The PAF complex regulation of Prmt5 facilitates the progression and maintenance of MLL fusion leukemia.

Acute myeloid leukemia (AML) is a disease associated with epigenetic dysregulation. 11q23 translocations involving the H3K4 methyltransferase MLL1 (KMT2A) generate oncogenic fusion proteins with deregulated transcriptional potential. The polymerase-associated factor complex (PAFc) is an epigenetic co-activator complex that makes direct contact with MLL fusion proteins and is involved in AML, however, its functions are not well understood. Here, we explored the transcriptional targets regulated by the PAFc that facilitate leukemia by performing RNA-sequencing after conditional loss of the PAFc subunit Cdc73. We found Cdc73 promotes expression of an early hematopoietic progenitor gene program that prevents differentiation. Among the target genes, we confirmed the protein arginine methyltransferase Prmt5 is a direct target that is positively regulated by a transcriptional unit that includes the PAFc, MLL1, HOXA9 and STAT5 in leukemic cells. We observed reduced PRMT5-mediated H4R3me2s following excision of Cdc73 placing this histone modification downstream of the PAFc and revealing a novel mechanism between the PAFc and Prmt5. Knockdown or pharmacologic inhibition of Prmt5 causes a G1 arrest and reduced proliferation resulting in extended leukemic disease latency in vivo. Overall, we demonstrate the PAFc regulates Prmt5 to facilitate leukemic progression and is a potential therapeutic target for AMLs.

Synthesis, conformation, and immunosuppressive activity of CLX and its analogues.

The CLX peptide isolated from flax seed has a sequence cyclo-(PPFFILLX), where X is a nonproteinaceous amino acid residue, (2S,4R) 4-amine-N-methylproline. Picur, B.; Lisowski, M.; Siemion, I.Z. Letters Pept Sci 1998, 5, 183-187. The structure of X strongly suggests that this natural amino acid plays a role of the dipeptide moiety with a nonplanar cis peptidomimetic bond. The X residue contains two asymmetrical carbons and thus can appear in four configurations: (2S,4R), (2S,4S), (2R,4S), and (2R,4R). All four diastereoisomers of X were synthesized and characterized as trifluoroacetates of 4-phtalimido-N-methylproline benzylamides. Their full physicochemical characteristics are presented in this article. The synthesis of linear and cyclic analogues of CLX containing all four possible diastereoisomers of X was performed. Additionally, analogues with gamma-aminobutyric acid (GABA) and glycyl-N-methyl-glycine dipeptide [G(Me)G] substituted for X were synthesized. The obtained peptides were purified using HPLC, examined by ESI/MS, and then studied by CD spectroscopy. They were also tested for immunosuppressive activity (PFC in vitro). All of them revealed diverse immunosuppressive activity, however, lower than that of cyclolinopeptide A (CLA) Wieczorek, Z.; Bengtsson, B.; Trojnar, J.; Siemion, I.Z. Peptide Res 1991, 4, 275-283. and cyclosporine A (CsA). Ellis, G.P.; West, G.B. Progress Med Chem 1988, 25, 1-33. The structure of CLX with (2S,4R) 4-amino-N-methylproline was determined by 2-D NMR methods. All amide bonds are in the trans configuration. The cis peptidomimetic group delta-CH(2)-N(CH(3))- is exposed to the outside of the CLX molecule. The peptide contains two loops similar to beta-turns of type IV. Chou, P.Y.; Fasman, G.D. J Mol Biol 1977, 115, 136-715 and has the extended shape flanked by F3 and L7 residues with significant side chain flexibility.

Canonical protein inhibitors of serine proteases.

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising alpha-helical, beta sheet, and alpha/beta proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.

Structure-function relationship of serine protease-protein inhibitor interaction.

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

Amide proton temperature coefficients as hydrogen bond indicators in proteins.

Correlations between amide proton temperature coefficients (deltasigmaHN/deltaT) and hydrogen bonds were investigated for a data set of 793 amides derived from 14 proteins. For amide protons showing temperature gradients more positive than -4.6 ppb/K there is a hydrogen bond predictivity value exceeding 85%. It increases to over 93% for amides within the range between -4 and -1 ppb/K. Detailed analysis shows an inverse proportionality between amide proton temperature coefficients and hydrogen bond lengths. Furthermore, for hydrogen bonds of similar bond lengths, values of temperature gradients in alpha-helices are on average I ppb/K more negative than in beta-sheets. In consequence. a number of amide protons in alpha-helices involved in hydrogen bonds shorter than 2 A show deltasigmaHN/deltaT < -4.6 ppb/K. Due to longer hydrogen bonds, 90% of amides in 3(10) helices and 98% in beta-turns have temperature coefficients more positive than -4.6ppb/K. Ring current effect also significantly influences temperature coefficients of amide protons. In seven out of eight cases non-hydrogen bonded amides strongly deshielded by neighboring aromatic rings show temperature coefficients more positive than -2 ppb/K. In general, amide proton temperature gradients do not change with pH unless they correspond to conformational changes. Three examples of pH dependent equilibrium showing hydrogen bond formation at higher pH were found. In conclusion, amide proton temperature coefficients offer an attractive and simple way to confirm existence of hydrogen bonds in NMR determined structures.

NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors.

The three-dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI-1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular beta-sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment. A detailed analysis of the protease binding loop conformation reveals that it is similar to those found in other canonical serine protease inhibitors. The AMCI-1 structure exhibits a common fold with a novel family of inhibitors from the intestinal parasitic worm Ascaris suum. The pH-induced conformational changes in the binding loop region observed in the Ascaris inhibitor ATI are absent in AMCI-1. Similar binding site sequences and structures strongly suggest that the lack of the conformational change can be attributed to a Glu-->Gln substitution at the P1' position in AMCI-1, compared to ATI. Analysis of amide proton temperature coefficients shows very good correlation with the presence of hydrogen bond donors in the calculated AMCI-1 structure.

Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks.

The solution structure of a novel 69 residue proteinase inhibitor, Linum usitatissimum trypsin inhibitor (LUTI), was determined using a method based on computer aided assignment of nuclear Overhauser enhancement spectroscopy (NOESY) data. The approach applied uses the program NOAH/DYANA for automatic assignment of NOESY cross-peaks. Calculations were carried out using two unassigned NOESY peak lists and a set of determined dihedral angle restraints. In addition, hydrogen bonds involving amide protons were identified during calculations using geometrical criteria and values of HN temperature coefficients. Stereospecific assignment of beta-methylene protons was carried out using a standard procedure based on nuclear Overhauser enhancement intensities and 3J(alpha)(beta) coupling constants. Further stereospecific assignment of methylene protons and diastereotopic methyl groups were established upon structure-based method available in the program GLOMSA and chemical shift calculations. The applied algorithm allowed us to assign 1968 out of 2164 peaks (91%) derived from NOESY spectra recorded in H2O and 2H2O. The final experimental data input consisted of 1609 interproton distance restraints, 88 restraints for 44 hydrogen bonds, 63 torsion angle restraints and 32 stereospecifically assigned methylene proton pairs and methyl groups. The algorithm allowed the calculation of a high precision protein structure without the laborious manual assignment of NOESY cross-peaks. For the 20 best conformers selected out of 40 refined ones in the program CNS, the calculated average pairwise rmsd values for residues 3 to 69 were 0.38 A (backbone atoms) and 1.02 A (all heavy atoms). The three-dimensional LUTI structure consists of a mixed parallel and antiparallel beta-sheet, a single alpha-helix and shows the fold of the potato 1 family of proteinase inhibitors. Compared to known structures of the family, LUTI contains Arg and Trp residues at positions P6' and P8', respectively, instead of two Arg residues, involved in the proteinase binding loop stabilization. A consequence of the ArgTrp substitution at P8' is a slightly more compact conformation of the loop relative to the protein core.

Isolation And Structure Elucidation Of 5,7-disubstituted Simple Coumarins In The Fruits Of Heracleum Mantegazzianum.

In addition to bergapten ( 1 ), xanthotoxin ( 2 ), isopimpinellin ( 3 ), imperatorin ( 4 ), and pimpinellin ( 5 ), for the first time 5,7-disubstituted simple coumarins, limettin (5,7-dimethoxycoumarin) ( 6 ) and a new compound 5-methoxy-7-(3,3-dimethylalliloxy)-coumarin ( 7 ), have been isolated from the fruits of Heracleum mantegazzianum Somm. et Lev. The new compound 7 is a derivative of anisocoumarin B. The structures of 6 and 7 were determined on the basis of 1D and 2D NMR spectra.

1H NMR Studies of Aqueous Micellar Solutions of N-Dodecyl- N,N-dimethyl-N-benzylammonium Chloride.

(1)H NMR studies of the micellization process in N-dodecyl-N,N-dimethyl-N-benzylammonium chloride (DBeAC) were carried out. The spectra were measured at concentrations below and above the critical micelle concentration (CMC). The chemical shifts and Nuclear Overhauser Effect (NOE) measurements were analyzed as a function of the concentration. This allowed us to provide experimental evidence that the benzyl group of DBeAC changes its position during micellization and, as a consequence, is located inside the micelle. Copyright 1999 Academic Press.