Phosphorylation Of Serine Residues Research Articles

A prismatic view of protein phosphorylation in health and disease.

The paramount relevance of protein phosphorylation in health and disease motivated us to gather several contributions from experts working in this area in order to portray the recent developments in the field. Our effort and the effort of 54 authors with their 12 contributions gave rise to this Research Topic, which represents a valuable forum where phosphorylation is discussed from different angles, including bioinformatics approaches and experimental methods that are currently used to decipher the complex mechanisms underlying this bewitching post-translational modification (PTM). The articles collected in this Research Topic illustrate very diverse aspects of phosphorylation, such as its biological effects and induced structural changes, the role of kinases and phosphatases as therapeutic targets, the use of phosphorylation profiles as biomarkers, how phosphorylation dys-regulation may cause disease, and more.

Insulin induces phosphorylation of serine residues of translationally controlled tumor protein in 293T cells.

Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.

Phosphorylation of Leghemoglobin at S45 is Most Effective to Disrupt the Molecular Environment of Its Oxygen Binding Pocket.

In leguminous plants, nitrogenase that catalyzes anaerobic symbiotic nitrogen fixation is protected by the sequestration of O2 by Leghemoglobin (LegH). The modulation of the oxygen binding capacity of Hemoglobin(Hb) by different post-translational modifications is well studied; whereas similar studies on LegH's O2 binding are not yet benchmarked. Our results show that in vitro serine phosphorylation of recombinant LegH from Lotus japonicus, a model legume, by a homologous kinase caused a reduction in its oxygen consumption as determined by Clark type electrode. Although mass spectrometry revealed a few phosphorylated serine residues in the LegH, molecular modeling study showed that particularly S45 is the most critical one, along with S55, however the latter with lesser impact on its molecular environment responsible for oxygen consumption. Separate S45D and S55D mutants of recombinant LegH also corroborated the results obtained from molecular modeling study. Thus, this work lays groundwork for further investigation of structural and functional role of serine phosphorylation as one of the mechanisms by which oxygen consumption by LegH may possibly be regulated during nodulation.

Regiospecific function of the C‐terminal domain of RNA polymerase II in yeast

The C‐terminal domain (CTD) of RPB1, the largest subunit of RNA polymerase II is comprised of repeating units of a seven amino acid sequence and is indispensible for function in eukaryotes. The aim of this study was to determine if repeats had specific function, dependent on their location within the sequence, or whether repeats were largely redundant. Using a library of precisely designed CTD mutants, a novel TET‐off system to control RPB1 expression, and phenotypic analysis in yeast we demonstrate that, in fact, different regions of the CTD exhibit different function. Specifically, we show differential sensitivity to various transcriptional inhibitors and can link phenotypes to the presence or absence of phosphorylatable serine residues. This study leads to a novel model for the assembly of CTD‐associating factors on the CTD during transcription. We also begin to use this library to identify changes in CTD‐associating protein recruitment to the CTD and to identify regions of the CTD important for processes such as mRNA processing and histone methylation.

Phosphorylation and Alternative Splicing of 7B2 Reduce Prohormone Convertase 2 Activation.

FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues within a variety of different bone proteins. In this work we demonstrate that FAM20C also phosphorylates threonines, specifically those within the N-terminal domain of the neuroendocrine chaperone 7B2. Analysis of the primary sequence of 7B2 revealed that three threonine residues in its N-terminal domain are located within FAM20C consensus motifs: Thr73, Thr99, and Thr111. The individual substitution of Thr73 and Thr111 residues by neutral alanines caused a marked decrease in the total phosphorylation of 7B2. Furthermore, the phosphomimetic substitution of Thr111 by Glu clearly diminished the ability of 7B2 to activate pro-prohormone convertase 2 (PC2) in 7B2-lacking SK-N-MC neuroblastoma cells, suggesting that the phosphorylation of this residue critically impacts the 7B2-proPC2 interaction. However, the phosphomimetic mutation did not alter 7B2's ability to function as an antiaggregant for human islet amyloid polypeptide. FAM20C-mediated phosphorylation of a common alternatively spliced variant of human 7B2 that lacks Ala100 (thus eliminating the Thr99 phosphorylation consensus site) was similar to the Ala-containing protein, but this variant did not activate proPC2 as efficiently as the Ala-containing protein. Although threonines within 7B2 were phosphorylated efficiently, FAM20C was incapable of performing the well-known regulatory threonine phosphorylation of the molecular chaperone binding immunoglobulin protein. Taken together, these results indicate that FAM20C plays a role in 7B2-mediated proPC2 activation by phosphorylating residue Thr111; and that 7B2 function is regulated by alternative splicing.

Glutamate-Dependent BMAL1 Regulation in Cultured Bergmann Glia Cells.

Glutamate, the major excitatory amino acid, activates a wide variety of signal transduction cascades. This neurotransmitter is involved in photic entrainment of circadian rhythms, which regulate physiological and behavioral functions. The circadian clock in vertebrates is based on a transcription-translation feedback loop in which Brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) acts as transcriptional activator of others clock genes. This protein is expressed in nearly all suprachiasmatic nucleus neurons, as well as in the granular layer of the cerebellum. In this context, we decided to investigate the role of glutamate in the molecular mechanisms involved in the processes of transcription/translation of BMAL1 protein. To this end, primary cultures of chick cerebellar Bergmann glial cells were stimulated with glutamatergic ligands and we found that BMAL1 levels increased in a dose- and time dependent manner. Additionally, we studied the phosphorylation of serine residues in BMAL1 under glutamate stimulation and we were able to detect an increase in the phosphorylation of this protein. The increased expression of BMAL1 is most probably the result of a stabilization of the protein after it has been phosphorylated by the cyclic AMP-dependent protein kinase and/or the Ca(2+)/diacylglycerol dependent protein kinase. The present results strongly suggest that glutamate participates in regulating BMAL1 in glial cells and that these cells might prove to be important in the control of circadian rhythms in the cerebellum.

Helicobacter pylori Induces Serine Phosphorylation of EGFR via Novel TAK1-p38 Activation Pathway in an HB-EGF-Independent Manner.

The interaction of Helicobacter pylori with gastric epithelial cells can result in the activation of transcription factor NF-κB via TGF-β-activated kinase 1 (TAK1). In this study, we have demonstrated the role of H. pylori in the activation of EGFR via TAK1-mediated phosphorylation of p38. Gastric epithelial AGS or MKN-45 cells were co-cultured with wild-type or cagA(-) H. pylori strains. H. pylori was added to the cells, and the activation of EGFR, p65 (NF-κB) subunit, p38, ERK, and TAK1 was examined by Western blotting. Infected cells were pretreated with or without ligands, chemical inhibitors, anti-HB-EGF antibody, and siRNAs to evaluate the effects on phosphorylation of various EGFR residues. Fluorescence microscopy and flow cytometry were performed to detect the internalization of EGFR. Incubating cells with wild-type and CagA(-) H. pylori strains resulted in the rapid and transient phosphorylation of serine residues of EGFR. RNAi experiments using siRNA against TAK1 and p38 pathways blocked the phosphorylation of serine residue. Immunofluorescence and flow cytometry revealed that EGFR was internalized in H. pylori-infected cells after EGFR phosphorylation in a p38-dependent manner. In contrast, pretreatment with gefitinib and anti-HB-EGF antibody did not block both the phosphorylation and internalization of EGFR. Helicobacter pylori induces internalization of EGFR via novel TAK1-p38-serine activation pathway which is independent of HB-EGF. The interaction between TAK1 and EGFR in H. pylori-infected cells might open new dimensions in understanding H. pylori-associated gastric carcinogenesis.

All-trans retinoic acid prevents oxidative stress-induced loss of renal tight junction proteins in type-1 diabetic model

We previously reported that diabetes decreased the expression of renal tight junction (TJ) proteins claudin-5 in glomerulus, and claudin-2 and occludin in proximal tubule through an oxidative stress dependent way. Now we investigated whether all-trans retinoic acid (atRA), a compound that plays a relevant role in kidney maintenance and that possesses antioxidant properties, prevents loss of TJ proteins in streptozotocin (STZ)-treated rats. atRA was administered daily by gavage (1mg/kg) from Days 3–21 after STZ administration. atRA attenuated loss of body weight, proteinuria and natriuresis but it did not prevent hyperglucemia. Other metabolic alterations, such as: increased kidney injury molecule (KIM)-1, oxidative stress, protein kinase C (PKC) beta 2, NADPH oxidase subunits (p47phox and gp91phox) expressions and endothelial nitric oxide synthase (eNOS) uncoupling, and decreased nitric oxide synthesis, nuclear factor-erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions were also attenuated by atRA. In vitro scavenging capacity assays showed that atRA scavenged peroxyl radicals (ROO•), singlet oxygen (1O2) and hypochlorous acid (HOCl) in a concentration-dependent manner. Decreased expressions of occludin, claudins-2 and -5 induced by diabetes were ameliorated by atRA. We also found that diabetes induced tyrosine nitration (3-NT), SUMOylation and phosphorylation in serine residues of claudin-2 and atRA prevented these changes. In conclusion, atRA exerted nephroprotective effects by attenuating oxidative stress and preventing loss of renal TJ proteins.

Aquaporin-0 Water Conduction Regulation by Calmodulin

Aquaporin-0 (AQP0) contributes to the nurturing and cleaning of the eye lens of waste product. It is a tetrameric protein composed of four identical monomers, each of which has its own water pore. AQP0 water conduction is regulated by pH, Ca2+ concentration, and the phosphorylation of Serine residues at the C-terminal. High cellular Ca2+ concentration enhances the binding of Calmodulin (CaM), a Ca2+ dependent protein, to AQP0 from cytoplasm. Molecular Dynamics (MD) simulations performed to investigate the effects of two Calmodulin binding to AQP0 tetramer revealed free energy barriers up to 6 kcal/mol for water conduction. MD simulations also exposed three distinct locations, where the water molecule's pathway changes between AQP0-CaM model versus AQP0 open model.

Crossbridge Arrangement in Cardiac Thick Filaments Isolated from cMyBP-C Phosphomimetic Mice

Although cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, the mechanism by which changes in phosphorylation of cMyBP-C affect crossbridge kinetics remains unknown. cMyBP-C mediated changes in thick filament structure, binding of cMyBP-C to the thin filament, or both remain viable alternatives. In this study, we isolated thick filaments from the hearts of phosphomimetic mice in which the three phosphorylatable serine residues (Ser273, Ser282 and Ser302) in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid to mimic the fully non-phosphorylated (CT-SA mice) or fully phosphorylated (CT-SD mice) state of cMyBP-C, respectively. Thus, the state of phosphorylation of cMyBP-C of every filament was known in this transgenic system. We found that the thick filaments from the CT-SA mouse hearts corresponding to the non-phosphorylated state of cMyBP-C had highly ordered crossbridge arrays, while the filaments from the CT-SD mouse hearts showed a strong tendency towards disorder of the myosin array. These results are consistent with our previous studies of filaments from cMyBP-C knockout mouse hearts. The results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed quasi-helical ordering of the myosin heads on the backbone consistent with slower crossbridge kinetics; while filaments with phosphorylated cMyBP-C show a greater tendency towards disorder of the heads consistent with increased flexibility of the myosin heads, extension of the heads from the backbone, and increased potential interaction with the actin thin filament. Work supported by NIH grants SC1 HL096017 (RWK), AR034711 (RC), R37 HL82900 and P01 HL094291 (RLM). RC also received funding through PPG grant P01 HL059408 (David Warshaw).

Peptidyl-Prolyl cis/trans Isomerase NIMA-Interacting 1 as a Therapeutic Target in Hepatocellular Carcinoma.

Phosphorylation of proteins on serine or threonine residues preceding proline is a pivotal signaling mechanism regulating cell proliferation. The recent identification and characterization of the enzyme peptidyl-prolyl cis/trans isomerase never in mitosis A (NIMA)-interacting 1 (PIN1) has led to the discovery of a new mechanism regulating phosphorylation in cell signaling. PIN1 specifically binds phosphorylated serine or threonine residues immediately preceding proline (pSer/Thr-Pro) and then regulates protein functions, including catalytic activity, phosphorylation status, protein interactions, subcellular location, and protein stability, by promoting cis/trans isomerization of the peptide bond. Recent results have indicated that such conformational changes following phosphorylation represent a novel signaling mechanism in the regulation of many cellular functions. Understanding this mechanism also provides new insight into the pathogenesis and treatment of human hepatocellular carcinoma. A better understanding of the role of PIN1 in the pathogenesis of hepatocellular carcinoma may lead to the identification of molecular targets for prevention and therapeutic intervention.

Ribosomal Protein S6 Phosphorylation: Four Decades of Research.

The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time about four decades ago. Since then, numerous studies have shown that this modification occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues. However, despite a large body of information on the respective kinases and the signal transduction pathways, the physiological role of rpS6 phosphorylation remained obscure until genetic manipulations were applied in both yeast and mammals in an attempt to block this modification. Thus, studies based on both mice and cultured cells subjected to disruption of the genes encoding rpS6 and the respective kinases, as well as the substitution of the phosphorylatable serine residues in rpS6, have laid the ground for the elucidation of the multiple roles of this protein and its posttranslational modification. This review focuses primarily on newly identified kinases that phosphorylate rpS6, pathways that transduce various signals into rpS6 phosphorylation, and the recently established physiological functions of this modification. It should be noted, however, that despite the significant progress made in the last decade, the molecular mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology are still poorly understood.

Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling

Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway.

Definition of smad3 phosphorylation events that affect malignant and metastatic behaviors in breast cancer cells.

Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer.

Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C.

We have used enzyme kinetics to investigate the molecular mechanism by which the N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, and C0C2) affect the activation of myosin ATP hydrolysis by F-actin and by native porcine thin filaments. N-Terminal domains of cMyBP-C inhibit the activation of myosin-S1 ATPase by F-actin. However, mouse and human C1C2 and C0C2 produce biphasic activating and inhibitory effects on the activation of myosin ATP hydrolysis by native cardiac thin filaments. Low ratios of MyBP-C N-terminal domains to thin filaments activate myosin-S1 ATP hydrolysis, but higher ratios inhibit ATP hydrolysis, as is observed with F-actin alone. These data suggest that low concentrations of C1C2 and C0C2 activate thin filaments by a mechanism similar to that of rigor myosin-S1, whereas higher concentrations inhibit the ATPase rate by competing with myosin-S1-ADP-Pi for binding to actin and thin filaments. In contrast to C0C2 and C1C2, the activating effects of the C0C1 domain are species-dependent: human C0C1 activates actomyosin-S1 ATPase rates, but mouse C0C1 does not produce significant activation or inhibition. Phosphorylation of serine residues in the m-linker between the C1 and C2 domains by protein kinase-A decreases the activation of thin filaments by huC0C2 at pCa > 8 but has little effect on the activation mechanism at pCa = 4. In sarcomeres, the low ratio of cMyBP-C to actin is expected to favor the activating effects of cMyBP-C while minimizing inhibition produced by competition with myosin heads.

A signal “on” photoelectrochemical biosensor for assay of protein kinase activity and its inhibitor based on graphite-like carbon nitride, Phos-tag and alkaline phosphatase

A highly sensitive and selective photoelectrochemical (PEC) biosensor is fabricated for the detection of protein kinase activity based on visible-light active graphite-like carbon nitride (g-C3N4) and the specific recognition utility of Phos-tag for protein kinase A (PKA)-induced phosphopeptides. For assembling the substrate peptides, g-C3N4 and gold nanoparticles (g-C3N4-AuNPs) complex is synthesized and characterized. When the immobilized peptides on g-C3N4–AuNPs modified ITO electrode are phosphorylated under PKA catalysis, they can be specifically identified and binded with biotin functionalized Phos-tag (Phos-tag–biotin) in the presence of Zn2+. Then, through the specific interaction between biotin and avidin, avidin functionalized alkaline phosphatase (avidin-ALP) is further assembled to catalyze its substrate of l-ascorbic acid-2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting an increased photocurrent compared with the absence of phosphorylation event. Based on the specific identification effect of Phos-tag, the fabricated biosensor presents excellent selectivity for capturing the phosphorylated serine residues in the substrate peptides. With the good photoactivity of g-C3N4 and ALP-catalyzed signal amplification, the fabricated biosensor presents high sensitivity and low detection limit (0.015unit/mL, S/N=3) for PKA. The applicability of this PEC biosensor is further testified by the evaluation of PKA inhibition by HA-1077 with the IC50 value of 1.18μM. This new strategy is also successfully applied to detect the change of PKA activity in cancer cell lysate with and without drug stimulation. Therefore, the developed PEC method has great potential in screening of kinase inhibitors and highly sensitive detection of kinase activity.

Structural Insights into the Mechanism of Negative Regulation of Single-box High Mobility Group Proteins by the Acidic Tail Domain

The Drosophila and plant (maize) functional counterparts of the abundant vertebrate chromosomal protein HMGB1 (HMG-D and ZmHMGB1, respectively) differ from HMGB1 in having a single HMG box, as well as basic and acidic flanking regions that vary greatly in length and charge. We show that despite these variations, HMG-D and ZmHMGB1 exist in dynamic assemblies in which the basic HMG boxes and linkers associate with their intrinsically disordered, predominantly acidic, tails in a manner analogous to that observed previously for HMGB1. The DNA-binding surfaces of the boxes and linkers are occluded in "auto-inhibited" forms of the protein, which are in equilibrium with transient, more open structures that are "binding-competent." This strongly suggests that the mechanism of auto-inhibition may be a general one. HMG-D and ZmHMGB1 differ from HMGB1 in having phosphorylation sites in their tail and linker regions. In both cases, in vitro phosphorylation of serine residues within the acidic tail stabilizes the assembled form, suggesting another level of regulation for interaction with DNA, chromatin, and other proteins that is not possible for the uniformly acidic (hence unphosphorylatable) tail of HMGB1.

Biochemical characterization of P-type copper ATPases.

Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS (transmembrane metal-binding sites)] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.e. Na+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD (N-terminal metal-binding domain), containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper.

Phosphorylation of αS1-casein is regulated by different genes

Casein phosphorylation is a posttranslational modification catalyzed by kinase enzymes that attach phosphate groups to specific AA in the protein sequence. This modification is one of the key factors responsible for the stabilization of calcium phosphate nanoclusters in casein micelles and for the internal structure of the casein micelles. αS1-Casein (αs1-CN) is of special interest because it constitutes up to 40% of the total casein fraction in milk, and it has 2 common phosphorylation states, with 8 (αS1-CN-8P) and 9 (αS1-CN-9P) phosphorylated serine residues. Factors affecting this variation in the degree of phosphorylation are not currently known. The objective of this research was to determine the genetic background of αS1-CN-8P and αS1-CN-9P. The genetic and phenotypic correlation between αS1-CN-8P and αS1-CN-9P was low (0.18 and 0.19, respectively). This low genetic correlation suggests a different genetic background. These differences were further investigated by means of a genome-wide association study, which showed that both αS1-CN-8P and αS1-CN-9P were affected by a region on Bos taurus autosome (BTA) 6, but only αS1-CN-8P was affected by a region on BTA11 that contains the gene that encodes for β-lactoglobulin (β-LG), and only αS1-CN-9P was affected by a region on BTA14 that contains the diacylglycerol acyltransferase 1 (DGAT1) gene. Estimated effects of β-LG protein genotypes showed that only αS1-CN-8P was associated with the β-LG A/B polymorphism (g.1772G>A and g.3054C>T); the AA genotype of β-LG was associated with a lower concentration of αS1-CN-8P (−0.32% wt/wt) than the BB genotype (+0.41% wt/wt). Estimated effects of DGAT1 K232A genotypes showed that only αS1-CN-9P was associated with the DGAT1 gene polymorphism; DGAT1 AA genotype was associated with a higher αS1-CN-9P concentration (+0.53% wt/wt) than the DGAT1 KK genotype (−0.44% wt/wt). The results give insight in phosphorylation of αS1-CN-8P and αS1-CN-9P, which seem to be regulated by a different set of genes.

A Theoretical Study of SRPK Interaction with the Flexible Domains of Hepatitis B Capsids

Hepatitis B virus (HBV) controls genome encapsidation and reverse transcription from a single-stranded RNA to a double-stranded DNA through the flexible C-terminal domain (CTD) of the capsid proteins. Although the microscopic structure of the nucleocapsid plays a critical role in the life cycle of HBV, the location of CTD residues at different stages of viral replication remains poorly understood. In this work, we report the radial distributions of individual amino-acid residues of the CTD tails for both empty and RNA-containing HBV capsids by using a coarse-grained model for the key biological components and the classical density functional theory. The density functional theory calculations reveal substantial exposure of the CTD residues outside the capsid, in particular when it is devoid of any nucleic materials. The outermost layer of the capsid surface mainly consists of residues from 170Ser-175Arg of the CTD tails, i.e., the serine-arginine protein kinase binding motif. The theoretical description corroborates recent in vitro studies that show a transient CTD distribution captured by serine-arginine protein kinase binding. We have also investigated the nucleocapsid structural changes due to phosphorylation of serine residues and shown a correlation between the CTD location and the internal distribution of RNA segments.