Update on the Roles of Rice MAPK Cascades

The mitogen-activated protein kinase (MAPK) cascades have been validated playing critical roles in diverse aspects of plant biology, from growth and developmental regulation, biotic and abiotic stress responses, to phytohormone signal transduction or responses. A classical MAPK cascade consists of a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK), and a MAPK. From the 75 MAPKKKs, eight MAPKKs, and 15 MAPKs of rice, a number of them have been functionally deciphered. Here, we update recent advances in knowledge of the roles of rice MAPK cascades, including their components and complicated action modes, their diversified functions controlling rice growth and developmental responses, coordinating resistance to biotic and abiotic stress, and conducting phytohormone signal transduction. Moreover, we summarize several complete MAPK cascades that harbor OsMAPKKK-OsMAPKK-OsMAPK, their interaction with different upstream components and their phosphorylation of diverse downstream substrates to fulfill their multiple roles. Furthermore, we state a comparison of networks of rice MAPK cascades from signal transduction crosstalk to the precise selection of downstream substrates. Additionally, we discuss putative concerns for elucidating the underlying molecular mechanisms and molecular functions of rice MAPK cascades in the future.     

Rethinking the Citric Acid Cycle: Connecting Pyruvate Carboxylase and Citrate Synthase to the Flow of Energy and Material

In 1937, Sir H. A Krebs first published the Citric Acid Cycle, a unidirectional cycle with carboxylic acids. The original concept of the Citric Acid Cycle from Krebs’ 1953 Nobel Prize lecture illustrates the unidirectional degradation of lactic acid to water, carbon dioxide and hydrogen. Here, we add the heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex, connecting the original Citric Acid Cycle to the flow of energy and material. The heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex catalyses the first reaction of the Citric Acid Cycle, the oxidation of lactate to pyruvate, and thus secures the provision of pyruvic acid. In addition, we modify Krebs’ original concept by feeding the cycle with oxaloacetic acid. Our concept enables the integration of anabolic processes and allows adaption of the organism to recover ATP faster.     

Additive-Free Enzymatic Phosphorylation and Ligation of Artificial Oligonucleotides with C-Nucleosides at the Reaction Points

We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D * , T * , G * , and C * , corresponding—from a viewpoint of hydrogen-bonding patterns—to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5′-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5′-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3′-natural ⋅ 5′-artificial, 3′-artificial ⋅ 5′-natural, and 3′-artificial ⋅ 5′-artificial terminal combination at the nicks.     

Prebiotic Pathway from Ribose to RNA Formation

At the focus of abiotic chemical reactions is the synthesis of ribose. No satisfactory explanation was provided as to the missing link between the prebiotic synthesis of ribose and prebiotic RNA (preRNA). Hydrogen cyanide (HCN) is assumed to have been the principal precursor in the prebiotic formation of aldopentoses in the formose reaction and in the synthesis of ribose. Ribose as the best fitting aldopentose became the exclusive sugar component of RNA. The elevated yield of ribose synthesis at higher temperatures and its protection from decomposition could have driven the polymerization of the ribose-phosphate backbone and the coupling of nucleobases to the backbone. RNA could have come into being without the involvement of nucleotide precursors. The first nucleoside monophosphate is likely to have appeared upon the hydrolysis of preRNA contributed by the presence of reactive 2′-OH moieties in the preRNA chain. As a result of phosphorylation, nucleoside monophosphates became nucleoside triphosphates, substrates for the selective synthesis of genRNA.     

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway—isopentenyl phosphate kinase from Thermoplasma acidophilum—towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide unnatural pyrophosphates and provide a critical first step in characterizing the rate-limiting steps in the artificial ADH pathway.     

Analysis of Site-Specific Phosphorylation of PTEN by Using Enzyme-Catalyzed Expressed Protein Ligation

The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C terminus. Prior enzymatic characterization of the four monophosphorylated (1p) PTENs by using classical expressed protein ligation (EPL) was complicated by the inclusion of a non-native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here, we apply subtiligase-mediated EPL to create wt 1p-PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with the role of Tyr379 in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p-PTENs, which are poorly recognized by an anti-phospho-PTEN antibody, three of the four wt 1p-PTENs are recognized by a commonly used anti-phospho-PTEN antibody.     

Piperine Targets Different Drug Resistance Mechanisms in Human Ovarian Cancer Cell Lines Leading to Increased Sensitivity to Cytotoxic Drugs

Our goal was to examine the anticancer effects of piperine against the resistant human ovarian cancer cells and to explore the molecular mechanisms responsible for its anticancer effects. Our study used drug-sensitive ovarian cancer cell line W1 and its sublines resistant to paclitaxel (PAC) and topotecan (TOP). We analyzed the cytotoxic effect of piperine and cytostatic drugs using an MTT assay. The impact of piperine on protein expression was determined by immunofluorescence and Western blot. We also examined its effect on cell proliferation and migration. We noticed a different level of piperine resistance between cell lines. Piperine increases the cytotoxic effect of PAC and TOP in drug-resistant cells. We observed an increase in PTPRK expression correlated with decreased pTYR level after piperine treatment and downregulation of P-gp and BCRP expression. We also noted a decrease in COL3A1 and TGFBI expression in investigated cell lines and increased COL3A1 expression in media from W1PR2 cells. The expression of Ki67 protein and cell proliferation rate decreased after piperine treatment. Piperine markedly inhibited W1TR cell migration. Piperine can be considered a potential anticancer agent that can increase chemotherapy effectiveness in cancer patients.     

Regulatory role of phosphoproteins in the development of bovine small intestine during early life

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1–2 and 10–12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1–2, 10–12, and 22–24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.