A refactored biosynthetic pathway for the production of glycosylated microbial sunscreens.

Mycosporine-like amino acids (MAAs) are a family of water-soluble and colorless secondary metabolites, with high extinction coefficients, that function as microbial sunscreens. MAAs share a cyclohexinimine chromophore that is diversified through amino acid substitutions and attachment of sugar moieties. The genetic and enzymatic bases for the chemical diversity of MAAs remain largely unexplored. Here we report a series of structurally distinct MAAs and evidence for an unusual branched biosynthetic pathway from a cyanobacterium isolated from lake sediment. We used a combination of high-resolution liquid chromatography-mass spectrometry (HR-LCMS) analysis and nuclear magnetic resonance (NMR) spectroscopy to identify diglycosylated-palythine-Ser (C22H36N2O15) as the dominant chemical variant in a series of MAAs from Nostoc sp. UHCC 0302 that contained either Ser or Thr. We obtained a complete 9.9 Mb genome sequence to gain insights into the genetic basis for the biosynthesis of these structurally distinct MAAs. We identified MAA biosynthetic genes encoded at two locations on the circular chromosome. Surprisingly, direct pathway cloning and heterologous expression of the complete mysABCJ 1 D 1 G 1 H biosynthetic gene cluster in Escherichia coli (E. coli) led to the production of 450 Da monoglycosylated-palythine-Thr (C18H30N2O11). We reconstructed combinations of the two distant biosynthetic gene clusters in refactored synthetic pathways and expressed them in the heterologous host. These results demonstrated that the MysD1 and MysD2 enzymes displayed a preference for Thr and Ser, respectively. Furthermore, one of the four glycosyltransferases identified, MysG1, was active in E. coli and catalysed the attachment of a hexose moiety to the palythine-Thr intermediate. Together these results provide the first insights into the enzymatic basis for glycosylation of MAAs and demonstrates how paralogous copies of the MysD enzymes allow the simultaneous biosynthesis of specific chemical variants to increase the structural variation in this family of microbial sunscreens.

Serine synthesis pathway enzyme PHGDH is critical for muscle cell biomass, anabolic metabolism, and mTORC1 signaling.

Cells use glycolytic intermediates for anabolism, e.g., via the serine synthesis and pentose phosphate pathways. However, we still understand poorly how these metabolic pathways contribute to skeletal muscle cell biomass generation. The first aim of this study was therefore to identify enzymes that limit protein synthesis, myotube size, and proliferation in skeletal muscle cells. We inhibited key enzymes of glycolysis, the pentose phosphate pathway, and the serine synthesis pathway to evaluate their importance in C2C12 myotube protein synthesis. Based on the results of this first screen, we then focused on the serine synthesis pathway enzyme phosphoglycerate dehydrogenase (PHGDH). We used two different PHGDH inhibitors and mouse C2C12 and human primary muscle cells to study the importance and function of PHGDH. Both myoblasts and myotubes incorporated glucose-derived carbon into proteins, RNA, and lipids, and we showed that PHGDH is essential in these processes. PHGDH inhibition decreased protein synthesis, myotube size, and myoblast proliferation without cytotoxic effects. The decreased protein synthesis in response to PHGDH inhibition appears to occur mainly mechanistic target of rapamycin complex 1 (mTORC1)-dependently, as was evident from experiments with insulin-like growth factor 1 and rapamycin. Further metabolomics analyses revealed that PHGDH inhibition accelerated glycolysis and altered amino acid, nucleotide, and lipid metabolism. Finally, we found that supplementing an antioxidant and redox modulator, N-acetylcysteine, partially rescued the decreased protein synthesis and mTORC1 signaling during PHGDH inhibition. The data suggest that PHGDH activity is critical for skeletal muscle cell biomass generation from glucose and that it regulates protein synthesis and mTORC1 signaling.NEW & NOTEWORTHY The use of glycolytic intermediates for anabolism was demonstrated in both myoblasts and myotubes, which incorporate glucose-derived carbon into proteins, RNA, and lipids. We identify phosphoglycerate dehydrogenase (PHGDH) as a critical enzyme in those processes and also for muscle cell hypertrophy, proliferation, protein synthesis, and mTORC1 signaling. Our results thus suggest that PHGDH in skeletal muscle is more than just a serine-synthesizing enzyme.

Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM.

S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called "last-resort" drugs. Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant S. aureus is high. M23B family peptidoglycan hydrolases, enzymes that can kill S. aureus by cleaving glycine-glycine peptide bonds in S. aureus cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. We report here the near complete 1H/13C/15N resonance assignment of the catalytic domain of LytM, residues 185-316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall.

Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11.

L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg-1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL-1 min-1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.

1H, 13C, and 15N NMR chemical shift assignment of LytM N-terminal domain (residues 26-184).

Antibiotic resistance is a growing problem and a global threat for modern healthcare. New approaches complementing the traditional antibiotic drugs are urgently needed to secure the ability to treat bacterial infections also in the future. Among the promising alternatives are bacteriolytic enzymes, such as the cell wall degrading peptidoglycan hydrolases. Staphylococcus aureus LytM, a Zn2+-dependent glycyl-glycine endopeptidase of the M23 family, is one of the peptidoglycan hydrolases. It has a specificity towards staphylococcal peptidoglycan, making it an interesting target for antimicrobial studies. LytM hydrolyses the cell wall of S. aureus, a common pathogen with multi-resistant strains that are difficult to treat, such as the methicillin-resistant S. aureus, MRSA. Here we report the 1H, 15N and 13C chemical shift assignments of S. aureus LytM N-terminal domain and linker region, residues 26-184. These resonance assignments can provide the basis for further studies such as elucidation of structure and interactions.

A Plastic Biosynthetic Pathway for the Production of Structurally Distinct Microbial Sunscreens.

Mycosporine-like amino acids (MAAs) are small, colorless, and water-soluble secondary metabolites. They have high molar extinction coefficients and a unique UV radiation absorption mechanism that make them effective sunscreens. Here we report the discovery of two structurally distinct MAAs from the lichen symbiont strain Nostoc sp. UHCC 0926. We identified these MAAs as aplysiapalythine E (C23H38N2O15) and tricore B (C34H53N4O15) using a combination of high-resolution liquid chromatography-mass spectrometry (HR-LCMS) analysis and nuclear magnetic resonance (NMR) spectroscopy. We obtained a 8.3 Mb complete genome sequence of Nostoc sp. UHCC 0926 to gain insights into the genetic basis for the biosynthesis of these two structural distinct MAAs. We identified MAA biosynthetic genes encoded in three separate locations of the genome. The organization of biosynthetic enzymes in Nostoc sp. UHCC 0926 necessitates a branched biosynthetic pathway to produce two structurally distinct MAAs. We detected the presence of such discontiguous MAA biosynthetic gene clusters in 12% of the publicly available complete cyanobacterial genomes. Bioinformatic analysis of public MAA biosynthetic gene clusters suggests that they are subject to rapid evolutionary processes resulting in highly plastic biosynthetic pathways that are responsible for the chemical diversity in this family of microbial sunscreens.

Aerobic exercise training and gut microbiome-associated metabolic shifts in women with overweight: a multi-omic study.

Physical activity is essential in weight management, improves overall health, and mitigates obesity-related risk markers. Besides inducing changes in systemic metabolism, habitual exercise may improve gut's microbial diversity and increase the abundance of beneficial taxa in a correlated fashion. Since there is a lack of integrative omics studies on exercise and overweight populations, we studied the metabolomes and gut microbiota associated with programmed exercise in obese individuals. We measured the serum and fecal metabolites of 17 adult women with overweight during a 6-week endurance exercise program. Further, we integrated the exercise-responsive metabolites with variations in the gut microbiome and cardiorespiratory parameters. We found clear correlation with several serum and fecal metabolites, and metabolic pathways, during the exercise period in comparison to the control period, indicating increased lipid oxidation and oxidative stress. Especially, exercise caused co-occurring increase in levels of serum lyso-phosphatidylcholine moieties and fecal glycerophosphocholine. This signature was associated with several microbial metagenome pathways and the abundance of Akkermansia. The study demonstrates that, in the absence of body composition changes, aerobic exercise can induce metabolic shifts that provide substrates for beneficial gut microbiota in overweight individuals.

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 µM to >7.2 µM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite.

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp α-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.

Branched-Chain Amino Acid Deprivation Decreases Lipid Oxidation and Lipogenesis in C2C12 Myotubes.

Impaired lipid metabolism is a common risk factor underlying several metabolic diseases such as metabolic syndrome and type 2 diabetes. Branched-chain amino acids (BCAAs) that include valine, leucine and isoleucine have been proven to share a role in lipid metabolism and hence in maintaining metabolic health. We have previously introduced a hypothesis suggesting that BCAA degradation mechanistically connects to lipid oxidation and storage in skeletal muscle. To test our hypothesis, the present study examined the effects of BCAA deprivation and supplementation on lipid oxidation, lipogenesis and lipid droplet characteristics in murine C2C12 myotubes. In addition, the role of myotube contractions on cell metabolism was studied by utilizing in vitro skeletal-muscle-specific exercise-like electrical pulse stimulation (EPS). Our results showed that the deprivation of BCAAs decreased both lipid oxidation and lipogenesis in C2C12 myotubes. BCAA deprivation further diminished the number of lipid droplets in the EPS-treated myotubes. EPS decreased lipid oxidation especially when combined with high BCAA supplementation. Similar to BCAA deprivation, high BCAA supplementation also decreased lipid oxidation. The present results highlight the role of an adequate level of BCAAs in healthy lipid metabolism.

Structure of SNX9 SH3 in complex with a viral ligand reveals the molecular basis of its unique specificity for alanine-containing class I SH3 motifs.

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]xØPxxP, the hydrophobic residue Ø being proline or leucine. We have studied the unusual Ø = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.

Interaction mechanism of endogenous PP2A inhibitor protein ENSA with PP2A.

The vast diversity of protein phosphatase 2A (PP2A) holoenzyme composition ensures its multifaceted role in the regulation of cellular growth and signal transduction. In several pathological conditions, such as cancer, PP2A is inhibited by endogenous inhibitor proteins. Several PP2A inhibitor proteins have been identified, one of which is α-endosulfine (ENSA). ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. The role of ENSA in PP2A inhibition is rather well characterized, but knowledge of the mechanism of inhibition is scarce. In this study, we have performed comprehensive structural characterization of ENSA, and its interaction with PP2A A- and various B56-subunit isoforms by combining NMR spectroscopy, small-angle X-ray scattering (SAXS) and interaction assays. The results clearly indicate that ENSA is an intrinsically disordered protein containing three transient α-helical structures. ENSA was observed to interact PP2A mainly via A-subunit, as the affinity with the A-subunit is significantly stronger than with any of the B56 subunits. Based on our results, it seems that ENSA follows the dock-and-coalesce mechanism in associating with PP2A A-subunit. Taken together, our results provide an essential structural and molecular framework to understanding molecular bases of ENSA-mediated PP2A inhibition, which is crucial for the development of new therapies for diseases linked to PP2A inhibition.

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products.

Serine proteases regulate many physiological processes and play a key role in a variety of cancers. Aeruginosins are a family of natural products produced by cyanobacteria that exhibit pronounced structural diversity and potent serine protease inhibition. Here, we sequenced the complete genome of Nodularia sphaerocarpa UHCC 0038 and identified the 43.7 kb suomilide biosynthetic gene cluster. Bioinformatic analysis demonstrated that suomilide belongs to the aeruginosin family of natural products. We identified 103 complete aeruginosin biosynthetic gene clusters from 12 cyanobacterial genera and showed that they encode an unexpected chemical diversity. Surprisingly, purified suomilide inhibited human trypsin-2 and -3, with IC50 values of 4.7 and 11.5 nM, respectively, while trypsin-1 was inhibited with an IC50 of 104 nM. Molecular dynamics simulations suggested that suomilide has a long residence time when bound to trypsins. This was confirmed experimentally for trypsin-1 and -3 (residence times of 1.5 and 57 min, respectively). Suomilide also inhibited the invasion of aggressive and metastatic PC-3M prostate cancer cells without affecting cell proliferation. The potent inhibition of trypsin-3, together with a long residence time and the ability to inhibit prostate cancer cell invasion, makes suomilide an attractive drug lead for targeting cancers that overexpress trypsin-3. These results substantially broaden the genetic and chemical diversity of the aeruginosin family and suggest that aeruginosins may be a source of selective inhibitors of human serine proteases.

Enteroviruses and coronaviruses: similarities and therapeutic targets.

Introduction: Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes.Areas covered: This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral proteases have been evaluated.Expert opinion: The main protease of coronaviruses and enteroviruses share similarities in their structure and function. These proteases process their viral polyproteins and thus drugs that bind to the active site have potential to target both virus groups. It is important to develop drugs that target more evolutionarily conserved processes and proteins. Moreover, it is a wise strategy to concentrate on processes that are similar between several virus families.

The structure and biosynthesis of heinamides A1-A3 and B1-B5, antifungal members of the laxaphycin lipopeptide family.

Laxaphycins are a family of cyclic lipopeptides with synergistic antifungal and antiproliferative activities. They are produced by multiple cyanobacterial genera and comprise two sets of structurally unrelated 11- and 12-residue macrocyclic lipopeptides. Here, we report the discovery of new antifungal laxaphycins from Nostoc sp. UHCC 0702, which we name heinamides, through antimicrobial bioactivity screening. We characterized the chemical structures of eight heinamide structural variants A1-A3 and B1-B5. These variants contain the rare non-proteinogenic amino acids 3-hydroxy-4-methylproline, 4-hydroxyproline, 3-hydroxy-d-leucine, dehydrobutyrine, 5-hydroxyl ß-amino octanoic acid, and O-carbamoyl-homoserine. We obtained an 8.6-Mb complete genome sequence from Nostoc sp. UHCC 0702 and identified the 93 kb heinamide biosynthetic gene cluster. The structurally distinct heinamides A1-A3 and B1-B5 variants are synthesized using an unusual branching biosynthetic pathway. The heinamide biosynthetic pathway also encodes several enzymes that supply non-proteinogenic amino acids to the heinamide synthetase. Through heterologous expression, we showed that (2S,4R)-4-hydroxy-l-proline is supplied through the action of a novel enzyme LxaN, which hydroxylates l-proline. 11- and 12-residue heinamides have the characteristic synergistic activity of laxaphycins against Aspergillus flavus FBCC 2467. Structural and genetic information of heinamides may prove useful in future discovery of natural products and drug development.

Higher glucose availability augments the metabolic responses of the C2C12 myotubes to exercise-like electrical pulse stimulation.

The application of exercise-like electrical pulse simulation (EL-EPS) has become a widely used exercise mimetic in vitro. EL-EPS produces similar physiological responses as in vivo exercise, while less is known about the detailed metabolic effects. Routinely, the C2C12 myotubes are cultured in high-glucose medium (4.5 g/L), which may alter EL-EPS responses. In this study, we evaluate the metabolic effects of EL-EPS under the high- and low-glucose (1.0 g/L) conditions to understand how substrate availability affects the myotube response to EL-EPS. The C2C12 myotube, media, and cell-free media metabolites were analyzed using untargeted nuclear magnetic resonance (NMR)-based metabolomics. Furthermore, translational and metabolic changes and possible exerkine effects were analyzed. EL-EPS enhanced substrate utilization as well as production and secretion of lactate, acetate, 3-hydroxybutyrate, and branched-chain fatty acids (BCFAs). The increase in BCFAs correlated with branched-chain amino acids (BCAAs) and BCFAs were strongly decreased when myotubes were cultured without BCAAs suggesting the action of acyl-CoA thioesterases on BCAA catabolites. Notably, not all EL-EPS responses were augmented by high glucose because EL-EPS increased phosphorylated c-Jun N-terminal kinase and interleukin-6 secretion independent of glucose availability. Administration of acetate and EL-EPS conditioned media on HepG2 hepatocytes had no adverse effects on lipolysis or triacylglycerol content. Our results demonstrate that unlike in cell-free media, the C2C12 myotube and media metabolites were affected by EL-EPS, particularly under high-glucose condition suggesting that media composition should be considered in future EL-EPS studies. Furthermore, acetate and BCFAs were identified as putative exerkines warranting more research.NEW & NOTEWORTHY The present study examined for the first time the metabolome of 1) C2C12 myotubes, 2) their growth media, and 3) cell-free media after exercise-like electrical pulse stimulation under distinct nutritional loads. We report that myotubes grown under high-glucose conditions had greater responsiveness to EL-EPS when compared with lower glucose availability conditions and increased media content of acetate and branched-chain fatty acids suggests they might act as putative exerkines warranting further research.

Decreased temperature increases the expression of a disordered bacterial late embryogenesis abundant (LEA) protein that enhances natural transformation.

Late embryogenesis abundant (LEA) proteins are important players in the management of responses to stressful conditions, such as drought, high salinity, and changes in temperature. Many LEA proteins do not have defined three-dimensional structures, so they are intrinsically disordered proteins (IDPs) and are often highly hydrophilic. Although LEA-like sequences have been identified in bacterial genomes, the functions of bacterial LEA proteins have been studied only recently. Sequence analysis of outer membrane interleukin receptor I (BilRI) from the oral pathogen Aggregatibacter actinomycetemcomitans indicated that it shared sequence similarity with group 3/3b/4 LEA proteins. Comprehensive nuclearcgq magnetic resonance (NMR) studies confirmed its IDP nature, and expression studies in A. actinomycetemcomitans harboring a red fluorescence reporter protein-encoding gene revealed that bilRI promoter expression was increased at decreased temperatures. The amino acid backbone of BilRI did not stimulate either the production of reactive oxygen species from human leukocytes or the production of interleukin-6 from human macrophages. Moreover, BilRI-specific IgG antibodies could not be detected in the sera of A. actinomycetemcomitans culture-positive periodontitis patients. Since the bilRI gene is located near genes involved in natural competence (i.e., genes associated with the uptake of extracellular (eDNA) and its incorporation into the genome), we also investigated the role of BilRI in these events. Compared to wild-type cells, the ΔbilRI mutants showed a lower transformation efficiency, which indicates either a direct or indirect role in natural competence. In conclusion, A. actinomycetemcomitans might express BilRI, especially outside the host, to survive under stressful conditions and improve its transmission potential.

The Interaction Mechanism of Intrinsically Disordered PP2A Inhibitor Proteins ARPP-16 and ARPP-19 With PP2A.

Protein phosphatase 2A (PP2A) activity is critical for maintaining normal physiological cellular functions. PP2A is inhibited by endogenous inhibitor proteins in several pathological conditions including cancer. A PP2A inhibitor protein, ARPP-19, has recently been connected to several human cancer types. Accordingly, the knowledge about ARPP-19-PP2A inhibition mechanism is crucial for the understanding the disease development and the therapeutic targeting of ARPP-19-PP2A. Here, we show the first structural characterization of ARPP-19, and its splice variant ARPP-16 using NMR spectroscopy, and SAXS. The results reveal that both ARPP proteins are intrinsically disordered but contain transient secondary structure elements. The interaction mechanism of ARPP-16/19 with PP2A was investigated using microscale thermophoresis and NMR spectroscopy. Our results suggest that ARPP-PP2A A-subunit interaction is mediated by linear motif and has modest affinity whereas, the interaction of ARPPs with B56-subunit is weak and transient. Like many IDPs, ARPPs are promiscuous binders that transiently interact with PP2A A- and B56 subunits using multiple interaction motifs. In summary, our results provide a good starting point for future studies and development of therapeutics that block ARPP-PP2A interactions.