Characterization of two classes of small molecule inhibitors of Arp2/3 complex.

Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

Monoclonal antibodies against IREM-1: potential for targeted therapy of AML.

IREM-1 is an inhibitory cell surface receptor with an unknown function and is expressed on myeloid cell lineages, including cell lines derived from acute myeloid leukemia (AML) patients. We have generated a series of monoclonal antibodies (mAbs) against the extracellular domain of IREM-1 and further assessed its expression in normal and AML cells. IREM-1 was restricted to cells from myeloid origin and extensive expression analysis in primary cells obtained from AML patients showed IREM-1 expression in leukemic blasts of 72% (39/54) of samples. We therefore searched for specific IREM-1 mAbs with activity in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Lead mAbs against IREM-1 showed specific cytotoxic activity against a variety of AML-derived cell lines and freshly isolated blasts from AML patients. Internalization of mAbs upon IREM-1 binding was also shown. In vivo anticancer activity of lead mAbs was observed in an established HL-60 xenograft model with a tumor growth delay of up to 40% and in a model using primary human AML cells, where treatment with anti-IREM-1 mAb resulted in a significant reduction of engrafted human cells. These results demonstrate IREM-1 as a potential novel target for immunotherapy of AML.

Evidence for a novel GTPase priming step in the SRP protein targeting pathway.

Protein targeting by the signal recognition particle (SRP) pathway requires the interaction of two homologous GTPases that reciprocally regulate each other's GTPase activity, the SRP signal peptide- binding subunit (SRP54) and the SRP receptor alpha-subunit (SRalpha). The GTPase domain of both proteins abuts a unique 'N domain' that appears to facilitate external ligand binding. To examine the relationship between the unusual regulation and unique architecture of the SRP pathway GTPases, we mutated an invariant glycine in Escherichia coli SRP54 and SRalpha orthologs ('Ffh' and 'FtsY', respectively) that resides at the N-GTPase domain interface. A G257A mutation in Ffh produced a lethal phenotype. The mutation did not significantly affect Ffh function, but severely reduced interaction with FtsY. Likewise, mutation of FtsY Gly455 produced growth defects and inhibited interaction with Ffh. The data suggest that Ffh and FtsY interact only in a 'primed' conformation which requires interdomain communication. Based on these results, we propose that the distinctive features of the SRP pathway GTPases evolved to ensure that SRP and the SR engage external ligands before interacting with each other.

Identification of a U8 snoRNA-specific binding protein.

Eukaryotic nucleoli contain a large and diverse population of small nucleolar ribonucleoprotein particles (snoRNPs) that play diverse and essential roles in ribosome biogenesis. We previously demonstrated that U8 snoRNP is essential for processing of both 5.8 and 28 S rRNA. The RNA component of the U8 RNP particle is necessary but not sufficient for processing. Using an electrophoretic mobility sift assay, we enriched for U8-specific binding proteins from Xenopus ovary extracts. UV cross-linking reactions with partially purified fractions implicated a 29-kDa protein directly binding to U8 RNA. This protein interacted specifically and with high affinity with U8 snoRNA; it did not bind other snoRNAs and is probably not a common component of all snoRNPs. This is the first report of a protein component specific to an snoRNP essential for processing of the large ribosomal subunit in vertebrates.

Covalent glycoinositolphospholipid binding to hemoglobin: a new post-translational modification of Hb occurring in hyperinsulinism with concomitant hypoglycemia.

In this work a novel hitherto unrecognised minor hemoglobin (Hb) fraction, which we detected previously in hemolysates of erythrocytes exposed to a high concentration of insulin under hypoglycemic conditions, both in vivo and in vitro, is analysed. The modification of Hb in HbA1x was shown to be due the addition of glycoinositolphospholipid (GPI) to the C termini of both beta polypeptide chains. A structurally related minor Hb fraction was identified in erythrocytes exposed in vitro to insulin-mimetic agent, trypsin. To our knowledge this is the first demonstration of such a modification of Hb, as well as the first demonstration of post-translational GPI binding to proteins in response to insulin. The mechanism proposed for GPI-Hb formation is briefly described.

The effect of phenobarbital on the nonenzymatic glycation of hemoglobin.

The effect of phenobarbital (PB) on nonenzymatic glycation of hemoglobin in vivo and in vitro was studied. In epileptic patients on long-term therapy with PB, the yield of glycated hemoglobin fraction HbA1c was found to be significantly higher than in normal control. In samples of Hb exposed to glucose in vitro in the presence of PB, HbA1c was also found to be significantly higher than in those exposed to glucose in the absence of PB. Enhanced reactivity of beta-NH2 terminus of Hb with glucose, was partly explained by observing that its pKa was decreased in the presence of PB. It was shown that the effect of PB on pKa of beta-NH2 terminus is one of the consequences of the Hb-PB interactions which were studied in this work by electrophoretic and NMR techniques.

Modified hemoglobin in hypoglycemia caused by hyperinsulinism.

Hemoglobin (Hb) was found to be modified in patients with prolonged hypoglycemia caused by hyperinsulinism. This modification was of a nonpolar nature, did not affect the overall charge of the Hb and caused its aggregation in aqueous solution. After normal concentrations of glucose and insulin were reestablished, this modification slowly disappeared. Hemoglobin from normal erythrocytes incubated in vitro with high concentrations of insulin showed similar behavior.