SummaryThe heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.

Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.

In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.

Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern depending on cell type. Haploid a and α cells bud in the axial budding pattern, which depends on a transient marker and requires proteins Bud3, Bud4, Axl1 and Axl2. Here we report that Bud4 functions as a platform that mediates the ordered assembly of the axial landmark at the division site during M and early G1. While Bud4 associates with Bud3 in all cell types and in the absence of Axl1 or Axl2, Bud4 interacts with Axl1 and Axl2 mainly in haploid cells and only in the presence of all other components of the landmark. Bud4 can bind to GTP or GDP, and a GTP binding-defective Bud4 fails to interact with Axl1 in vitro. The same bud4 mutation leads to mis-localization of Axl1 and disrupts the axial budding pattern, indicating that GTP binding to Bud4 is important for its role in bud-site selection. We also show the cell-type-specific association of the axial landmark with Bud5, a GDP/GTP exchange factor for Rsr1. Despite their expression in all cell types, Bud4 and Axl2 associate with Bud5 specifically in haploid cells and in the presence of Axl1, whose expression is limited to a and α cells. Together, our findings suggest that Bud4 plays a critical role in the assembly of the axial landmark and its link to the Rsr1 GTPase module.

Background: Myostatin is a strong inhibitor of muscle growth and a therapeutic target for the treatment of muscle wasting. Results: Follistatin-like 3, a myostatin inhibitor, interacts uniquely with myostatin as compared with other ligands through its N-terminal domain. Conclusion:The N-terminal domains of follistatin-type molecules may be specificity determinants in ligand binding. Significance: Follistatin-type molecules form unique, specific interactions with different TGF-␤ family ligands.

Background: NBL1 is a moderate antagonist important for modulating bone morphogenetic protein (BMP) signaling in vivo. Results: Using x-ray crystallography and mutagenesis, regions important for BMP inhibition within NBL1 were identified. Conclusion: Modifications to the BMP binding epitope of NBL1 account for differences in its anti-BMP activity. Significance: This suggests that DAN proteins can be modified to be more effective antagonists for therapeutic purposes.

Background: GASP-1 and GASP-2 are highly specific antagonists for the TGF-␤ ligand myostatin, a negative regulator of muscle growth. Results: GASP-1 and GASP-2 form asymmetric and symmetric complexes with myostatin, respectively. Conclusion: Despite the different binding modes, the GASP proteins retain a high specificity for myostatin. Significance: Inhibition of myostatin can be achieved using different binding modes and may facilitate future development of novel anti-myostatin therapeutics.

Background: Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the a chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIa mAb. Objective: This study sought to use humanized mice to extend these results to humans. Methods: We actively immunized huFcεRIa/F709 mice, which express human (hu) instead of mouse FcεRIa and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIa mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cellstimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIa mAbs that bind FcεRIa regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIa binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. Results: Anti-huFcεRIa mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of na€ ıve, IgE-sensitized huFcεRIa mice and huFcεRIa/F709 mice that were egg-allergic with anti-FcεRIa mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIa mAbs also safely removed 98% of mast cell IgE and prevented IgE-mediated anaphylaxis. Conclusions: Rapid desensitization with anti-FcεRIa mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.