Serine Residues Research Articles

Electrochemical C-O and C-N Arylation using Alternating Polarity in flow for Compound Libraries.

Etherification and amination of aryl halide scaffolds are commonly used reactions in parallel medicinal chemistry to rapidly scan structure-activity relationships with abundant building blocks. Electrochemical methods for aryl etherification and amination demonstrate broad functional group tolerance and extended nucleophile scope compared to traditional methods. Nevertheless, there is a need for robust and scale-transferable workflows for electrochemical compound library synthesis. Herein we describe a platform for automated electrochemical synthesis of C-X arylation (X=NH, OH) in flow to access compound libraries. A comprehensive Design of Experiment (DoE) study identifies an optimal protocol which generates high yields across>30 aryl halide scaffolds, diverse amines (including electron-deficient sulfonamides, sulfoximines, amides, and anilines) and alcohols (including serine residues within peptides). Reaction sequences are automated on commercially available equipment to generate libraries of anilines and aryl ethers. The unprecedented application of potentiostatic alternating polarity in flow is essential to avoid accumulating electrode passivation. Moreover, it enables reactions to be performed in air, without supporting electrolyte and with high reproducibility over consecutive runs. Our method represents a powerful means to rapidly generate nucleophile independent C-X arylation compound libraries using flow electrochemistry.

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.

The O-GlcNAc database: introducing new features and tools developed from community feedback.

O-GlcNAc is a reversible post-translational modification found on serine and threonine residues of nucleocytoplasmic proteins. Four years ago, we released the O-GlcNAc Database( oglcnac.mcw.edu ), a comprehensive catalog of O-GlcNAcylated proteins that has become one of the most cited resources in the field, with hundreds of unique users per month. We are now presenting an updated O-GlcNAc Database, which includes nearly 20,000 O-GlcNAcylated proteins and 48 species, marking substantial growth in data volume and scope. This paper presents the most noteworthy features implemented over the last year, often originating from feedback from the O-GlcNAc community. Among these features, we provide a brief overview of the database content, introduce our new protein viewer mode, and discuss the implementation of subcellular localization information and its applications in the O-GlcNAc score. We also provide an interface to use CytOVS, a tool designed to evaluate and sort O-GlcNAcome datasets derived from MS experiments. In conclusion, this new and improved O-GlcNAc Database represents a significant advancement in providing a comprehensive and expanded resource for researchers in the field of O-GlcNAc biology.

Singlet oxygen-mediated photochemical cross-linking of an engineered fluorescent flavoprotein iLOV

Genetically encoded photoactive proteins are integral tools in modern biochemical and molecular biological research. Within this tool box, truncated variants of the phototropin two light-oxygen-voltage flavoprotein have been developed to photochemically generate singlet oxygen (1O2) invitro and invivo, yet the effect of 1O2 on these genetically encoded photosensitizers remains underexplored. In this study, we demonstrate that the "improved" light-oxygen-voltage flavoprotein is capable of photochemical 1O2 generation. Once generated, 1O2 induces protein oligomerization via covalent cross-linking. The molecular targets of protein oligomerization by cross-linking are not endogenous tryptophans or tyrosines, but rather primarily histidines. Substitution of surface-exposed histidines for serine or glycine residues effectively eliminates protein cross-linking. When used in biochemical applications, such protein-protein cross-links may interfere with native biological responses to 1O2, which can be ameliorated by substitution of the surface exposed histidines of improved" light-oxygen-voltage or other 1O2-generating flavoproteins.

Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death

The intracellular bacterial pathogen Legionella pneumophila utilizes the Dot/Icm system to translocate over 330 effectors into the host cytosol. These virulence factors modify a variety of cell processes, including pathways involved in cell death and survival, to promote bacterial proliferation. Here, we show that the effector LegK3 is a eukaryotic-like Ser/Thr kinase that functions to suppress host apoptosis. Mechanistically, LegK3 directly phosphorylates multiple caspases involved in apoptosis signaling, including Caspase-3, Caspase-7, and Caspase-9. LegK3-induced phosphorylation of these caspases occurs at serine (Ser29 in Caspase-3 and Ser199 in Caspase-7) or threonine (Thr102 in Caspase-9) residues located in the prodomain or interdomain linkers. These modifications interfere with the suitability of the caspases as the substrates of initiator caspases or upstream regulators without impacting their proteolytic activity. Collectively, our study reveals a novel strategy used by L. pneumophila to maintain the integrity of infected cells for its intracellular growth.

Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: Characterization of the enzyme's in vitro truncation by thiol endopeptidase activity

Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.

Functional profiling of serine, threonine and tyrosine sites.

Systematic perturbation of amino acids at endogenous loci provides diverse insights into protein function. Here, we performed a genome-wide screen to globally assess the cell fitness dependency of serine, threonine and tyrosine residues. Using an adenine base editor, we designed a whole-genome library comprising 817,089 single guide RNAs to perturb 584,337 S, T and Y sites. We identified 3,467 functional substitutions affecting cell fitness and 677 of them involving phosphorylation, including numerous phosphorylation-mediated gain-of-function substitutions that regulate phosphorylation levels of itself or downstream factors. Furthermore, our findings highlight that specific substitution types, notably serine to proline, are crucial for maintaining domain structure broadly. Lastly, we demonstrate that 309 enriched hits capable of initiating cell overproliferation might be potential cancer driver mutations. This study represents an extensive functional profiling of S, T and Y residues and provides insights into the distinctive roles of these amino acids in biological mechanisms and tumor progression.

Morpholine-tethered Novel Hydrazones as Promising Non-peptidic Prolyl Oligopeptidase (POP) Inhibitors: Synthesis In Vitro and In Silico Studies.

Prolyl oligopeptidase (POP) is a pivotal druggable target implicated in diverse biological processes and linked to the development of various ailments, including neurodegenerative disorders. While conventional peptide-based inhibitors have been a centerpiece, their limitations, such as restricted bioavailability, necessitate exploration of non-peptidic inhibitors for their therapeutic potential. This study focuses on designing, synthesizing, and assessing morpholine-based hydrazones targeting the catalytic serine residue of POP. The hydrazones (5a-o), reported as moderately potent analogs compared to the renowned Z-Pro-Prolinal, demonstrated in vitro POP inhibition with IC50 values ranging from 13.60 ± 2.51 to 36.51 ± 1.82 μM. The derivative 5h, with an IC50 of 13.60 ± 2.51 μM, emerged as the most potent inhibitor. Moreover, the in vitro kinetic study of compound 5h indicated that it exhibited concentration-dependent type of inhibition. in silico docking studies of 5h revealed robust interactions in the POP enzyme's active site, yielding a docking score of -6.30 Kcal/- mol, consistent with experimental results. All findings underscored the potential of synthesized derivatives for drug development.

DCLK1 induces a pro-tumorigenic phenotype to drive gastric cancer progression.

Doublecortin-like kinase 1 (DCLK1) is a proposed driver of gastric cancer (GC) that phosphorylates serine and threonine residues. Here, we showed that the kinase activity of DCLK1 orchestrated cancer cell-intrinsic and-extrinsic processes that led to pro-invasive and pro-metastatic reprogramming of GC cells. Inhibition of the kinase activity of DCLK1 reduced the growth of subcutaneous xenograft tumors formed from MKN1 human gastric carcinoma cells in mice and decreased the abundance of the stromal markers α-Sma, vimentin, and collagen. Similar effects were seen in mice with xenograft tumors formed from MKN1 cells expressing a kinase-inactive DCLK1 mutant (MKN1D511N). MKN1D511N cells also had reduced in vitro migratory potential and stemness compared with control cells. Mice orthotopically grafted with MKN1 cells overexpressing DCLK1 (MKN1DCLK1) showed increased invasiveness and had a greater incidence of lung metastases compared with those grafted with control MKN1 cells. Mechanistically, we showed that the chemokine CXCL12 acted downstream of DCLK1 in cultured MKN1 cells and in mice subcutaneously implanted with gastric tumors formed by MKN1DCLK1 cells. Moreover, inhibition of the kinase activity of DCLK1 or the expression of DCLK1D511N reversed the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a broadly acting and potentially targetable promoter of GC.

Disruption of herpes simplex virus type 2 pUL21 phosphorylation impairs secondary envelopment of cytoplasmic nucleocapsids.

It is well known that post-translational modification of proteins by phosphorylation can regulate protein function. Here, we determined that phosphorylation of the multifunctional HSV-2 tegument protein pUL21 requires the viral serine/threonine kinase pUL13. In addition, we identified serine residues within HSV-2 pUL21 that can be phosphorylated. Phenotypic analysis of mutant HSV-2 strains with deficiencies in pUL21 phosphorylation revealed reductions in both cell-cell spread of virus infection and virus replication. Deficiencies in pUL21 phosphorylation also compromised the secondary envelopment of cytoplasmic nucleocapsids, a critical final step in the maturation of all herpes virions. Unlike HSV-2 pUL21, phosphorylation of HSV-1 pUL21 was not detected. This fundamental difference between HSV-2 and HSV-1 may underlie our previous observations that the requirements for pUL21 differ between HSV species.

RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.

Molecular dynamics modelling of the stress–strain response of [formula omitted]-sheet nanocrystals

Molecular dynamics simulations were conducted on two model antiparallel β-sheet crystallites [GA]n and [GAS]n to study deformation in chain, sheet stacking, and hydrogen bonding directions under uniaxial loading. In chain direction, both models were mechanically stable, even beyond the 570 K amorphousation temperature of silk fiber; however, [GA]n model displayed higher yield strain, stress, elastic modulus, and resilience than [GAS]n. In transverse directions, they had similar stress–strain behavior and demonstrated significant anisotropic mechanical behavior. Hence, inclusion of an amino acid with a rich side chain group extending between β-sheets reduces the stiffness of crystallite in chain direction. Serine and alanine residues maintained existing H-bonds and established new ones during stretching in chain direction and shrinking in transverse directions which affected the mechanical response near the yield point. Comparison between β-sheet crystallite and PPTA (Kevlar) showed that the mechanical performance of these crystal polymers were very similar in chain direction, but contrarily β-sheet crystallite had higher stiffness in H-bonding and sheet stacking directions than PPTA. This study may provide a guideline in designing of polyaminoacid based biocompatible materials with superior mechanical performance.

CLONING OF CDNA-GENE OF ARABIDOPSIS THALIANA RIBOSOMAL PROTEIN S6, ITS EXPRESSION IN ESCHERICHIA COLI AND PURIFICATION OF ATRPS6B1 RECOMBINANT PROTEIN

Living organisms must adapt their growth and development in response to external factors like stress and nutrient availability throughout their lifespan. Consequently, they have evolved various regulatory pathways to enhance environmental perception and facilitate necessary metabolic adjustments. These pathways involve key proteins that, under stress and nutrient limitation, initiate anabolic and catabolic cellular processes. One critical pathway present in all eukaryotes involves the protein kinase TOR (Target of rapamycin), a large kinase that governs numerous biological processes, including the activation of S6K kinase, which phosphorylates the S6 protein (RPS6). Crystal structures have revealed the precise location of RPS6 within the 40S ribosomal subunit, highlighting structural features such as the C-terminal helix containing multiple phosphorylation sites. RPS6 primarily regulates mechanisms controlling cell growth and division. In plants, eS6 is encoded by two conserved genes, and its activation and phosphorylation are closely linked to S6Ks. Recent studies emphasize the significance of S6 protein phosphorylation as a key event in signaling pathways related to cell growth and survival factors, crucially regulating translation initiation and protein synthesis. S6 protein can be phosphorylated on various serine and threonine residues by S6K kinase, and further research shows its interaction with other molecules and proteins, expanding its functional roles in cell signaling. Further exploration of ribosomal protein S6 could unveil new insights into its molecular interactions, roles in cellular physiology and pathophysiology, and potential applications in enhancing plant biomass and yield. This study involved cloning and site-directed mutagenesis of cDNA for the second isoform of AtRPS6 protein (AtRPS6B), followed by expression of natural and phosphomimetic forms of the protein in E. coli ArcticExpress (DE3) cells. The proteins were then purified using metal-chelate affinity chromatography (IMAC), and their presence and degree of purification were confirmed via Western blotting. These findings contribute to the broader research context surrounding ribosomal protein S6. RPS6, a key effector in the TOR signaling pathway, plays a crucial role in regulating ribosome biosynthesis by controlling transcription and translation processes. Phosphorylation of RPS6, indicative of S6K activity, is associated with actively dividing cells. However, the precise role of RPS6 in ribosome biosynthesis regulation remains a subject of ongoing investigation. The data obtained from the study on AtRPS6B expand our understanding of the molecular mechanisms underlying ribosomal biosynthesis regulation. These insights may significantly contribute to our knowledge of the interplay between signaling pathways, ribosomal proteins, cellular metabolism, and offer new perspectives for therapeutic interventions targeting cellular processes associated with ribosomes.

Inorganic Polyphosphate Is in the Surface of Trypanosoma cruzi but Is Not Significantly Secreted.

Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi. PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi. We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-β), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.

The multifaceted role of different SnRK gene family members in regulating multiple abiotic stresses in plants.

Abiotic stresses are a major constraint for agricultural productivity and food security in today's era of climate change. Plants can experience different types of abiotic stresses, either individually or in combination. Sometimes, more than one stress event may occur simultaneously or one after another during the lifecycle of the plant. In general, key survival strategies for stress tolerance may differ from one stress to another. However, at the molecular level, evolutionarily conserved protein kinase SUCROSE NONFERMENTING 1 (SNF1)-related protein kinase (SnRK) gene family members, comprising SnRK1, SnRK2, and SnRK3 gene families, play a key role in different types of stress and adaptive responses. SnRK gene family members can act as master regulators and regulate the central metabolism of plants, which determines the energy distribution in either survival or growth/developmental processes. The key mechanism of SnRK-mediated regulation is associated with the phosphorylation of downstream genes, which either induces or dampens the function of target proteins. This may be crucial for maintaining differential morpho-physiological and biochemical processes in plants, including potassium signalling, ROS homeostasis, sugar signalling, and energy homeostasis. Furthermore, phosphorylation sites associated with different targets were also reviewed, which showed that SnRK-mediated phosphorylation of Serine and Threonine residues of the target protein is a site-specific event, where the target consists of specific amino acid sequences, including RXXS/T, Serine-threonine rich regions, or AMPK/SNF1 types. Here, we review different classes of SnRK gene family members and their multifaceted roles in understanding the commonality of SnRK-mediated responses to multiple abiotic stresses in plants.

Abstract P222: O-GlcNAcylation induces aortic stiffness in aging female rats via CD47-SIRPα -1 signaling

A reduction in aortic compliance serves as an early indicator of incipient vascular disease. While extracellular matrix modifications, such as elastin fragmentation, are the major mediators of aortic stiffness, the mechanisms that underlie these phenotypes remain to be elucidated. O-GlcNAc, the attachment of β-N-acetylglucosamine to serine and threonine residues of proteins, is a highly dynamic and widespread post-translational modification. This modification plays a pivotal role in modulating the function, activity, localization, and stability of target proteins across nuclear, cytoplasmic, and mitochondrial compartments. Primarily involved in intracellular signaling and transcriptional regulation, O-GlcNAc responds to nutrient availability and cell stress. However, chronic O-GlcNAc is consistently linked to vascular impairments. Indeed, our preliminary data has demonstrated that O-GlcNAc is elevated in aorta of Fisher-334 female at 16 months of age (16MO) than 3MO and pharmacological induction of O-GlcNAc (TMG 100 uM 24h) in 3MO rats can cause aortic stiffness via increased elastase activity and elastin fragmentation. However, how this inherently intracellular cellular signaling could impact aorta remodeling is unknown. CD47 (Cluster of Differentiation 47), also known as integrin-associated protein, is a transmembrane protein that acts as a “do not eat me” signal to immune cells. RNAseq data from our lab suggested that TMG (1 uM, 24 h) increased CD47 expression in vascular smooth muscle cells (n =3, p=0.08). Therefore, we hypothesized that CD47 could be an important downstream mediator of O-GlcNAc-induced aortic stiffness. Our findings revealed that CD47 inhibitors, [(RRx 10 uM) and SEN 177 (100 nM)] effectively mitigated the aortic stiffness and elastase activity (Ctrl 2302±38; TMG 4058±328; RRx 1776±121; Sen177 3032±50 A.U.) induced by TMG, suggesting that CD47 is a novel mechanism of O-GlcNAc-induced aortic stiffness. As aging is a major risk factor for aortic stiffness, we also revealed that CD47 was increased in the aorta of 16MO rats compared to 3MO controls (0.1±0.03 vs. 0.3±0.06). Potential ligands for CD47 include thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). We observed that in 16MO rats, circulating SIRPα is increased, but not TSP1, suggesting a receptor-ligand interaction that is applicable in aging. Overall, these data reveal a previously unidentified and significant role for CD47 in O-GlcNAc-induced aortic stiffness.

Phylogenomics and functional analysis of Glycerol-3-Phosphate Acyltransferase (GPAT) Genes: A critical role in lipid biosynthesis.

The tung tree (Vernicia fordii Hemsl.), an economically important woody plant, is widely planted for the production of high-quality tung oil. Glycerol-3-phosphate acyltransferases (GPATs), the rate-limiting enzymes in triacylglycerol synthesis, play an important role in seed oil biosynthesis. In this study, we performed a genome-wide analysis of VfGPATs. A total of 9 VfGPATs were identified from the whole tung genome, and phylogenetic analysis divided the VfGPATs into three major clades: clade II (VfGPAT9), clade III (VfATS1) and clade IV (VfGPAT1 ~ 8). Subcellular localization analysis revealed that five VfGPATs (1, 5, 6, 8, and 9) are localized in the endoplasmic reticulum, and four VfGPATs (3-1, 3-2, 3-3, and ATS1) are localized in the chloroplast. Overexpression of VfGPATs in Arabidopsis thaliana revealed that the oil content in VfGPAT8- and VfGPAT9-transgenic plants were significantly increased by 26.60 and 55.94% compared to the wild-type. Transient expression of VfGPAT8 + VfFADX and VfGPAT9 + VfFADX could promote the synthesis of α-eleostearic acid and enhance the accumulation of lipid droplets in tobacco (Nicotiana benthamiana) leaves. We further tested the enzymatic activities of VfGPAT8 and VfGPAT9 with the yeast double mutant strain ZAFU1. The results showed that VfGPAT8 complemented the phosphatidate biosynthetic defect in the double mutant, while VfGPAT9 could not, suggesting that VfGPAT8 has a high acetyltransferase activity. However, altering serine (S) residue at position 113 of VfGPAT9 to threonine (T) could restore its enzymatic activity. This study provided important insights into the evolutionary history of VfGPATs and will promote the genetic improvement of tung trees and related species.

Fatiguing exercise reduces cellular passive Young's modulus in human vastus lateralis muscle.

Previous studies demonstrated that acute fatiguing exercise transiently reduces whole-muscle stiffness, which might contribute to increased risk of injury and impaired contractile performance. We sought to elucidate potential intracellular mechanisms underlying these reductions. To that end, the cellular passive Young's modulus was measured in muscle fibres from healthy, young males and females. Eight volunteers (four male and four female) completed unilateral, repeated maximal voluntary knee extensions until task failure, immediately followed by bilateral percutaneous needle muscle biopsy of the post-fatigued followed by the non-fatigued control vastus lateralis. Muscle samples were processed for mechanical assessment and separately for imaging and phosphoproteomics. Fibres were passively (pCa 8.0) stretched incrementally to 156% of initial sarcomere length to assess Young's modulus, calculated as the slope of the resulting stress-strain curve at short (sarcomere length=2.4-3.0µm) and long (sarcomere length=3.2-3.8µm) lengths. Titin phosphorylation was assessed by liquid chromatography followed by high-resolution mass spectrometry. The passive modulus was significantly reduced in post-fatigued versus control fibres from male, but not female, participants. Post-fatigued samples showed altered phosphorylation of five serine residues (four located within the elastic region of titin) but did not exhibit altered active tension or sarcomere ultrastructure. Collectively, these results suggest that acute fatigue is sufficient to alter phosphorylation of skeletal titin in multiple locations. We also found reductions in the passive modulus, consistent with prior reports in the literature investigating striated muscle stiffness. These results provide mechanistic insight contributing to the understanding of dynamic regulation of whole-muscle tissue mechanics in vivo. HIGHLIGHTS: What is the central question of this study? Previous studies have shown that skeletal muscle stiffness is reduced following a single bout of fatiguing exercise in whole muscle, but it is not known whether these changes manifest at the cellular level, and their potential mechanisms remain unexplored. What is the main finding and its importance? Fatiguing exercise reducescellular stiffnessin skeletal muscle from males but not females, suggesting thatfatigue alters tissue compliance in asex-dependent manner. The phosphorylation status of titin, a potential mediator of skeletal muscle cellular stiffness, is modified by fatiguing exercise. Previous studies have shown that passive skeletal muscle stiffness is reduced following a single bout of fatiguing exercise. Lower muscle passive stiffness following fatiguing exercise might increase risk for soft-tissue injury; however, the underlying mechanisms of this change are unclear. Our findings show that fatiguing exercise reduces the passive Young's modulus in skeletal muscle cells from males but not females, suggesting that intracellular proteins contribute to reduced muscle stiffness following repeated loading to task failure in a sex-dependent manner. The phosphorylation status of the intracellular protein titin is modified by fatiguing exercise in a way that might contribute to altered muscle stiffness after fatiguing exercise. These results provide important mechanistic insight that might help to explain why biological sex impacts the risk for soft-tissue injury with repeated or high-intensity mechanical loading in athletes and the risk of falls in older adults.

The effect of phosphorylation efficiency on the oncogenic properties of the protein E7 from high-risk HPV

The Human papillomavirus (HPV) causes tumors in part by hijacking the host cell cycle and forcing uncontrolled cellular division. While there are >200 genotypes of HPV, 15 are classified as high-risk and have been shown to transform infected cells and contribute to tumor formation. The remaining low-risk genotypes are not considered oncogenic and result in benign skin lesions. In high-risk HPV, the oncoprotein E7 contributes to the dysregulation of cell cycle regulatory mechanisms. High-risk E7 is phosphorylated in cells at two conserved serine residues by Casein Kinase 2 (CK2) and this phosphorylation event increases binding affinity for cellular proteins such as the tumor suppressor retinoblastoma (pRb). While low-risk E7 possesses similar serine residues, it is phosphorylated to a lesser degree in cells and has decreased binding capabilities. When E7 binding affinity is decreased, it is less able to facilitate complex interactions between proteins and therefore has less capability to dysregulate the cell cycle. By comparing E7 protein sequences from both low- and high-risk HPV variants and using site-directed mutagenesis combined with NMR spectroscopy and cell-based assays, we demonstrate that the presence of two key nonpolar valine residues within the CK2 recognition sequence, present in low-risk E7, reduces serine phosphorylation efficiency relative to high-risk E7. This results in significant loss of the ability of E7 to degrade the retinoblastoma tumor suppressor protein, thus also reducing the ability of E7 to increase cellular proliferation and reduce senescence. This provides additional insight into the differential E7-mediated outcomes when cells are infected with high-risk verses low-risk HPV. Understanding these oncogenic differences may be important to developing targeted treatment options for HPV-induced cancers.

Phosphorylation patterns in the AT1R C-terminal tail specify distinct downstream signaling pathways.

Different ligands stabilize specific conformations of the angiotensin II type 1 receptor (AT1R) that direct distinct signaling cascades mediated by heterotrimeric G proteins or β-arrestin. These different active conformations are thought to engage distinct intracellular transducers because of differential phosphorylation patterns in the receptor C-terminal tail (the "barcode" hypothesis). Here, we identified the AT1R barcodes for the endogenous agonist AngII, which stimulates both G protein activation and β-arrestin recruitment, and for a synthetic biased agonist that only stimulates β-arrestin recruitment. The endogenous and β-arrestin-biased agonists induced two different ensembles of phosphorylation sites along the C-terminal tail. The phosphorylation of eight serine and threonine residues in the proximal and middle portions of the tail was required for full β-arrestin functionality, whereas phosphorylation of the serine and threonine residues in the distal portion of the tail had little influence on β-arrestin function. Similarly, molecular dynamics simulations showed that the proximal and middle clusters of phosphorylated residues were critical for stable β-arrestin-receptor interactions. These findings demonstrate that ligands that stabilize different receptor conformations induce different phosphorylation clusters in the C-terminal tail as barcodes to evoke distinct receptor-transducer engagement, receptor trafficking, and signaling.