present study supports the experimentally proposed dynamic model. Results of this study can be important for computational elucidation of the functional modulation of PC4.

A consensus phosphoserine within the large cytoplasmic loop of insect nAChR α8 subunits modulated interaction between 14-3-3ε and nAChRs to regulate neonicotinoid efficacy

Neonicotinoids are insect-selective nicotinic acetylcholine receptors (nAChRs) agonists that are used extensively for plant protection and animal health care. Some chaperone proteins, such as 14-3-3 proteins, importantly modulate nAChRs to display the physiological and pharmacological properties. Here we found that there is a 14-3-3 binding motif RSPSTH within the cytoplasmic loop of most insect α8 subunits. In the motif, a potential phosphorylated serine residue, serine 337, was a putative protein kinase A (PKA) substrate. Using Locusta migratoria α8 subunit as a representative, here we demonstrated that Loc14‐3-3ε interacted with the unique phosphoserine (α8S337) of Locα8 subunit to regulate agonist efficacy on hybrid Locα8/β2 nAChRs in Xenopus oocytes. Co-expression of Loc14-3-3ε caused a dramatic rise of maximal inward currents (Imax) of Locα8/β2 for acetylcholine and imidacloprid to 2.9-fold and 3.1-fold of that of Locα8/β2 alone. The S337A substitution of Locα8 reduced the Imax rise when Locα8S337A/β2 and Loc14-3-3ε were co-expressed. The increased agonist currents by exogenous Loc14-3-3ε on Locα8/β2 could be almost abolished by either PKA inhibitor KT5720 or 14-3-3 inhibitor difopein. The findings revealed that serine 337 within motif RSPSTH was important for the interaction between insect nAChRs and 14-3-3ε, and inhibiting the interaction would change the pharmacological property of insect nAChRs to agonist such as neonicotinoids which may provide insights to develop new targets for insecticide design.

Carnosic acid attenuated cytochrome c release through the mitochondrial structural protein Mic60 by PINK1 in SH-SY5Y cells.

Mitochondrial dysfunction has been implicated in Parkinson's disease. Mic60 is a critical component of mitochondrial crista remodeling and participates in maintaining mitochondrial structure and function. This study investigated whether the carnosic acid (CA) of rosemary protects the mitochondria of SH-SY5Y cells against the neurotoxicity of 6-hydroxydopamine (6-OHDA) by regulating Mic60. Our results showed that CA pretreatment reversed the reduction in the Mic60 and citrate synthase proteins, as well as the protein induction of PKA caused by 6-OHDA. Moreover, Mic60 and PINK1 siRNAs blocked the ability of CA to lessen the release of mitochondrial cytochrome c by 6-OHDA. As shown by immunoprecipitation assay, in 6-OHDA-treated cells, the interaction of Mic60 with its phosphorylated threonine residue was decreased, but the interaction with its phosphorylated serine residue was increased. PINK1 siRNA and forskolin, a PKA activator, reversed these interactions. Moreover, forskolin pretreatment prevented CA from rescuing the interaction of PINK1 and Mic60 and the reduction in cytochrome c release and mitophagy impairment in 6-OHDA-treated cells. In conclusion, CA prevents 6-OHDA-induced cytochrome c release by regulating Mic60 phosphorylation by PINK1 through a downregulation of PKA. The regulation of Mic60 by CA can be considered as a protective mechanism for the prevention of Parkinson's disease.

Insulin: A connection between pancreatic β cells and the hypothalamus

Insulin is a hormone secreted by pancreatic β cells. The concentration of glucose in circulation is proportional to the secretion of insulin by these cells. In target cells, insulin binds to its receptors and activates phosphatidylinositol-3-kinase/protein kinase B, inducing different mechanisms depending on the cell type. In the liver it activates the synthesis of glycogen, in adipose tissue and muscle it allows the capture of glucose, and in the hypothalamus, it regulates thermogenesis and appetite. Defects in insulin function [insulin resistance (IR)] are related to the development of neurodegenerative diseases in obese people. Furthermore, in obesity and diabetes, its role as an anorexigenic hormone in the hypothalamus is diminished during IR. Therefore, hyperphagia prevails, which aggravates hyper-glycemia and IR further, becoming a vicious circle in which the patient cannot regulate their need to eat. Uncontrolled calorie intake induces an increase in reactive oxygen species, overcoming cellular antioxidant defenses (oxidative stress). Reactive oxygen species activate stress-sensitive kinases, such as c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, that induce phos-phorylation in serine residues in the insulin receptor, which blocks the insulin signaling pathway, continuing the mechanism of IR. The brain and pancreas are organs mainly affected by oxidative stress. The use of drugs that regulate food intake and improve glucose metabolism is the conventional therapy to improve the quality of life of these patients. Currently, the use of antioxidants that regulate oxidative stress has given good results because they reduce oxidative stress and inflammatory processes, and they also have fewer side effects than synthetic drugs.

Tropomyosin R160A is crucial for TnI phosphorylation-induced effects on calcium sensitivity.

The thin filament, consisting of the three-protein complex troponin (TnI, TnT, and TnC) and tropomyosin (Tpm) bound to filamentous actin, acts as the main transducer that relates sarcoplasmic calcium levels to the contraction-relaxation cycles of the myocardium. In the absence of calcium, TnI biases Tpm in a position on actin that blocks myosin binding whereas calcium binding to TnC causes a propagated conformational change in Tn/Tpm that relieves this inhibition. During heart failure and adrenergic stimulation, phosphorylation of the N-terminal region of troponin I (TnI) at residues S23 and S24, has been shown to play a role in heart muscle adaptation through a decrease in calcium sensitivity. Previous modeling studies performed in thin filament models have suggested that positively charged groups on Tpm, namely R160, could interact with the phosphorylated serine residues on TnI. Here we tested the hypothesis that interactions between Tpm-R160 and phosphorylated S23/24 participate in phosphorylation-induced effects on calcium sensitivity (Pavadai et al., 2022). Tpm mutated from wild-type R160 to alanine (Tpm-R160A) and phosphomimetic TnI affecting both serine 23 and 24 (S23/24D) were generated and expressed recombinantly. Using in vitro motility (IVM) assays, we quantified the effects of these mutations alone and in combination. Regulated IVM experiments using thin filaments reconstituted with phosphomimetic S23/24D-TnI and wild-type Tpm revealed a decrease in calcium sensitivity, showing that phosphorylation is successfully mimicked by mutating serine to an aspartic acid,consistent with previous studies on human cardiac tissue. However, when TnI-S23/24D is paired with mutant R160A-Tpm, the phosphorylation-induced changes in calcium sensitivity are diminished. These results are consistent with Tpm-R160 participating in the lusitropy observed when the N-terminal of TnI is phosphorylated.

Characterization of mutants deficient in N-terminal phosphorylation of the chloroplast ATP synthase subunit β.

Understanding the regulation of photosynthetic light harvesting and electron transfer is of great importance to efforts to improve the ability of the electron transport chain to supply downstream metabolism. A central regulator of the electron transport chain is ATP synthase, the molecular motor that harnesses the chemiosmotic potential generated from proton-coupled electron transport to synthesize ATP. ATP synthase is regulated both thermodynamically and post-translationally, with proposed phosphorylation sites on multiple subunits. In this study we focused on two N-terminal serines on the catalytic subunit β in tobacco (Nicotiana tabacum), previously proposed to be important for dark inactivation of the complex to avoid ATP hydrolysis at night. Here we show that there is no clear role for phosphorylation in the dark inactivation of ATP synthase. Instead, mutation of one of the two phosphorylated serine residues to aspartate to mimic constitutive phosphorylation strongly decreased ATP synthase abundance. We propose that the loss of N-terminal phosphorylation of ATPβ may be involved in proper ATP synthase accumulation during complex assembly.

Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.

Calcium interactions in amelogenin-derived peptide assembly.

Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between 31P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR 31P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register β-sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.

Phosphorylation of serine residues S252, S268/S269, and S879 in p120 catenin activates migration of presomitic mesoderm in gastrulating zebrafish embryos.

Cadherin-associated protein p120 catenin regulates cell adhesion and migration in cell cultures and is required for axial elongation in embryos. Its roles in adhesion and cell migration are regulated by phosphorylation. We determined the effects of phosphorylation of six serine and three threonine residues in p120 catenin during zebrafish (Danio rerio) embryogenesis. We knocked down endogenous p120 catenin-δ1 with an antisense RNA-splice-site morpholino (Sp-MO) causing defects in axis elongation. These defects were rescued by co-injections of mRNAs for wildtype mouse p120 catenin-δ1-3A or various mutated forms. Several mRNAs containing serine or threonine codons singly or doubly mutated to phosphomimetic glutamic acid rescued, and some nonphosphorylatable mutants did not. We discovered that phosphorylation of serine residue S252 or S879 is required for convergent extension of zebrafish embryos, since rescue occurred only when these residues were mutated to glutamic acid. In addition, the phosphorylation of either S268 or S269 is required, not both, consistent with the presence of only a single one of these residues in two isoforms of zebrafish and Xenopus laevis. In summary, phosphorylation of multiple serine and threonine residues of p120 catenin activates migration of presomitic mesoderm of zebrafish embryos facilitating elongation of the dorsal axis.