Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Bovine mRNAs in Cow's Milk Exosomes Are Bioavailable and Translated into Peptides in Non-bovine Systems (P06-030-19)

Objectives Exosomes are natural nanoparticles that participate in cell-to-cell communication by transferring regulatory molecules such as microRNAs from donor cells to recipient cells. Previously, we have demonstrated that exosomes and microRNAs are not exclusively obtained from endogenous synthesis but may also be absorbed from milk. We assessed whether 1) exosomes contain mRNAs (i.e., RNAs other than microRNAs), 2) mRNAs are bioavailable in mice, and 3) bovine mRNAs are translated into peptides. Methods For mRNA analysis, exosomes were isolated from bovine milk using differential ultracentrifugation, and RNA cargos were analyzed by RNA-sequencing. For bioavailability analysis, a synthetic fragment of fluorophore (IRDye)-labeled bovine CSN3 mRNA was transfected into bovine milk exosomes, which were administered to Balb/c mice by oral gavage. Tissues distribution of CSN3 mRNA was assessed 24 h after gavage by using a LiCor Odyssey CLx imager. For analysis of mRNA translation, mRNA from bovine milk exosomes were translated using the rabbit reticulocyte lysate system and BODIPY-labeled lysine. Peptides were separated by two-dimensional gel electrophoresis and fluorescence was visualized using a Typhoon FLA 7000 scanner. Statistical analysis, NA. Results We detected > 3600 bovine mRNAs in exosomes. Most mRNAs were truncated; 107 mRNAs contained their natural ATG start codons. Thirteen of these mRNAs, including CSN3, encoded bovine proteins and peptides with amino acid sequences distinct from those in human and murine orthologs (Table 1). Mice absorbed CSN3 mRNA encapsulated in milk exosomes, which accumulated primarily in the liver (Fig. 1). Nine bovine peptide spots were detected by two-dimensional electrophoresis (Fig. 2). Conclusions Bovine milk exosomes contain mRNAs which are bioavailable and translated into peptides in non-bovine systems. We speculate that food mRNAs might play a role in food allergies and immune tolerance. Future studies: Identification of peptides by LC/MS-MS is in progress. We will assess the relevance of mRNA translation for allergies and tolerance in animal models. Funding Sources NIFA, NIH, Gates Foundation, PureTech, Inc. and USDA Hatch and Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs

Abstract 957: A Novel Interaction of Cardiac Troponin T with PKA Regulatory Subunits I and II

Increasing evidence suggests that cardiac myofilament activity is modulated by posttransla-tional modifications of cardiac troponin (cTn). In an attempt to identify novel proteins that associate with and modify cTn, we used the full-length human cardiac TnT as bait in the yeast two-hybrid system, and a human heart cDNA library as prey. Our screen of > 2×10 6 cDNA clones with hcTnT as bait, identified >1000 clones that grew on high-stringency plates, and 210 that turned the cells blue in the presence of α-gal, indicating positive interactions with hcTnT. DNA sequencing of the 210 clones led to the identification of 10 gene products: cTnI (identified in 180 clones), PKA-RIα, MLC2v, Dystrophin, PDZ and LIM domain 5, CGI-94, Muscle Creatine Kinase, ANF, Mitofusin-2 and an uncharacterized gene. HA-tagged clones (above) and myc-tagged cTnT were individually expressed using a rabbit reticulocyte lysate system and immunoprecipitated (IP) by agarose conjugated anti-myc and anti-HA antibodies. Therefore, the interaction of these proteins with cTnT was verified in a mammalian cellular milieu. Our finding suggests that cTnT, through its interaction with PKA regulatory domains (R), anchors PKA to the myofilament in close proximity to cTnI. This would explain the rapid, PKA dependent, phosphorylation of cTnI during increase intracellular cAMP levels following beta-adrenergic stimulation. While, PKA type I holoenzyme is predominantly cytoplasmic, the majority of PKA type II associates with cytoskeletal structures. Binding of PKA-RI and PKA-RII to intact and truncated forms of cTnT (amino acids 1–117; 118 –288; 1–180; 181–288) were determined by yeast two-hybrid and by IP assays. Our results demonstrate, for the first time, that both PKA-RI and RII associate with cTnT, and PKA-RI binds to a specific region of cTnT-1–180 fragment, whereas PKA-RII binds to a region of cTnT-181–288 fragment. PKA-dependent phosphorylation of cTnI-Ser 23/24 has profound effects on cardiac dynamics: modulating cross-bridge kinetics, energy consumption and calcium sensitivity. Therefore, it is important to have a mechanistic understanding of events that mediate and affect PKA-dependent cTnI phosphorylation.

A Homodimeric Sporamin-Type Trypsin Inhibitor with Antiproliferative, HIV Reverse Transcriptase-Inhibitory and Antifungal Activities from Wampee (Clausena lansium) Seeds

A homodimeric trypsin inhibitor with a molecular mass of 54 kDa was isolated from the seeds of Clausena lansium (Lour) Skeels with a very simple procedure comprising extraction with an aqueous buffer and ion exchange chromatography on CM-cellulose. It inhibited trypsin with an IC50 of 2.2 nM but was without any inhibitory effect on chymotrypsin and proteinase K. The uptake of MTT by human leukemia HL60 and hepatoma Hep G2 cells was inhibited with an IC50 of 100 uM. Translation in the cellfree rabbit reticulocyte lysate system was inhibited with an IC50 of 3.6 uM. The activity of HIV-1 reverse transcriptase was reduced in the presence of the trypsin inhibitor. The trypsin inhibitor exerted antifungal activity toward Physalospora piricola but not Mycosphaerella arachidicola, Botrytis cinerea, Fusarium oxysporum or Coprinus comatus.

Agonistic and antagonistic properties of progesterone metabolites at the human mineralocorticoid receptor

OBJECTIVE: Progesterone binds to the human mineralocorticoid receptor (hMR) with nearly the same affinity as do aldosterone and cortisol, but confers only low agonistic activity. It is still unclear how aldosterone can act as a mineralocorticoid in situations with high progesterone concentrations, e.g. pregnancy. One mechanism could be conversion of progesterone to inactive compounds in hMR target tissues. DESIGN: We analyzed the agonist and antagonist activities of 16 progesterone metabolites by their binding characteristics for hMR as well as functional studies assessing transactivation. METHODS: We studied binding affinity using hMR expressed in a T7-coupled rabbit reticulocyte lysate system. We used co-transfection of an hMR expression vector together with a luciferase reporter gene in CV-1 cells to investigate agonistic and antagonistic properties. RESULTS: Progesterone and 11beta-OH-progesterone (11beta-OH-P) showed a slightly higher binding affinity than cortisol, deoxycorticosterone and aldosterone. 20alpha-dihydro(DH)-P, 5alpha-DH-P and 17alpha-OH-P had a 3- to 10-fold lower binding potency. All other progesterone metabolites showed a weak affinity for hMR. 20alpha-DH-P exhibited the strongest agonistic potency among the metabolites tested, reaching 11.5% of aldosterone transactivation. The agonistic activity of 11beta-OH-P, 11alpha-OH-P and 17alpha-OH-P was 9, 5.1 and 4.1% respectively. At a concentration of 100 nmol/l, progesterone, 17alpha-OH-P and 20alpha-DH-P inhibit nearly 75, 40 and 35% of the transactivation by aldosterone respectively. All other progesterone metabolites tested demonstrate weaker affinity, and agonistic and antagonistic potency. CONCLUSIONS: The binding affinity for hMR and the agonistic and antagonistic activity diminish with increasing reduction of the progesterone molecule at C20, C17 and at ring A. We assume that progesterone metabolism to these compounds is a possible protective mechanism for hMR. 17alpha-OH-P is a strong hMR antagonist and could exacerbate mineralocorticoid deficiency in patients with congenital adrenal hyperplasia.

Flammulin: A novel ribosome-inactivating protein from fruiting bodies of the winter mushroom Flammulina velutipes

A protein with a molecular weight of 40 kDa, capable of inhibiting cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 0.25 nM, was isolated from fruiting bodies of the mushroom Flammulina velutipes. The protein, designated flammulin, was devoid of ribonuclease activity. Flammulin was unadsorbed on DEAE-cellulose at neutral pH and low ionic strength and adsorbed on CM-Sepharose and Affi-gel blue gel under similar conditions. Its N-terminal sequence demonstrates sites of similarity to those of plant ribosome-inactivating proteins (RIPs).Key words: mushroom, ribosome-inactivating protein, fruiting body.

Characterization of an Internal Ribosomal Entry Segment in the 5′ Leader of Murine Leukemia Virus envRNA

ABSTRACT The 5′ untranslated region, also called the leader, of oncoretroviruses and lentiviruses is long and is formed of several structured domains critically important in virus replication. The 5′ leader of murine leukemia virus (MLV) RNA contains an internal ribosomal entry segment (IRES) which promotes synthesis of Gag and glyco-Gag polyprotein precursors. In the present study we investigated the translational features of the 5′ leader of MLV subgenomic RNA (env RNA) encoding the Env polyprotein precursor. When theenv leader was inserted between two genes, such aslacZ and the neomycin resistance cassette, in a dicistronic vector, it allowed IRES-dependent translation of the 3′ cistron in the rabbit reticulocyte lysate system and in murine cells. The drug rapamycin and the foot-and-mouth disease virus L protease, known to inhibit cap-dependent translation, caused an enhancement of the translation driven by the env leader sequence, consistent with an IRES activity promoting Env expression. Analysis of several deletion mutants led us to localize the minimal env IRES between the splice junction and the env AUG start codon.

Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins

ABSTRACT The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

The Cysteine-Rich Region of the Entamoeba histolytica Adherence Lectin (170-Kilodalton Subunit) Is Sufficient for High-Affinity Gal/GalNAc-Specific Binding In Vitro

ABSTRACT Adherence of Entamoeba histolytica trophozoites to colonic mucin, epithelium, and other target cells is mediated by the amebic Gal/GalNAc lectin. We constructed in vitro expression vectors containing full-length (residues 1 to 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin,hgl2. In vitro transcription followed by translation using a nuclease-treated rabbit reticulocyte lysate system was carried out. Immunoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipitation with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which recognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hgl2 enhanced immunoreactivity (P < 0.05) with the conformationally dependent MAb 3F4. Binding of PDI-refolded full-length (P < 0.001) and cysteine-rich (P = 0.005) Hgl2 to CHO cells was galactose dependent and competitively inhibited by native hololectin (50% inhibitory concentration of 39.6 ng/ml). The cysteine-poor region (1 to 353) did not bind CHO cells. Both full-length (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjugate GalNAc19BSA in a GalNAc-specific manner. The smaller cysteine-rich fragment (480 to 900) also exhibited GalNAc-specific binding but to a lesser extent (P < 0.05) than residues 1 to 1280 and 356 to 1143. Neither the cysteine-poor fragment (1 to 480), luciferase (protein control), nor control translation reactions (without hgl2 lectin mRNA) bound GalNAc19BSA. Binding to GalNAc19BSA was shown to be dependent on the concentration of GalNAc19BSA coated in each well or 35S-lectin added (KD = 0.85 ± 0.37 pM). Binding was competitively inhibited by the terminal GalNAc-containing glycoprotein asialofetuin (P < 0.005). Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subunit contains one or more carbohydrate-binding domains.