Histone arginine methylation keeps RUNX1 target genes in an intermediate state.

The coordinated recruitment of epigenetic regulators of gene expression by transcription factors such as RUNX1 (AML1, acute myeloid leukemia 1) is crucial for hematopoietic differentiation. Here, we identify protein arginine methyltransferase 6 (PRMT6) as a central functional component of a RUNX1 corepressor complex containing Sin3a and HDAC1 in human hematopoietic progenitor cells. PRMT6 is recruited by RUNX1 and mediates asymmetric histone H3 arginine-2 dimethylation (H3R2me2a) at megakaryocytic genes in progenitor cells. H3R2me2a keeps RUNX1 target genes in an intermediate state with concomitant H3K27me3 and H3K4me2 but not H3K4me3. Upon megakaryocytic differentiation PRMT6 binding is lost, the H3R2me2a mark decreases and a coactivator complex containing WDR5/MLL and p300/pCAF is recruited. This leads to an increase of H3K4me3 and H3K9ac, which result in augmented gene expression. Our results provide novel mechanistic insight into how RUNX1 activity in hematopoietic progenitor cells maintains differentiation genes in a suppressed state but poised for rapid transcriptional activation.

Immunomodulation in a novel model of experimental chronic pancreatitis.

Chronic pancreatitis is a disease that involves the lymphocytic inflammation of the pancreatic gland, the destruction and fibrous transformation of the endocrine and ductal structures. An involvement of the immune system in the disease progression is assumed and possibly allows immune modulation as a novel treatment strategy. We used a new model of experimental chronic pancreatitis to examine the effect of immune modulation with the mTOR-inhibitor rapamycin on clinical, chemical and histological parameters of chronic pancreatitis. Pancreatitis was induced by injecting 8 mg/kg bodyweight DBTC intravenously in male Sprague Dawley rats. 24 and 72 hours later, 20 µg/kg bodyweight cerulein was injected intraperitoneally to simulate recurrent attacks of pancreatitis typical for the clinical course. 48 hours after the DBTC injection, rats were randomly allocated to placebo or sirolimus (1.5 mg/kg bw i.p.). The treatment was repeated every 24hours for 5 days. The rats were sacrificed 7, 14, 21 and 35 days after DBTC injection. Histologic examination revealed a reduced acute pancreatic damage in the treatment group in the first week and less chronic changes in the further course. ALT and amylase increased in Placebo animals over the observation period and was lower in sirolimus treated animals. Oral glucose tolerance test showed that all placebo animals were diabetic four weeks after DBTC while sirolimus treated animals were normoglycemic. An early, limited treatment with immunomodulatory and antifibrotic agents like sirolimus can positively influence the detrimental course of experimental chronic pancreatitis and may offer a treatment alternative in humans.

Histone deacetylase inhibitors sensitize glioblastoma cells to TRAIL-induced apoptosis by c-myc-mediated downregulation of cFLIP.

Glioblastoma is the most common primary brain tumor with a very poor prognosis, calling for novel treatment strategies. Here, we provide first evidence that histone deacetylase inhibitors (HDACI) prime glioblastoma cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis at least in part by c-myc-mediated downregulation of cellular FLICE-inhibitory protein (cFLIP). Pretreatment with distinct HDACI (MS275, suberoylanilide hydroxamic acid, valproic acid) significantly enhances TRAIL-induced apoptosis in several glioblastoma cell lines. Monitoring a panel of apoptosis-regulatory proteins revealed that MS275 reduces the expression of cFLIP(L) and cFLIP(S). This leads to decreased recruitment of cFLIP(L) and cFLIP(S) and increased activation of caspase-8 to the TRAIL death-inducing signaling complex, resulting in enhanced cleavage of caspase-8, -9 and -3 and caspase-dependent apoptosis. Also, MS275 promotes TRAIL-triggered processing of Bid, activation of Bax, loss of mitochondrial membrane potential and release of cytochrome c. MS275-mediated downregulation of cFLIP occurs at the mRNA level independent of proteasome- or caspase-mediated degradation, and is preceded by upregulation of nuclear levels of c-myc, a transcriptional repressor of cFLIP. Notably, MS275 causes increased binding of c-myc to the cFLIP promoter and reduces cFLIP promoter activity. Indeed, knockdown of c-myc partially rescues cFLIP(L) from MS275-inferred downregulation and significantly decreases TRAIL- and MS275-induced apoptosis. Also, overexpression of cFLIP(L) or cFLIP(S) significantly reduces MS275- and TRAIL-induced apoptosis. Importantly, MS275 sensitizes primary cultured glioblastoma cells towards TRAIL and cooperates with TRAIL to reduce long-term clonogenic survival of glioblastoma cells and to suppress glioblastoma growth in vivo underscoring the clinical relevance of this approach. Thus, these findings demonstrate that HDACI represent a promising strategy to prime glioblastoma for TRAIL-induced apoptosis by targeting cFLIP.

Single doses of FK506 and OKT3 reduce severity in early experimental acute pancreatitis.

OBJECTIVE: To find out if two immunomodulatory drugs used in organ transplantation (FK506 (tacrolimus) and OKT3 (Orthoclone) would reduce early inflammatory complications in experimental acute pancreatitis. DESIGN: Laboratory study. SETTING: University hospital, Germany. ANIMALS: 36 Balb/c mice. INTERVENTIONS: Pancreatitis induced by 7 intraperitoneal injections of cerulein 50 microg/kg at hourly intervals followed by FK506 0.32 mg/kg, OKT3 0.6 mg/kg, or 0.9% sodium chloride (controls) (n = 12 in each group). 12 hours after induction of pancreatitis the animals were killed. MAIN OUTCOME MEASURES: Serum amylase activity and interleukin-6 (IL-6) concentrations; histological damage to pancreas and lungs, apoptotic cells in pancreas; and myeloperoxidase activity in lungs. RESULTS: No animal died during the experiment. At 12h serum amylase activity and IL-6 concentrations were increased in all 3 groups, but highest in the OKT3 group. The pancreatic histological score, apoptosis, and inflammatory infiltration were lower in the two experimental groups than controls, but the degree of vacuolisation of acinar cells was similar. Packed cell volume was higher in the control than the experimental groups, and pulmonary damage and myeloperoxidase activity were less in the experimental groups than the controls. CONCLUSION: Single therapeutic doses of FK506 and OKT3 reduced the early severity of pancreatitis, pulmonary damage, and haemoconcentration in mice. Single doses of FK506 or OKT3 may therefore be effective in preventing the early complications of pancreatitis.

The structure of the C949S mutant human alpha(2)-macroglobulin demonstrates the critical role of the internal thiol esters in its proteinase-entrapping structural transformation.

A three-dimensional reconstruction of a protein-engineered mutant alpha(2)-macroglobulin (alpha(2)M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed alpha(2)Ms. The native alpha(2)M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed alpha(2)M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed alpha(2)Ms were seen in electron micrographs and the C949S mutant and alpha(2)M-methylamine structures are highly similar, we conclude that the intact thiol esters maintain native alpha(2)M in a quasi-stable state. In their absence, alpha(2)M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.

Three-dimensional reconstructions of calcium/calmodulin-dependent (CaM) kinase IIalpha and truncated CaM kinase IIalpha reveal a unique organization for its structural core and functional domains.

Studies of the structural organization of calcium/ calmodulin-dependent protein kinase IIalpha (CaM KIIalpha) and truncated CaM KIIalpha by three-dimensional electron microscopy and protein engineering show that the structures consist of 12 subunits that are organized in two stacked hexameric rings with 622 symmetry. The body of CaM KIIalpha is gear-shaped, consisting of six slanted flanges, and has six foot-like processes attached by narrow appendages to both ends of the flanges. Truncated CaM KIIalpha that lacks functional domains has a structure that is very similar to the body of CaM KIIalpha. Thus, the functional domains reside in the foot-like processes, and the association domain comprises the gear-shaped core. The ribbon diagram of the bilobate structure of CaM KI fits nicely in the envelope of the foot-like component and indicates that the crevice between the two lobes comprising the functional domains is near the middle portion of the foot. The clustering of the functional domains provides a favorable arrangement for the autophosphorylation reaction, and the unusual arrangement of the catalytic domain on extended tethers appears to be significant for the remarkable functional diversity of CaM KIIalpha in cellular regulation.

Three-dimensional structure of the human plasmin alpha2-macroglobulin complex.

The three-dimensional reconstructions of the human plasmin alpha2-macroglobulin binary complex were computed from electron microscopy images of stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a molecule with approximate dimensions of 170 (length) x 140 (width) x 140 A (depth). The asymmetric plasmin structure imparts significant asymmetry to the plasmin alpha2-macroglobulin complex not seen in the structures resulting from the reaction of alpha2-macroglobulin with methylamine or chymotrypsin. The structure shows, when combined with other studies, that the C-terminal catalytic domain of the rod-shaped plasmin molecule is entrapped inside of the alpha2-macroglobulin cavity, whereas its N-terminal kringle domains protrude outside one end between the two arm-like features of the transformed alpha2-macroglobulin structure. This arrangement ensures that the catalytic site of plasmin is prevented from degrading plasma proteins. The internalized C-terminal portion of the plasmin structure resides primarily on the major axis of alpha2-macroglobulin, suggesting that after the initial cleavage of the two bait domains and the thiol esters, the rod-shaped plasmin molecule enters the alpha2-macroglobulin cavity through the large openings afforded by the half-transformed structure. This mode of entrapment requires the untwisting and the separation of the two strands that constitute the alpha2-macroglobulin structure.

On the unique structural organization of the Saccharomyces cerevisiae pyruvate dehydrogenase complex.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE2) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP.E3). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E2 scaffold. However, this investigation shows that the 12 large openings in the tE2 core permit the entrance of tBP, BP, and BP.E3 into a large central cavity where the BP component apparently binds near the tip of the tE2 trimer. The bone-shaped E3 molecule is anchored inside the central cavity through its interaction with BP. One end of E3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP.E3. Thus, E2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E3.BP components are determined by the geometric constraints of the underlying scaffold.

Utility of Butvar support film and methylamine tungstate stain in three-dimensional electron microscopy: agreement between stain and frozen-hydrated reconstructions.

A random conical tilt reconstruction of negatively stained Saccharomyces cerevisiae fatty acid synthase was used as a model to compute a three-dimensional reconstruction from untilted stain specimens of the molecules in multiple orientations using a three-dimensional projection alignment method. The resulting structure (24 A resolution) has a more uniform resolution than the initial structure and the handedness revealed in the random conical tilt method is preserved. In a similar approach, this model was used to compute a 21-A-resolution frozen-hydrated structure from untilted specimens of the molecules in multiple orientations. Even though the reconstructions are in close agreement, the stain structure appears to enhance the protein density associated with less robust features. These procedures significantly reduce the time and effort required to obtain a three-dimensional reconstruction from frozen-hydrated data with a resolution that is comparable to the best obtained by more laborious methods. The agreement between the stain and frozen-hydrated reconstructions affords convincing evidence concerning the validity of the structure and the information afforded by the two reconstructions significantly enhances the structural analysis of the molecule.

Structure-function relationships of the Saccharomyces cerevisiae fatty acid synthase. Three-dimensional structure.

The three-dimensional structure of the Saccharomyces cerevisie fatty acid synthase was computed from electron microscopy of stain images. The barrel-shaped structure (point group symmetry 32) has major and minor axes of approximately 245 x 220 A, respectively, and consists of two different subunits organized in an alpha6beta6 complex (Mr = 2.5 x 10(6)). Two sets of three beta subunits form triangle-shaped caps that enclose the ends of the barrel. The wall of the barrel appears to consist of three N-shaped alpha subunit pairs each with an over and underlying arch-shaped beta subunit. Inside the molecule there are three major interconnected cavities that are tilted approximately 20 degrees with respect to its major axis. An axle-shaped structure extends the length of the cavity on the 3-fold axis and is connected to the two ends of the barrel. The cavities are partially divided on the equator of the molecule by three spokes that extend from the axle on the 2-fold axis to the exterior wall. We propose that these six cavities constitute the six equivalent sites of fatty acid synthesis resulting in an extraordinary structure-function relationship with the 42 catalytic sites involved in fatty acid synthesis inside the molecule. The six cavities each have two funnel-shaped openings ( approximately 20 A in diameter) which may serve to permit the diffusion of substrates and products in and out of these functional units. The subunits appear to be arranged in a manner that affords extensive intermolecular interactions contributing to the stability of this macromolecular complex.

Stereoselective syntheses of 3-mercaptoproline derivatives protected for solid phase peptide synthesis.

The incorporation of cis- and trans-3-mercaptoproline (3-MPc and 3-MPt) into analogs of biologically active peptides has been shown to be an effective means for reducing the conformational mobility of the peptide backbone. We report herein a novel stereoselective synthetic route to L-1 and L-2, derivatives of 3-MPt and 3-MPc suitably protected for solid phase peptide synthesis. The optically active starting material was the previously reported cis-3-hydroxyprolinol derivative L-3. Oxidation of the C1 alcohol to the carboxylic acid, formation of the methyl ester and deprotection of the C3 alcohol yielded L-6 in an overall yield of 68%. Reaction of the secondary alcohol with thiolacetic acid under Mitsunobu conditions gave the thiolacetate L-7 in 77% yield with clean inversion of configuration. Conversion of L-7 to L-1 was accomplished in a one-pot sequence consisting of three steps: hydrolysis of the thiolacetate, formation of the thioether and hydrolysis of the methyl ester. The overall yield of L-1 from L-3 was 38%. Synthesis of L-2 required an epimerization of L-6, which was accomplished using a standard Mitsunobu inversion to give the trans-3-hydroxyproline derivative L-8. Transformation of L-8 to L-2 followed that described for overall yield of L-2 from L-3 was 18%. The availability of pure enantiomers of 3-MPt and 3-MPc protected for SPPS will greatly facilitate their use as conformational constraints for studying peptide-receptor interactions.

The novel three-dimensional structure of native human alpha 2-macroglobulin and comparisons with the structure of the methylamine derivative.

A three-dimensional reconstruction of alpha 2-macroglobulin (alpha 2 M) was computed from stain images. The structure appears to have point group symmetry 222 and, as also revealed by a tilt experiment, has the gross shape of a oval that displays a approximately 90 degrees twist in the body of the molecule. The reconstruction reveals a novel structure that consists of two Z-shaped components arranged in opposite orientation. These shapes are interconnected by two bridges at the elbow bends of the Z and by two archlike features that join their ends. The molecule has dimensions of approximately 190 x 125 x 120 A that encloses a 90 degrees twisted ellipsoidal shaped central cavity of 70 x 35 A. The cavity has four small openings arranged in a staggered configuration that extend to the outside. Serial slices of alpha 2 M and alpha 2 M-methylamine show that the bodies of the structures appear to be twisted in the opposite orientation. It is proposed that the four thioester bonds in the native molecule are responsible for maintaining its twisted configuration and that their cleavage with methylamine results in the structure becoming twisted in the opposite orientation. A comparison of average images derived from unstained particles of monoclonal Fab-labeled alpha 2 M and alpha 2 M-methylamine is consistent with this proposal. This unusual change in the handedness of alpha 2 M may have an important role in the encapsulation of the proteinase.

Conformationally readdressed CCK-B/delta-opioid peptide ligands.

The sequence of a cholecystokinin (CCK) related peptide was modified to obtain analogues, which interact selectively either with CCK-B, or with delta-opioid receptors. Two kinds of peptides were designed, namely, the cyclic peptides of the H-Tyr-cyclo (D-Pen-Gly-Trp-L/D-3-transmercaptoproline)-Asp-Phe-NH2 sequence (compounds 1a and 1b, respectively), and the linear peptides of the H-Tyr-D-Val-Gly-Trp-L/D-3-trans-methylmercaptoproline-Asp-Phe- NH2 sequence (compounds 2a and 2b, respectively). The only difference between the chemical structures of the linear analogues compared to the cyclic ones is that one covalent bond has been eliminated and a sulfur atom is replaced by a methyl group. Molecular modeling showed that, among low-energy conformers of cyclic compounds 1, there are three-dimensional structures compatible to the model for delta-receptor-bound conformer, suggested earlier [G. V. Nikiforovich, V.J. Hruby, O. Prakash, and C.A. Gehrig (1991) Biopolymers, vol. 31, pp. 941-955]. Results of binding assays fully supported the rationale for the design of compounds 1 and 2. The cyclic analogue 1a has Ki values of 4.5 and > 5000 nM at delta- and mu-opioid receptors, respectively; and IC50 values of 1.6 and > 10,000 nM for CCK-A and CCK-B receptors, respectively. The results of this study demonstrate a possibility to redirect a peptide sequence that interacts with one type of receptors (CCK-B receptors) toward interaction with another type (delta-opioid receptors) belonging to a different physiological system. This redirection could be performed by changing the conformational properties of the peptide with very minimal changes in its chemical structure.

Ac-[3- and 4-alkylthioproline31]-CCK4 analogs: synthesis and implications for the CCK-B receptor-bound conformation.

It has been reported that substitution of the Met31 residue in Boc-CCK4 (Boc-Trp30-Met31-Asp32-Phe33-NH2, CCK33 numbering) by trans-3-propyl-L-proline yields a highly potent and selective CCK-B agonist. To further explore the structural requirements of the Met31 side chain in the receptor-bound conformation of CCK4, we have synthesized several Ac-CCK4 analogs containing substitution of Met31 by 3- and 4-(alkylthio)-substituted proline derivatives. To this end we have developed novel synthetic routes to enantiomerically pure N-Boc-4-cis- and -trans-(methylthio)prolines and racemic N-Boc-3-cis and -trans-[(4-methylbenzyl)thio]prolines. The protected mercaptoprolines were incorporated into Ac-CCK4 analogs using SPPS and were alkylated using various electrophiles following cleavage from the solid support. Binding assays reveal that 3-(alkylthio)prolines analogs have higher affinities at the CCK-B receptor than the corresponding 4-(alkylthio)proline analogs, and that trans-3-(alkylthio)proline analogs had higher affinities than corresponding cis-3-(alkylthio)proline analogs. Within both the cis- and trans-3-(alkylthio)proline series, the order of potency was found to be Me < Et < n-Pr. The trans-3-(n-propylthio)-L-proline analog demonstrates a higher affinity than that reported for Boc-CCK4[trans-3-propyl-L-Pro31]. Comparison of the low-energy structures calculated for several high-affinity Ac-CCK4 analogs reveal a common geometry which we propose to be the CCK-B receptor-bound conformation. This model shows grouping of the hydrophobic side chains of Trp, Met, and Phe at one side of the molecule and the hydrophilic side chain of Asp and the C-terminal carboxamide at the other side.

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Three-dimensional structures of the human alpha 2-macroglobulin-methylamine and chymotrypsin complexes.

The three-dimensional structures of chymotrypsin- and methylamine-treated negatively stained human alpha 2-macroglobulin have been determined by weighted back projection from electron microscope data. Projections of the reconstructions show good concordance with two-dimensional averages of both stained and frozen-hydrated molecules. The reconstructions reveal that the H-shaped front projection of the molecule is related to the smaller ellipsoidal end view by a rotation of 90 degrees about the crossbar (minor axis) of the H. This finding is in agreement with tilt studies. The reconstruction of the alpha 2-macroglobulin-methylamine reveals an hour-glass shaped void which is filled by the two proteinase molecules in the reconstruction of alpha 2-macroglobulin-chymotrypsin. Protein plugs which appear to block the exterior entrances to the cavity may function to prevent access of proteins to the encapsulated proteinase and serve to block its escape. Extensive thresholding of each reconstruction leaves a "backbone" consisting of two side-by-side rod-like structures, suggesting that this is the arrangement of the two protomeric units which form the molecule. Both structures show some departure from the expected symmetry. The asymmetries are robust features of the reconstructions and may reflect structurally asymmetric features of the transformation from the native to the chymotrypsin-treated form of the molecule.

Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure.

The yeast fatty acid synthase (M(r) = 2.5 x 10(6)) is organized in an alpha 6 beta 6 complex. In these studies, the synthase structure has been examined by negative-stain and cryo-electron microscopy. Side and end views of the structure indicate that the molecule, shaped similar to a prolate ellipsoid, has a high-density band of protein bisecting its major axis. Stained and frozen-hydrated average images of the end views show an excellent concordance and a hexagonal ring having three each alternating egg- and kidney-shaped features with low-protein-density protrusions extending outward from the egg-shaped features. Images also show that the barrel-like structure is not hollow but has a Y-shaped central core, which appears to make contact with the three egg-shaped features. Numerous side views of the structure give good evidence that the beta subunits have an archlike shape. We propose a model for the synthase that has point-group symmetry 32 and six equivalent sites of fatty acid synthesis. The protomeric unit is alpha 2 beta 2. The ends of each of the two archlike beta subunits interact with opposite sides of the two dichotomously arranged disclike alpha subunits. Three such protomeric units form the ring. We propose that the six fatty acid synthesizing centers are composed of two complementary half-alpha subunits and a beta subunit, an arrangement having all the partial activities of the multifunctional enzyme required for fatty acid synthesis.